Patent application title: NOGO Receptor Homologs
Inventors:
Stephen M. Strittmatter (Guilford, CT, US)
Richard L. Cate (Cohasset, MA, US)
Dinah W.y. Sah (Boston, MA, US)
Dinah W.y. Sah (Boston, MA, US)
IPC8 Class: AA61K39395FI
USPC Class:
4241391
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds antigen or epitope whose amino acid sequence is disclosed in whole or in part (e.g., binds specifically-identified amino acid sequence, etc.)
Publication date: 2011-06-02
Patent application number: 20110129477
Abstract:
The invention relates generally to genes that encode proteins that
inhibit axonal growth. The invention relates specifically to genes
encoding NgR protein homologs in humans and mice. The invention also
includes compositions and methods for modulating the expression and
activity of Nogo and the NgR proteins. Specifically, the invention
includes peptides, proteins and antibodies that block Nogo-mediated
inhibition of axonal extension. The compositions and methods of the
invention are useful in the treatment of cranial or cerebral trauma,
spinal cord injury, stroke or a demyelinating disease.Claims:
1-3. (canceled)
4. An isolated nucleic acid encoding the polypeptide of SEQ ID NO: 2.
5-16. (canceled)
17. An isolated polypeptide comprising an amino sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:14.
18-22. (canceled)
23. An isolated antibody that binds to the polypeptide of claim 17.
24-25. (canceled)
26. A method of decreasing inhibition of axonal growth of a CNS neuron, comprising the step of contacting the neuron with an effective amount of the polypeptide of claim 17.
27. A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of the polypeptide of claim 17.
28. A method of decreasing inhibition of axonal growth of a CNS neuron comprising the step of contacting the neuron with an effective amount of the antibody according to claim 23.
29. A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of the antibody according to claim 23.
30. A method for identifying a molecule that binds the polypeptide of claim 17 comprising: (a) providing the polypeptide of claim 17; (b) contacting the polypeptide with a candidate molecule; (c) detecting binding of the candidate molecule to said polypeptide.
Description:
FIELD OF THE INVENTION
[0001] The invention relates to neurology and molecular biology. More particularly, the invention relates to CNS neurons and axonal growth
BACKGROUND
[0002] Among the mechanisms through which the cells of an organism communicate with each other and obtain information and stimuli from their environment is through cell membrane receptor molecules expressed on the cell surface. Many such receptors have been identified, characterized, and sometimes classified into major receptor superfamilies based on structural motifs and signal transduction features. The receptors are a first essential link for translating an extracellular signal into a cellular physiological response.
[0003] Receptors on neurons are particularly important in the development of the nervous system during embryogenesis. The neurons form connections with target cells during development through axonal extension of the neurons toward the target cells in a receptor-mediated process. Axons and dendrites have a specialized region of their distal tips known as the growth cone. Growth cones enable the neuron to sense the local environment through a receptor-mediated process and direct the movement of the axon or dendrite of the neuron toward the neuron's target cell. This process is known as elongation. Growth cones can be sensitive to several guidance cues, for example, surface adhesiveness growth factors, neurotransmitters and electric fields. The guidance of growth at the cone depends on various classes of adhesion molecules, intercellular signals, as well as factors that stimulate and inhibit growth cones.
[0004] Interestingly, damaged neurons do not elongate in the central nervous system (CNS) following injury due to trauma or disease, whereas axons in the peripheral nervous system (PNS) regenerate readily. The fact that damaged CNS neurons fail to elongate is not due to an intrinsic property of CNS axons, but rather due to the CNS environment that is not permissive for axonal elongation. Classical grafting experiments by Aguayo and colleagues (e.g., Richardson et al., (1980) Nature 284, 264-265) demonstrated that CNS axons can in fact elongate over substantial distances within peripheral nerve grafts, and that CNS myelin inhibits CNS axon elongation. Therefore, given the appropriate environment, CNS axons can regenerate, implying that CNS axonal injury can potentially be addressed by appropriate manipulation of the CNS environment.
[0005] The absence of axon regeneration following injury can be attributed to the presence of axon growth inhibitors. These inhibitors are predominantly associated with myelin and constitute an important barrier to regeneration. Axon growth inhibitors are present in CNS-derived myelin and the plasma membrane of oligodendrocytes that synthesize myelin in the CNS (Schwab et al., (1993) Annu. Rev. Neurosci. 16, 565-595). Myelin-associated inhibitors appear to be a primary contributor to the failure of CNS axon regeneration in vivo after an interruption of axonal continuity, whereas other non-myelin associated axon growth inhibitors in the CNS may play a lesser role. These inhibitors block axonal regeneration following neuronal injury due to trauma, stroke or viral infection.
[0006] Numerous myelin-derived axon growth inhibitors have been characterized (see, for review, David et al., (1999) WO995394547; Bandman et al., (1999) U.S. Pat. No. 5,858,708; Schwab, (1996) Neurochem. Res. 21, 755-761). Several components of CNS white matter, NI35, NI250 (Nogo) and Myelin-associated glycoprotein (MAG), which have inhibitory activity for axonal extension, have been described as well (Schwab et al., (1990) WO9005191; Schwab et al., (1997) U.S. Pat. No. 5,684,133). In particular, Nogo is a 250 kDa myelin-associated axon growth inhibitor that was originally characterized based on the effects of the purified protein in vitro and monoclonal antibodies that neutralize the protein's activity (Schwab (1990) Exp. Neurol. 109, 2-5). The Nogo cDNA was first identified through random analysis of brain cDNA and had no suggested function (Nagase et al., (1998) DNA Res. 5, 355-364). The identification of this Nogo cDNA as the cDNA encoding the 250 kDa myelin-associated axon growth inhibitor was discovered only recently (GrandPre et al., (2000) Nature 403, 439-444; Chen et al., (2000) Nature 403, 434-439; Prinjha at al., (2000) Nature 403, 383-384).
[0007] Importantly, Nogo has been shown to be the primary component of CNS myelin responsible for inhibiting axonal elongation and regeneration. Nogo's selective expression by oligodendrocytes and not by Schwann cells (the cells that myelinate P.S. axons) is consistent with the inhibitory effects of CNS myelin, in contrast to P.S. myelin (GrandPre et al., (2000) Nature 403, 434-439). In culture, Nogo inhibits axonal elongation and causes growth cone collapse (Spillmann et al., (1998) J. Biol. Chem. 272, 19283-19293). Antibodies (e.g., IN-1) against Nogo have been shown to block most of the inhibitory action of CNS myelin on neurite growth in vitro (Spillmann et al., (1998) J. Biol. Chem. 272:19283-19293). These experiments indicate that Nogo is the main component of CNS myelin responsible for inhibition of axonal elongation in culture. Furthermore, in vivo, the IN-1 antibody has been shown to enhance axonal regeneration after spinal cord injury, resulting in recovery of behaviors such as contact placing and stride length (Schnell and Schwab (1990) Nature 343, 269-272; Bregman et al., (1995) Nature 378, 498-501). Thus, there is substantial evidence that Nogo is a disease-relevant molecular target. Agents that interfere with the binding of Nogo to its receptor would be expected to improve axonal regeneration in clinical states in which axons have been damaged, and improve patient outcome.
[0008] Modulation of Nogo has been described as a means for treatment of regeneration for neurons damaged by trauma, infarction and degenerative disorders of the CNS (Schwab et al., (1994) WO9417831; Tatagiba et al., (1997) Neurosurgery 40, 541-546) as well as malignant tumors in the CNS such as glioblastoma (Schwab et al., (1993) U.S. Pat. No. 5,250,414); Schwab et al., (2000) U.S. Pat. No. 6,025,333).
[0009] Antibodies which recognize Nogo have been suggested to be useful in the diagnosis and treatment of nerve damage resulting from trauma, infarction and degenerative disorders of the CNS (Schnell & Schwab, (1990) Nature 343, 269-272, Schwab et al., (1997) U.S. Pat. No. 5,684,133). For CNS axons, there is a correlation between the presence of myelin and the inhibition of axon regeneration over long distances (Savio and Schwab (1990) Proc. Natl. Acad. Sci. 87, 4130-4133; Keirstead et al., (1992) Proc. Natl. Acad. Sci. 89, 11664-11668). After Nogo is blocked by antibodies, neurons can again extend across lesions caused by nerve damage (Schnell and Schwab (1990) Nature 343, 269-272).
SUMMARY OF THE INVENTION
[0010] Genes encoding homologs (NgR2 and NgR3) of a Nogo receptor (NgR1) in mice and humans have been discovered. Various domains in the polypeptides encoded by the NgR2 and NgR3 genes have been identified and compared to domains in mouse and human NGR1 polypeptides. This comparison has led to identification of a consensus sequence (NgR consensus sequence) that characterizes a family of proteins (NgR family). Based on these and other discoveries, the invention features molecules and methods for modulating axonal growth in CNS neurons.
[0011] The invention provides a polypeptide that contains a polypeptide containing a tryptophan rich LRRCT domain consisting of the amino acid sequence:
TABLE-US-00001 [SEQ ID NO: 19] N X1 W X2 C X3 C R A R X4 L W X5 W X6 X7 X8 X9 R X10 S S S X11 V X12 C X13 X14 P X15 X16 X17 X18 X19 X20 D L X21 X22 L X23 X24 X25 D X26 X27 X28 C
[0012] wherein X is any protein amino acid or a gap, and the polypeptide does not include amino acid sequence from residue 260 to 309 of SEQ ID NO: 5 (human NGR1) or SEQ ID NO: 17 (mouse NgR1).
[0013] Preferably, X17 and X23 are (independently) arginine or lysine. In some embodiments, the amino acid sequence of the LRRCT domain is residues 261-310 of SEQ ID NO:2, or residues 261-310 of SEQ ID NO. 2 with up to 10 conservative amino acid substitutions. In some embodiments, the polypeptide contains the following NTLRRCT amino acid sequence:
TABLE-US-00002 [SEQ ID NO: 18] C P X1 X2 C X3 C Y X4 X5 P X6 X7 T X8 S C X9 X10 X11 X12 X13 X14 X15 X16 P X17 X18 X19 P X20 X21 X22 X23 R X24 F L X25 X26 N X27 I X28 X29 X30 X31 X32 X33 X34 F X35 X36 X37 X38 X39 X40 X41 X42 L W X43 X44 S N X45 X46 X47 X48 I X49 X50 X51 X52 F X53 X54 X55 X56 X57 L E X58 L D L X59 D N X60 X61 L X62 X63 X64 X65 P X66 T F X67 G L X68 X69 L X70 X71 L X72 L X73 X74 C X75 L X76 X77 X78 X79 X80 X81 F X82 G L X83 X84 L Q Y L Y L Q X85 N X86 X87 X88 X89 L X90 D X91 X92 F X93 D L X94 N L X95 H L F L H G N X96 X97 X98 X99 X100 X101 X102 X103 X104 F R G L X105 X106 L D R L L L H X107 N X108 X109 X110 X111 V H X112 X113 A F X114 X115 L X116 R L X117 X118 L X119 L F X120 N X121 L X122 X123 L X124 X125 X126 X127 L X128 X129 L X130 X131 L X132 X133 L R L N X134 N X135 W X136 C X137 C R X138 R X139 L W X140 W X141 X142 X143 X144 R X145 S S S X146 V X147 C X148 X149 P X150 X151 X152 X153 X154 X155 D L X156 X157 L X158 X159 X160 D X161 X162 X163 C
wherein X is any amino acid residue or a gap and wherein the polypeptide is not the polypeptide of SEQ ID NO: 5 (human NGR1) or SEQ ID NO: 17 (mouse NgR1). For example, X6, X37 and X38 may represent a gap. Specific examples of polypeptides of the invention are SEQ ID NO: 2 (human NgR2), SEQ ID NO: 4 (mouse NgR3), and SEQ ID NO: 14 (human NgR3). In some embodiments, the polypeptide contains: (a) a NTLRRCT domain, and (b) less than a complete CTS domain, provided that a partial CTS domain, if present, consists of no more than the first 39 amino acids of the CTS domain. While the polypeptide may contain a functional GPI domain, a functional GPI domain may be absent, e.g., when a soluble polypeptide is desired. A polypeptide of the invention optionally includes an amino acid sequence of a heterologous polypeptide, e.g., an Fc portion of an antibody.
[0014] The invention also provides a nucleic acid encoding an above-described polypeptide; a vector containing the nucleic acid, which nucleic acid may be operably linked to an expression control sequence; and a transformed host cell containing the vector. A method of producing a polypeptide of the invention is also provided. The method includes introducing a nucleic acid encoding the above-described polypeptide into a host cell, culturing the cell under conditions suitable for expression of the polypeptide, and recovering the polypeptide.
[0015] The invention also provides an antisense molecule whose nucleotide sequence is complementary to a nucleotide sequence encoding a polypeptide selected from the group consisting of: a polypeptide consisting of residues 311-395 of SEQ ID NO: 2, a polypeptide consisting of residues 256-396 of SEQ ID NO: 14 and a polypeptide consisting of residues 321-438 of SEQ ID NO: 4, wherein the nucleic acid is from 8 to 100 nucleotides in length, e.g., about 20, 30, 40, 50, 60, 70, 80 or 90 nucleotides. The invention also provides a nucleic acid encoding such an antisense molecule.
[0016] The invention also provides an antibody that binds to an above-described polypeptide. Polypeptides or antibodies of the invention can be formulated into pharmaceutical compositions containing the polypeptide or antibody and a pharmaceutically acceptable carrier.
[0017] The invention also provides a method for decreasing inhibition of axonal growth of a CNS neuron. The method includes the step of contacting the neuron with an effective amount of a polypeptide or antibody of the invention.
[0018] The invention also provides a method for treating a central nervous system disease, disorder or injury. The method includes administering to a mammal, e.g., a human, an effective amount of a polypeptide or antibody of the invention. Exemplary diseases, disorders and injuries that may be treated using molecules and methods of the invention include, but are not limited to, cerebral injury, spinal cord injury, stroke, demyelinating diseases, e.g., multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy and Krabbe's disease.
[0019] The invention also provides a method for identifying a molecule that binds a polypeptide of the invention. The method includes the steps of: (a) providing a polypeptide of the invention; (b) contacting the polypeptide with the candidate molecule; and (c) detecting binding of the candidate molecule to the polypeptide.
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In case of conflict, the present application, including definitions, will control. All publications, patent and other references mentioned herein are incorporated by reference.
[0021] The materials, methods and examples presented below are illustrative only, and not intended to be limiting. Other features and advantages of the invention will be apparent from the detail description and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1A-1B shows an alignment of NgR2 (SEQ ID NO:2) and NgR3 (SEQ ID NO:4) with the known NgR, NgR1 (SEQ ID NO:5) and the Consensus Sequence (SEQ ID NO:6).
[0023] FIG. 2. mNgR3 does not bind hNogoA(1055-1120). COS-7 cells were transfected with vectors encoding myc-NgR1 or myc-NgR3, fixed, and stained with anti-myc antibodies or AP-hNogoA(1055-1120).
[0024] FIG. 3. An alignment of the amino acid sequences of human NgR1, murine NGR1, murine NgR3, human NgR3 and human NgR2. Numbering begins with amino acid #1 of murine NgR3. The consensus sequence is listed below. The LRR NT domain is indicated by a shaded box; domains LLR 1, LLR 3, LLR 5, and LLR 7 are indicated by open boxes; LLR 2, LLR 4, LLR 6 and LLR 8 are indicated by shaded boxes; and the LLR CT domain is indicated by a shaded box. Amino acids in bold in LLR 8 indicate a conserved glycosylation sites. A dot indicates conserved cystine residue in LRR4. Box at C terminus indicates putative GPI signals.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) encoding NgR homologs, referred to herein as NgR. Unless indicated otherwise, as used herein, the abbreviation in lower case (NgR) refers to a gene, cDNA, RNA or nucleic acid sequence, whereas the upper case version (NgR) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence. Specific proteins are designated by number, e.g., "NgR2" is a human NgR homolog, "NgR3" is a murine-derived NgR homolog, and "NgR1" is the known NgR identified by Dr. Stephen Strittmatter. Known NgRs are herein referred to as "NgRs." DNA polynucleotides of the invention include genomic DNA, cDNA and DNA that has been chemically synthesized in whole or in part.
[0026] Standard reference works setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York (1998); Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed., DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).
[0027] As used herein, the term "axon" refers to a long cellular protrusion from a neuron, whereby action potentials are conducted, either to or from the cell body.
[0028] As used herein, the term "axonal growth" refers to an extension of the long process or axon, originating at the cell body and proceeded by the growth cone.
[0029] As used herein, the term "central nervous system disorder" refers to any pathological state associated with abnormal function of the central nervous system (CNS). The term includes, but is not limited to, altered CNS function resulting from physical trauma to cerebral tissue, viral infection, autoimmune mechanisms and genetic mutation.
[0030] As used herein, the term "demyelinating disease" refers to a pathological disorder characterized by the degradation of the myelin sheath of the oligodendrocyte cell membrane.
[0031] As used herein, the term "growth cone" refers to a specialized region at the tip of a growing neurite that is responsible for sensing the local environment and moving the axon toward its appropriate synaptic target cell.
[0032] As used herein, the term "growth cone movement" refers to the extension or collapse of the growth cone toward a neuron's target cell.
[0033] As used herein, the term "neurite" refers to a process growing out of a neuron. As it is sometimes difficult to distinguish a dendrite from in axon in culture, the term "neurite" is used for both.
[0034] As used herein, the term "oligodendrocyte" refers to a neuroglial cell of the CNS whose function is to myelinate CNS axons.
[0035] "Synthesized" as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. "Wholly" synthesized DNA sequences are therefore produced entirely by chemical means, and "partially" synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. By the term "region" is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. The term "domain" is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region. Examples of NgR protein domains include, but are not limited to, the signal peptide, extracellular (i.e., N-terminal) domain, and leucine-rich repeat domains.
[0036] As used herein, the term "activity" refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e., having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event. Such activities may be measured by assays such as competitive inhibition of NGR1 binding to Nogo assays wherein, for example, unlabeled, soluble NgR2 is added to an assay system in increasing concentrations to inhibit the binding of Nogo to NGR1 expressed on the surface of CHO cells. As another example, one may assess the ability of neurons to extend across lesions caused by nerve damage (as in Schnell and Schwab (1990) Nature 343, 269-272) following inhibition of Nogo by various forms of NgR2 and/or NgR3 as a biological indicator of NgR function.
[0037] As used herein, the term "antibody" is meant to refer to complete, intact antibodies, and Fab, Fab', F(ab)2, and other fragments thereof. Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies, anti-anti-idiotypic antibodies, and humanized antibodies.
[0038] As used herein, the term "binding" means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.
[0039] As used herein, the term "compound" means any identifiable chemical or molecule, including, but not limited to, small molecules, peptides, proteins, sugars, nucleotides or nucleic acids, and such compound can be natural or synthetic.
[0040] As used herein, the term "complementary" refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.
[0041] As used herein, the term "contacting" means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention. The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc. Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding the NgR or fragment thereof.
[0042] As used herein, the phrase "homologous nucleotide sequence," or "homologous amino acid sequence," or variations thereof, refers to sequences characterized by an identity at the nucleotide level, or a homology at the amino acid level, of at least the specified percentage. Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding NgR1. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity. A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known NgRs. Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math, 1981, 2, 482-489, which is incorporated herein by reference in its entirety).
[0043] As used herein, the term "isolated" nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that is substantially free of nucleic acids encoding other proteins with which it is associated in nature, i.e., a nucleic acid that has been removed from its native environment. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic. DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NgR nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
[0044] As used herein, the term "heterologous" refers to a nucleotide or amino acid sequence that is a different, or non-corresponding sequence, or a sequence derived from a different species. For example, a mouse NgR nucleotide or amino acid sequence is heterologous to a human NgR nucleotide or amino acid sequence, and a human NgR nucleic or amino acid sequence is heterologous to a human immunoglobulin nucleotide or amino acid sequence.
[0045] As used herein, a "soluble NgR polypeptide" is a NgR polypeptide that does not anchor itself in a membrane. Such soluble polypeptides include, for example, NgR2 and NgR3 polypeptides that lack a sufficient portion of their GPI anchor signal to anchor the polypeptide or are modified such that the GPI anchor signal is not adequate to result in replacement of the peptide with a GPI anchor. In preferred embodiments, up to 5, 10, 20 or 25 amino acids are removed from the C-terminus of NgR2 or NgR3 to make the respective proteins soluble. As used herein soluble NgR polypeptides include full-length or truncated (e.g., with internal deletions) NgR.
[0046] Soluble NgR polypeptides may include the entire NgR protein up to the putative GPI signal sequence (e.g., amino acid 1 to about amino acid 395 of NgR2, and from amino acid 1 to about amino acid 438 of NgR3). In other embodiments, the signal peptide of the proteins may be removed or truncated (e.g., all or part of the signal sequence of NgR2, which spans amino acid 1 to about amino acid 30 of SEQ ID NO:2, may be removed; all or part of the signal sequence of NgR3, which spans amino acid 1 to about amino acid 40 of SEQ ID NO:4, may be removed). In some embodiments, the mature NgR2 (SEQ ID NO:8) and the mature NgR3 (SEQ ID NO:9) are used.
[0047] Soluble NgR polypeptides include at least one of the putative ligand-binding portions of NgR, including the first cysteine-rich region (SEQ ID NO:10, the leucine repeat region (SEQ ID NO:12) and the second cysteine-rich region (SEQ ID NO:11). In some embodiments, soluble NgR polypeptides consist of amino acid 1 through about amino acid 395 of SEQ ID NO:2, or amino acid 1 through about amino acid 438 of SEQ ID NO:4.
[0048] In other embodiments, the soluble NgR polypeptides are fusion proteins that contain amino acids 30 through about amino acid 395 of mature NgR2 or amino acid 40 through about amino acid 438 of NgR3, the C-terminal 10 amino acids of a human IgG 1 hinge region containing the two cysteine residues thought to participate in interchain disulfide bonding, and the CH2 and CH3 regions of a human IgGI heavy chain constant domain. This type of recombinant protein is designed to modulate inhibition of axonal elongation through inhibition of the Nogo ligand binding to NGR1, or by inhibiting the ligand of the NgR from interacting with cell surface NgR The NgR portion of the fusion binds to the Nogo ligand and the IgG1 portion binds to the FcγRI (macrophage) and FcγIII (NK cells and neutrophils) receptors.
[0049] The production of the soluble polypeptides useful in this invention may be achieved by a variety of methods known in the art. For example, the polypeptides may be derived from intact transmembrane NgR molecules by proteolysis using specific endopeptidases in combination with exopeptidases, Edman degradation, or both. The intact NgR molecule, in turn, may be purified from its natural source using conventional methods. Alternatively, the intact NgR may be produced by known recombinant DNA techniques using cDNAs, expression vectors and well-known techniques for recombinant gene expression.
[0050] Preferably, the soluble polypeptides useful in the present invention are produced directly, thus eliminating the need for an entire NgR as a starting material. This may be achieved by conventional chemical synthesis techniques or by well-known recombinant DNA techniques wherein only those DNA sequences which encode the desired peptides are expressed in transformed hosts. For example, a gene which encodes the desired soluble NgR polypeptide may be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired soluble NgR polypeptide. Specific DNA sequences coding for the desired peptide also can be derived from the full-length DNA sequence by isolation of specific restriction endonuclease fragments or by PCR synthesis of the specified region from cDNA.
[0051] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs:1, 3 or a complement of either of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NOs:1 or 3 as a hybridization probe, NgR nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g. as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).
[0052] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NgR nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
[0053] As used herein, the terms "modulates" or "modifies" means an increase or decrease in the amount, quality, or effect of a particular activity or protein.
[0054] As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes.
[0055] As used herein, the term "probe" refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
[0056] The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition.
[0057] The term "treating" refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
[0058] The term "therapeutic effect" refers to the inhibition or activation factors causing or contributing to the abnormal condition. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
[0059] The term "abnormal condition" refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival. An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism.
[0060] Abnormal cell proliferative conditions, for example, include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus and inflammation.
[0061] Abnormal differentiation conditions include, for example, neurodegenerative disorders, slow wound healing rates and slow tissue grafting healing rates.
[0062] Abnormal cell signaling conditions include, for example, psychiatric disorders involving excess neurotransmitter activity.
[0063] Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
[0064] The term "administering" relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.
[0065] The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human.
[0066] By "amplification" it is meant increased numbers of DNA or RNA in a cell compared with normal cells. "Amplification" as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, compared to the basal level.
[0067] The amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. The nucleotide sequences are presented by single strand only, in the 5' to 3' direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by three letters code.
Nucleic Acids
[0068] Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or "spliced out." RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a NgR polypeptide, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts. Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild-type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants arising from in vitro manipulation).
[0069] The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding NgR (conventionally followed by second-strand synthesis of a complementary strand to provide a double-stranded DNA).
[0070] Preferred DNA sequences encoding a human NgR polypeptide is set out in SEQ ID NOs:1 and 13. A preferred DNA of the invention comprises a double stranded molecule comprising the coding molecule (i.e., the "coding strand") along with the complementary molecule (the "non-coding strand" or "complement") having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA. Also preferred are other polynucleotides encoding NgR polypeptides, as shown in SEQ ID NO:3, which comprises murine NgR homolog, NgR3.
[0071] Also preferred are nucleotide sequences that encode at least a portion of a NgR polypeptide that has at least one biological function of a NgR. More preferred are nucleotide sequences that encode a portion of NgR that encodes at least the mature NgR without the hydrophobic C-terminal GPI signal. Also preferred are nucleotide sequences that encode the portion of NgR that encodes at least the ligand-binding region of NgR.
[0072] The invention further embraces other species, preferably mammalian, homologs of the human NgR DNA. Species homologs, sometimes referred to as "orthologs," in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention. Generally, percent sequence "homology" with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the NgR sequences set forth in SEQ ID NOs:1, 3 or 13, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
[0073] The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related NgR polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR). Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to NgR and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of NgR. Non-human species genes encoding proteins homologous to NgR can also be identified by Southern and/or PCR analysis and are useful in animal models for NgR disorders. Knowledge of the sequence of a human NgR DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding NgR expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express NgR Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a NgR locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.
[0074] The disclosure herein of a full-length polynucleotide encoding a NgR polypeptide makes readily available to the worker of ordinary skill in the art every possible fragment of the full-length polynucleotide. The invention, therefore, provides fragments of NgR-encoding polynucleotides comprising at least 6, and preferably at least 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding NgR. Preferably, fragments of polynucleotides of the invention comprise sequences unique to the NgR-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., "specifically") to polynucleotides encoding NgR (or fragments thereof). Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent and enzymatic labeling.
[0075] Fragments of polynucleotides are particularly useful as probes for detection of full-length or fragment of NgR polynucleotides. One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding NgR, or used to detect variations in a polynucleotide sequence encoding NgR.
[0076] The invention also embraces DNAs encoding NgR polypeptides that hybridize under moderately stringent or high stringency conditions to the noncoding strand, or complement, of the polynucleotide in any of SEQ ID NOs:1 or 3
[0077] Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1?6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 mg/ml denatured salmon sperm DNA at 65° C. This hybridization is followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOs:1 or 3 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). As used herein, "stringent hybridization conditions" means: 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% (wt/vol) dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.
Vectors
[0078] Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above. Vectors are used herein either to amplify DNA or RNA encoding NgR and/or to express DNA which encodes NgR. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), that serve equivalent functions.
[0079] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein, (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67, 3140), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
[0080] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69, 301-315) and pET 11d (Studier et al., GENE EXRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
[0081] One strategy to maximize recombinant protein expression in E. coli is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS ENZYMOLOGY 185, Acadermic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20, 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
[0082] In another embodiment, the NgR expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6, 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30, 933-943), pJRY88 (Schultz et al., (1987) Gene 54, 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0083] Alternatively, NgR can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., (1983) Mol. Cell. Biol. 3, 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170, 31-39).
[0084] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6, 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al., (Eds.) MOLECULAR CLONING: A LABORATORY MANUAL. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0085] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43, 235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33, 729-740; Queen and Baltimore (1983) Cell 33, 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86, 5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230, 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264, 166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249, 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546).
[0086] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense NgR mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue-specific or cell-type-specific expression of antisense RNA The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., Antisense RNA as a molecular tool for genetic analysis, REVIEWS-TRENDS IN GENETICS, Vol. 1(1) 1986.
[0087] Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses and retroviruses. Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Other expression vectors include, but are not limited to, pSPORT® vectors, pGEM® vectors (Promega), pPROEXvectors® (LTI, Bethesda, Md.), Bluescript® vectors (Stratagene), pQE® vectors (Qiagen), pSE420® (Invitrogen) and pYES2® (Invitrogen).
[0088] Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding NgR is operably linked or connected to suitable control sequences capable of effecting the expression of the NgR in a suitable host. DNA regions are operably linked or connected when they are functionally related to each other. For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence. Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include, but are not limited to a transcriptional promoter, enhancers, an optional operator sequence to control transcription, polyadenylation signals, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation. Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NgR proteins, mutant forms of NgR, fusion proteins, etc.).
[0089] Preferred vectors preferably contain a promoter that is recognized by the host organism. The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (THE BACTERIOPHAGE LAMBDA, Hershey, A. D. (Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; LAMBDA II, Hendrix, R. W. (Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacZ promoters of E. coli and the SV40 early promoter (Benoist et al., (1981) Nature 290, 304-310, which is incorporated herein by reference in its entirety). Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein.
[0090] Additional regulatory sequences can also be included in preferred vectors. Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno sequence of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarno sequence may be directly followed by DNA encoding NgR and result in the expression of the mature NgR protein.
[0091] Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.
[0092] An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and NgR DNA. An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat. No. 4,399,216).
[0093] Nucleotide sequences encoding NgR may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al., (1983) Mol. Cell. Biol. 3:280, Cosman et al. (1986) Mol. Immunol. 23:935, Cosman et al., (1984) Nature 312:768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
Host Cells and Transformed Host Cells
[0094] According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded NgR polypeptide. Preferably, the cell produces little or no endogenous NgR polypeptide. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector. Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate and mammalian cells systems.
[0095] Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with NgR. Host cells of the invention are also useful in methods for the large-scale production of NgR polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
[0096] Knowledge of NgR DNA sequences allows for modification of cells to permit, or increase, expression of endogenous NgR Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring NgR promoter with all or part of a heterologous promoter so that the cells express NgR at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous NgR encoding sequences. (See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92120808, and PCT International Publication No. WO 91/09955.) It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA If linked to the NgR coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the NgR coding sequences in the cells.
[0097] The DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or "knock-out" strategies; see Capecchi, Science 244:1288-1292 (1989)) of animals that fail to express functional NgR or that express a variant of NgR. Such animals (especially small laboratory animals such as rats, rabbits and mice) are useful as models for studying the in vivo activities of NgR and modulators of NgR.
[0098] Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces and Staphylococcus.
[0099] If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. Preferably, eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, Eds. (1973), which is incorporated herein by reference in its entirety).
[0100] In addition, a yeast cell may be employed as a host cell. Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination and a selectable marker gene Shuttle vectors for replication in both yeast and E. coli are also included herein.
[0101] Alternatively, insect cells may be used as host cells. In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, O'Rielly et al. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference in its entirety). In addition, the MAXBAC® complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.
[0102] Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
[0103] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
[0104] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NgR or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
[0105] In a preferred embodiment, the polypeptides of the invention, including forms of NgR2 and NgR3, soluble forms of NgR, chimeric NgR polypeptides, NgR/Ig fusions and fragments and variations of each of the above are expressed in Chinese Hamster Ovary (CHO) cells.
[0106] In order to introduce the DNA fragment coding for the NgR protein or polypeptide into the CHO cell to express the recombinant NgR protein or polypeptide, it is necessary to construct the expression vector.
[0107] The vectors for CHO expression include, but are not limited to, pA1-11, pXT1, pRc/CMV, pRc/RSV and pcDNAINeo. The promoter is not specifically limited provided it effectively promotes expression in CHO cells. Examples of suitable promoters are: SRα, SV40, LTR, CMV, and HSV-TK. Of these, CMV and Srα promoters are preferred.
[0108] In addition to the above-mentioned promoters, the expression vectors may contain enhancers, splicing signals, polyadenylation signals, selectable markers and an SV40 replication origin. Suitable selectable markers include, but are not limited to the dihydrofolate reductase (DHFR) gene which provides resistance to methotrexate (MTX), the ampicillin resistance gene, and the neomycin resistance gene.
[0109] Examples of the expression vectors each containing the DNA coding for NgR, portions, fragments and soluble constructs thereof, include the vector (such as one described above), into which the promoter is operably linked (preferably upstream) to the nucleotide sequence encoding the desired NgR construct; a polyadenylation signal downstream from the nucleotide sequence encoding the NgR construct; and, preferably, the vector includes an operable DHFR gene. Preferably, the ampicillin resistant gene is also operably contained in the vector.
[0110] CHO cell lacking the DHFR gene (Urlaub, G. et al., (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220) and CHO-K1 (Proc. Natl. Acad. Sci. USA 60, 1275 (1968)) are suitable for use.
[0111] The NgR expression vectors prepared as above are introduced into CHO cells by any known method, including, but not limited to the calcium phosphate method (Graham and van der Eb (1973) Virol. 52, 456-467) and electroporation (Nuemann et al., (1982) EMBO J. 1, 841-845).
[0112] Transformants carrying the expression vectors are selected based on the above-mentioned selectable markers. Repeated clonal selection of the transformants using the selectable markers allows selection of stable cell lines having high expression of the NgR constructs. Increased MTX concentrations in the selection medium allows gene amplification and greater expression of the desired protein. The CHO cell containing the recombinant NgR can be produced by cultivating the CHO cells containing the NR expression vectors constitutively expressing the NgR constructs.
[0113] Media used in cultivating CHO cells includes DMEM medium supplemented with about 0.5 to 20% fetal calf serum, DMEM medium and RPMI1640 medium. The pH of the medium is preferably about 6 to 8. Cultivation is preferably at about 30 to 40° C. for about 15 to 72 hours with aeration.
[0114] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NgR protein. Accordingly, the invention further provides methods for producing NgR protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NgR has been introduced) in a suitable medium such that NgR protein is produced. In another embodiment, the method further comprises isolating NgR from the medium or the host cell.
[0115] In situations where the NgR polypeptide will be found primarily intracellularly, intracellular material (including inclusion bodies for Gram-negative bacteria) can be extracted from the host cell using any standard technique known to one of ordinary skill in the art. Such methods would encompass, by way of example and not by way of limitation, lysing the host cells to release the contents of the periplasm/cytoplasm by French press, homogenization and/or sonication followed by centrifugation.
[0116] If the NgR polypeptide has formed inclusion bodies in the cytosol, such inclusion bodies may frequently bind to the inner and/or outer cellular membranes. Upon centrifugation, the inclusion bodies will be found primarily in the pellet material. The pellet material can then be treated at pH extremes or with one or more chaotropic agents such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris-carboxyethyl phosphine at acid pH to release, break apart and solubilize the inclusion bodies. Once solubilized, NgR polypeptide can be analyzed using gel electrophoresis, immunoprecipitation or the like. Various methods of isolating the NgR polypeptide would be apparent to one of ordinary skill in the art, for example, isolation may be accomplished using standard methods such as those set forth below and in Marston et al (1990) Meth. Enzymol. 182, 264-275 (incorporated by reference herein in its entirety).
[0117] If isolated NgR polypeptide is not biologically active following the isolation procedure employed, various methods for "refolding" or converting the polypeptide to its tertiary structure and generating disulfide linkages, can be used to restore biological activity. Methods known to one of ordinary skill in the art include adjusting the pH of the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization but usually at a lower concentration and is not necessarily the same chaotrope as used for the solubilization. It may be required to employ a reducing agent or the reducing agent plus its oxidized form in a specific ratio, to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s). Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol (DTT)/dithiane DTT, 2-mercaptoethanol (bME)/dithio-b(ME). To increase the efficiency of the refolding, it may be necessary to employ a cosolvent, such as glycerol, polyethylene glycol of various molecular weights and arginine.
Transgenic Animals
[0118] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NgR-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NgR sequences have been introduced into their genome or homologous recombinant animals in which endogenous NgR sequences have been altered. Such animals are useful for studying the function and/or activity of NgR and for identifying and/or evaluating modulators of NgR activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NgR gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
[0119] A transgenic animal of the invention can be created by introducing NgR-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NgR DNA sequence of SEQ ID NOs:1 or 3 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homolog of the human NgR gene, such as a mouse NgR gene, can be isolated based on hybridization to the human NgR cDNA (described further above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the NgR transgene to direct expression of NgR protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan 1986, in MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NgR transgene in its genome and/or expression of NgR mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding NgR can further be bred to other transgenic animals carrying other transgenes.
[0120] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NgR gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NgR gene. The NgR gene can be a human gene (e.g., SEQ ID NOs:1 or 13), but more preferably, is a non-human homolog of a human NgR gene. For example, a mouse homolog of human NgR gene of SEQ ID NOs:1 or 13 can be used to construct a homologous recombination vector suitable for altering an endogenous NgR gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NgR gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
[0121] Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NgR gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NgR protein). In the homologous recombination vector, the altered portion of the NgR gene is flanked at its 5' and 3' ends by additional nucleic acid of the NgR gene to allow for homologous recombination to occur between the exogenous NgR gene carried by the vector and an endogenous NgR gene in an embryonic stem cell. The additional flanking NgR nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector. See e.g., Thomas et al. (1987) Cell 51:503 for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NgR gene has homologously recombined with the endogenous NgR gene are selected (see e.g., Li et al. (1992) Cell 69:915).
[0122] The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See e.g., Bradley 1987, In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A Practical Approach, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Curr. Opin. Biotechnol. 2:823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
[0123] In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
[0124] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
Antisense
[0125] Also provided by the invention are antisense polynucleotides that recognize and hybridize to NgR polynucleotides. Full-length and fragment antisense polynucleotides are provided. Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to NgR RNA (as determined by sequence comparison of DNA encoding NgR to DNA encoding other known molecules). Identification of sequences unique to NgR encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs. After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Antisense polynucleotides are particularly relevant to regulating expression of NgR by those cells expressing NgR mRNA.
[0126] Antisense oligonucleotides, or fragments of a nucleotide sequence set forth in SEQ ID NO:1, 3, 13 or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding NgR are useful as diagnostic tools for probing gene expression in various tissues. For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NgR coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NgR protein of SEQ ID NO:2, 4 or 14 or antisense nucleic acids complementary to a NgR nucleic acid sequence of SEQ ID NOs:1, 3 or 13 are additionally provided.
[0127] In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding NgR. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human NgR corresponds to the coding region SEQ ID NO:1, 3 or 13). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding NgR. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
[0128] Antisense oligonucleotides are preferably directed to regulatory regions of a nucleotide sequence of SEQ ID NO:1, 3, 13 or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like. Given the coding strand sequences encoding NgR disclosed herein (e.g., SEQ ID NO:1, 3 or 13), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NgR mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NgR mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NgR mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
[0129] Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, S-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
[0130] The antisense nucleic acid molecules of the invention (preferably oligonucleotides of 10 to 20 nucleotides in length) are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NgR protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Suppression of NgR expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant NgR expression. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
[0131] Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine or cholesterol moieties at their 5' end.
[0132] An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
[0133] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., (1987) Nucleic Acids Res. 15, 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBS Lett. 215, 327-330).
[0134] The NgR sequences taught in the present invention facilitate the design of novel transcription factors for modulating NgR expression in native cells and animals, and cells transformed or transfected with NgR polynucleotides. For example, the Cys2-His2 zinc finger proteins, which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences. These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression. Knowledge of the particular NgR target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al., (1999) Proc. Natl. Acad. Sci. USA 96, 2758-2763; Liu et al., (1997) Proc. Nail. Acad. Sci. USA 94, 5525-5530; Greisman et al. (1997) Science 275, 657-661; Choo et al., (1997) J. Mol. Biol. 273, 525-532). Each zinc finger domain usually recognizes three or more base pairs. Since a recognition sequence of 18 base pairs is generally sufficient in length to render it unique in any known genome, a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al., (1999), above). The artificial zinc finger repeats, designed based on the promoter of NgR sequences, are fused to activation or repression domains to promote or suppress NgR expression (Liu et al., (1997), above). The promoter of NgR may be obtained by standard methods known to one of ordinary skill in the art with the disclosure contained herein and knowledge of the NgR sequence. Alternatively, the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al., (1997) Proc. Natl. Acad. Sci. USA 94, 3616-3620. Such proteins and polynucleotides that encode them, have utility for modulating NgR expression in vivo in both native cells, animals and humans; and/or cells transfected with NgR-encoding sequences. The novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein. Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McColl et al., (1997) Proc. Natl. Acad. Sci. USA 96, 9521-9526); Wu et al., (1995) Proc. Natl. Acad. Sci. USA 92, 344-348). The present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate NgR expression in cells (native or transformed) whose genetic complement includes these sequences.
Ribozymes and PNA Moieties
[0135] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes, described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can be used to catalytically cleave NgR mRNA transcripts to thereby inhibit translation of NgR RNA. A ribozyme having specificity for a NgR-encoding nucleic acid can be designed based upon the nucleotide sequence of a NgR DNA disclosed herein (i.e., SEQ ID NOs:1, 3 or 13). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NgR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, Ng mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261, 1411-1418.
[0136] Alternatively, NgR gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NgR (e.g., the NgR promoter and/or enhancers) to form triple helical structures that prevent transcription of the NgR gene in target cells. See generally, Helene (1991) Anticancer Drug Des. 6: 569-584; Helene. et al., (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) BioEssays 14, 807-815.
[0137] In various embodiments, the nucleic acids of NgR can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., (1996) Bioorg. Med. Chem. Lett. 4, 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al., (1996) above; Perry-O'Keefe et al., (1996) Proc. Nail. Acad. Sci. USA 93, 14670-14675.
[0138] PNAs of NgR can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NgR can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup (1996), above); or as probes or primers for DNA sequence and hybridization (Hyrup et al., (1996), above; Perry-O'Keefe (1996), above).
[0139] In another embodiment, PNAs of NgR can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NgR can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), above and Finn et al. (1996) Nucleic Acids Res. 24, 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucleic Acids Res. 17, 973-988). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996), above). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg. Med. Chem. Lett. 5:1119-1124.
[0140] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84, 648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol et al., (1988) Biotechniques 6, 958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5, 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
[0141] Automated sequencing methods can be used to obtain or verify the nucleotide sequence of NgR. The NgR nucleotide sequences of the present invention are believed to be 100% accurate. However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors. Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art. An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.
Polypeptides
[0142] The invention also provides purified and isolated mammalian NgR polypeptides encoded by a polynucleotide of the invention. Presently preferred is a human NgR polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:14. Another preferred embodiment is a mouse NgR polypeptide comprising the amino acid sequence of NgR3, as set forth in SEQ ID NO:4.
[0143] One aspect of the invention pertains to isolated NgR proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NgR antibodies. Preferably, fragments of NgR proteins comprise at least one biological activity of NgR. In one embodiment, native NgR proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NgR proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NgR protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
[0144] The invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% or at least 45% identity and/or homology to the preferred polypeptide of the invention. In addition, the invention embraces polypeptides having the consensus sequence shown in SEQ ID NO:6, shown in Table 5) excluding the previously characterized NgR ("NgR1"), and polypeptides comprising at least about 90% of the consensus sequence.
[0145] The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
[0146] In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment (Dayhoff, in ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference).
[0147] A determination of homology or identity is typically made by a computer homology program known in the art. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math, 1981, 2, 482-489, which in incorporated herein by reference in its entirety). Employing the GAP software provided in the GCG program package, (see Needleman and Wunsch (1970) J. Mol. Biol. 48, 443-453) the following settings for nucleic acid sequence comparison may be used: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOs:1, 3 or 13. BestFit was originally written for Version 1.0 by Paul Haeberli from a careful reading of the papers by Needleman and Wunsch (1970), above, and Smith and Waterman (1981), above. The following Bestfit settings for nucleic acid sequence comparison may be used: GAP creation penalty of 8.0 and GAP extension penalty of 2, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, with the CDS (encoding) part of the amino acid sequence shown in SEQ ID NOs:2, 4 or 14.
[0148] Alternatively, homology may be determined by hybridization analysis wherein a nucleic acid sequence is hybridized to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below.
[0149] Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention.
[0150] An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NgR protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NgR protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NgR protein having less than about 30% (by dry weight) of non-NgR protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NgR protein, still more preferably less than about 10% of non-NgR protein, and most preferably less than about 5% non-NgR protein. When the NgR protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
[0151] The language "substantially free of chemical precursors or other chemicals" includes preparations of NgR protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NgR protein having less than about 30% (by dry weight) of chemical precursors or non-NgR chemicals, more preferably less than about 20% chemical precursors or non-NgR chemicals, still more preferably less than about 10% chemical precursors or non-NgR chemicals, and most preferably less than about 5% chemical precursors or non-NgR chemicals.
[0152] Biologically active portions of a NgR protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the NgR protein, e.g., the amino acid sequence shown in SEQ ID NO:2, 4 or 14 that include fewer amino acids than the full length NgR proteins, and exhibit at least one activity of a NgR protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the NgR protein. A biologically active portion of a NgR protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
[0153] A biologically active portion of a NgR protein of the present invention may contain at least one of the features that is conserved between the NgR proteins (e.g., a conserved cysteine as the N-terminus of the mature protein, four conserved cysteines in the N-terminus before a leucine-rich region, four conserved cysteines C-terminal with respect to a leucine repeat region, eight leucine-rich repeats, and a hydrophobic C-terminus). An alternative biologically active portion of a NgR protein may contain at least two of the above-identified domains. Another biologically active portion of a NgR protein may contain at least three of the above-identified domains. Yet another biologically active portion of a NgR protein of the present invention may contain at least four of the above-identified domains.
[0154] Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NgR protein.
[0155] In an embodiment, the NgR protein has an amino acid sequence shown in SEQ ID NO:2, 4 or 14. In other embodiments, the NgR protein is substantially homologous to SEQ ID NO:2, 4 or 14 and retains the functional activity of the protein of SEQ ID NO:2, 4 or 14, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below.
[0156] Accordingly, in another embodiment, the NgR protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:14 and retains the functional activity of the NgR proteins of SEQ ID NO:2, 4 or 14.
[0157] Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of NgR polypeptides are embraced by the invention.
[0158] The invention also embraces variant (or analog) NgR polypeptides. In one example, insertion variants are provided wherein one or more amino acid residues supplement a NgR amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the NgR amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.
[0159] Insertion variants include NgR polypeptides wherein one or more amino acid residues are added to a NgR acid sequence or to a biologically active fragment thereof.
[0160] Variant products of the invention also include mature NgR products, i.e., NgR products wherein leader or signal sequences are removed, with additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins. NgR products with an additional methionine residue at position -1 (Met-1-NgR) are contemplated, as are variants with additional methionine and lysine residues at positions -2 and -1 (Met-2-Lys-1-NgR). Variants of NgR with additional Met, Met-Lys, Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.
Polypeptide Variants
[0161] The invention also embraces NgR variants having additional amino acid residues which result from use of specific expression systems.
[0162] As used herein, a NgR "chimeric protein" or "fusion protein" comprises a NgR polypeptide operatively linked to a non-NgR polypeptide. A "NgR polypeptide" refers to a polypeptide having an amino acid sequence corresponding to NgR, whereas a "non-NgR polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not homologous to the NgR protein, e.g., a protein that is different from the NgR protein and that is derived from the same or a different organism. Within a NgR fusion protein the NgR polypeptide can correspond to all or a portion of a NgR protein. In one embodiment, a NgR fusion protein comprises at least one biologically active portion of a NgR protein. In another embodiment, a NgR fusion protein comprises at least two biologically active portions of a NgR protein. In yet another embodiment, a NgR fusion protein comprises at least three biologically active portions of a NgR protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the NgR polypeptide and the non-NgR polypeptide are fused in-frame to each other. The non-NgR polypeptide can be fused to the N-terminus or C-terminus of the NgR polypeptide.
[0163] For example, in one embodiment a NgR fusion protein comprises a NgR domain operably linked to the extracellular domain of a second protein. Such fusion proteins can be further utilized in screening assays for compounds which modulate NgR activity (such assays are described in detail below).
[0164] For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide.
[0165] In another embodiment, the fusion protein is a NgR protein containing a heterologous signal sequence at its N-terminus. For example, the native NgR signal sequence (i.e., amino acids 1-30 of SEQ ID NO:2 and amino acids 1-40 of SEQ ID NO:4) can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion NgR can be increased through use of a heterologous signal sequence.
[0166] In yet another embodiment, the fusion protein is a NgR-immunoglobulin fusion protein in which the NgR sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family. The NgR-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between NgR ligand and a NgR protein on the surface of a cell, to thereby suppress NgR-mediated signal transduction in vivo. NgR-immunoglobulin fusion proteins can be used to affect the bioavailability of a NgR cognate ligand. Inhibition of the NgR ligand/NgR interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the NgR-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NgR antibodies in a subject, to purify NgR ligands, and in screening assays to identify molecules that inhibit the interaction of NgR with NgR ligand.
[0167] A NgR chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NgR-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NgR protein.
[0168] Variants resulting from expression in other vector systems are also contemplated.
[0169] Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of NgR is/are fused to another polypeptide.
[0170] In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a NgR polypeptide are removed. Deletions can be effected at one or both termini of the NgR polypeptide, or with removal of one or more non-terminal amino acid residues of NgR. Deletion variants, therefore, include all fragments of a NgR polypeptide.
[0171] The invention also embraces polypeptide fragments of the sequence set forth in SEQ ID NO:2, 4 or 14 wherein the fragments maintain biological (e.g., ligand binding and/or intracellular signaling) immunological properties of a NgR polypeptide. Fragments comprising at least 4, 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ID NO:2, 4 or 14 are contemplated by the invention. Preferred polypeptide fragments display antigenic properties unique to, or specific for, human NgR and its allelic and species homologs. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.
[0172] In still another aspect, the invention provides substitution variants of NgR polypeptides. Substitution variants include those polypeptides wherein one or more amino acid residues of a NgR polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below.
TABLE-US-00003 TABLE 1 X.sub.aa# (based on a NTLRRCT Column I Column II domain) (R1, R2, R3) (R2 + R3 only) X1 G, R, M X2 A, D, C X3 V, T X4 N, P, S X5 E, A, S X6 nothing, K nothing X7 V, M, P X8 T, V V X9 Q, P Q X10 Q, A Q X11 Q, H, N X12 G, N N X13 L, F F X14 Q, A, S X15 A, S X16 V, I X17 V, T, E, L X18 S, G X19 L, I X20 A, E, V, P X21 A, S, D X22 S, T X23 Q, E X24 IVL X25 Q, H Q X26 N, G N X27 R, L X28 T, G, R, S X29 F, L, T, H X30 L, V L X31 Q, R, P X32 Q, P, A P X33 G, A G X34 H, T, S X35 S, G, R X36 P, S, A X37 C, nothing nothing X38 R, nothing nothing X39 A, N X40 M, L X41 V, L, T X42 T, I T X43 L, I X44 Y, F, H X45 N, V N X46 I, L X47 T, S, A X48 F, Y, T, R X49 A, H, Y, D X50 P, A P X51 N, S, G, A X52 T, A T X53 E, R, T X54 G, H X55 F, L X56 V, Q, H X57 H, A, L X58 E, Q E X59 G, S G X60 R, A R X61 Q, H Q X62 R, H H X63 T, S X64 L, V L X65 A, E, D X66 E, D, A X67 Q, H Q X68 V, E, G X69 K, R X70 H, Q X71 A, S, T X72 Y, H X73 Y, D Y X74 K, R X75 G, Q X76 S, Q S X77 A, S, E X78 P, G P X79 A, G, P X80 G, N X81 I, V, L X82 G, R X83 H, V, A X84 S, A S X85 D, E X86 H, S, A X87 I, L X88 E, L, Q X89 Y, H, A X90 Q, P Q X91 D, N X92 I, L, T X93 V, A, R X94 V, A, G X95 S, T S X96 K, R X97 L, I L X98 W, R, S X99 S, L X100 L, V L X101 G, T, P X102 Q, P, E X103 G, H, R X104 I, T, V, A X105 V, G, H X106 N, S X107 E, G, Q X108 Q, R X109 L, V X110 Q, A X111 W, G, H X112 H, R, P X113 K, A, H X114 H, R X115 D, G X116 H, R, S, G X117 T, M X118 T, I X119 F, Y X120 N, A X121 S, N X122 T, A, S X123 E, S, A X124 Q, P X125 G, T X126 D, E D X127 C, A X128 P, D X129 V, G, P, R X130 A, S X131 E, Q Q X132 F, Y F X133 G, A, D X134 A, P X135 D, A, V X136 G, D X137 A, E X138 S, P X139 E, A X140 L, F X141 R, Q X142 R, K R X143 R, K R X144 F, A X145 G, V X146 A, D, E X147 T, P X148 A, V, S X149 T, S, L X150 E, G, P, Q X151 L, E, R X152 R, L R X153 G, D X154 Q, H, A X155 Q, R X156 K, R X157 L, A, R X158 R, A R X159 V, A, E X160 E, A, N X161 F, L F X162 R, Q X163 N, A, G
[0173] Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.
TABLE-US-00004 TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R Aromatic H F W Y Other N Q D E
[0174] Alternatively, conservative amino acids can be grouped as described in Lehninger, [BIOCHEMISTRY, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77] as set out in Table 3, immediately below.
TABLE-US-00005 TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C. Sulfur-containing: M D. Boderline: G Uncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sylfhydryl: C D. Boderline: G Positively Charged (Basic): K R H Negatively Charged (Acidic): D E
[0175] As still another alternative, exemplary conservative substitutions are set out in Table 4, below.
TABLE-US-00006 TABLE 4 Conservative Substitutions III Original Exemplary Residue Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala
[0176] In addition, amino acid residues that are conserved among family members of the NgR proteins of the present invention, as indicated by the alignment presented herein, are also predicted to be particularly unamenable to alteration. For example, NgR proteins of the present invention can contain at least one domain that is a typically conserved region in NgRs. Examples of these conserved domains include, e.g., leucine-rich repeat domain. Amino acid residues that are not conserved or are only semi-conserved among members of the NgR proteins may be readily amenable to alteration.
[0177] Full-length NgRs have an LRR region characterized by the amino acid consensus sequence shown in SEQ ID NO:19. At least some full-length NgRs also include a CT signaling (CTS) domain and a GPI domain.
[0178] The NgR domain designations used herein are defined as follows:
TABLE-US-00007 mNgR1 hNgR1 SEQ ID hNgR2 hNgR3 mNgR3 Domain SEQ ID: 5 NO: 17 SEQ ID: 2 SEQ ID: 14 SEQ ID: 4 Signal Seq. 1-26 1-26 1-30 -- 1-40 LRRNT 27-56 27-56 31-59 -- 41-69 LRR1 57-81 57-81 60-82 5-27 70-92 LRR2 82-105 82-105 83-106 28-51 93-106 LRR3 106-130 106-130 107-131 52-76 106-141 LRR4 131-154 131-154 132-155 77-100 142-165 LRR5 155-178 155-178 156-179 101-124 166-189 LRR6 179-202 179-202 180-203 125-148 190-213 LRR7 203-226 203-226 204-227 149-172 214-237 LRR8 227-250 227-250 228-251 173-196 238-261 LRRCT 260-309 260-309 261-310 206-255 271-320 CTS 310-445 310-445 311-395 256-396 321-438 (CT Signaling) GPI 446-473 456-473 396-420 370-392 439-462
[0179] In some embodiments of the invention, the above domains are modified. Modification can be in a manner that preserves domain functionality. Modification can include addition, deletion or substitution of certain amino acids. Exemplary modifications include conservative amino acid substitutions. Preferably such substitutions number 20 or fewer per 100 residues. More preferably, such substitutions number 10 or fewer per 100 residues. Further exemplary modifications include addition of flanking sequences of up to five amino acids at the N terminus and/or C terminus of one or more of the domains.
[0180] In some embodiments, the isolated nucleic acid molecule encodes a polypeptide at least about 70%, 80%, 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:2, 4 or 14.
[0181] Mutations can be introduced into SEQ ID NOS:1, 3 or 13 by standard techniques, e.g., site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can be made at one or more amino acid residues predicted to be non-essential. Alternatively, mutations can be introduced randomly along a NgR coding sequence. This can be accomplished, e.g., by saturation mutagenesis. The resulting mutants can be screened for NgR biological activity. Biological activities of Ng may include but are not limited to: (1) protein:protein interactions, e.g., with other NgRs or other cell-surface proteins involved in Nogo-related signaling; (2) complex formation with a NgR ligand; (3) binding to an anti-NgR antibody.
[0182] It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs. Similarly, the invention further embraces NgR polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. Variants that display ligand binding properties of native NgR and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant NgR activity.
[0183] Chemically modified NgR polypeptide compositions in which the NgR polypeptide is linked to a polymer are included within the scope of the present invention. The polymer may be water soluble to prevent precipitation of the protein in an aqueous environment, such as a physiological environment. Suitable water-soluble polymers may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer polyoxyethylated polyols (e.g. glycerol) and polyvinyl alcohol. The selected polymer is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled. Polymers may be of any molecular weight, and may be branched or unbranched, and mixtures of such polymers may also be used. When the chemically modified NgR polymer is destined for therapeutic use, pharmaceutically acceptable polymers will be selected for use.
[0184] When the polymer is to be modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is to be modified by reductive alkylation, the polymer should have a single reactive aldehyde group. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono Cl-ClO alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714, incorporated by reference herein in its entirety).
[0185] Pegylation of NgR polypeptides may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3, 4-10 (1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated by reference herein in its entirety). Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of polypeptides such as NgR is polyethylene glycol (PEG). As used herein, "polyethylene glycol" is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl-ClO) alkoxy- or aryloxy-polyethylene glycol.
[0186] Chemical derivatization of NgR polypeptides may be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule. Methods for preparing pegylated NgR polypeptides will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby NgR polypeptide becomes attached to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result.
[0187] Pegylated and other polymer:NgR polypeptides may generally be used to treat conditions that may be alleviated or modulated by administration of the NgR polypeptides described herein. However, the chemically-derivatized polymer:NgR polypeptide molecules disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to the nonderivatized molecules. The NgR polypeptides, fragments thereof, variants and derivatives, may be employed alone, together, or in combination with other pharmaceutical compositions. The cytokines, growth factors, antibiotics, antiinflammatories and/or chemotherapeutic agents as is appropriate for the indication being treated.
[0188] The present invention provides compositions comprising purified polypeptides of the invention. Preferred compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil and cocoa butter.
[0189] Variants that display ligand binding properties of native NgR and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant NgR activity.
[0190] With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode NgR from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference in its entirety.
[0191] For example, DNA that encodes NgR may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the NgR gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al. (1989) above.
[0192] A nucleic acid molecule comprising any of the NgR nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein. See U.S. Pat. Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis. The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase-mediated replication of a desired nucleic acid molecule.
[0193] A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in for example, Berger et al., Guide to Molecular Cloning Techniques, METHODS IN ENZYMOLOGY 152 Academic Press, San Diego, Calif., which is incorporated herein by reference in its entirety.
[0194] The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping.
Antibodies
[0195] Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for NgR or fragments thereof. Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fab', F(ab')2, and Fv, are also provided by the invention. The term "specific for," when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind NgR polypeptides exclusively (i.e., are able to distinguish NgR polypeptides from other known NgR polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between NgR and such polypeptides).
[0196] The antigenic peptide of NgR comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2, 4 or 14 and encompasses an epitope of NgR such that an antibody raised against the peptide forms a specific immune complex with NgR. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of NgR that are located on the surface of the protein, e.g., hydrophilic regions.
[0197] It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. in ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the NgR polypeptides of the invention are also contemplated, provided that the antibodies are specific for NgR polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
[0198] For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly expressed NgR protein or a chemically synthesized NgR polypeptide. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum or similar immunostimulatory agents. If desired, the antibody molecules directed against NgR can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
[0199] The term "monoclonal antibody" or "monoclonal antibody composition," as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of NgR. A monoclonal antibody composition thus typically displays a single binding affinity for a particular NgR protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular NgR protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see Kohler and Milstein (1975) Nature 256, 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor et al., (1983) Immunol. Today 4, 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole et al., (1985) in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote et al., (1983) Proc Natl. Acad. Sci. USA 80, 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole et al., (1985), above).
[0200] According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a NgR protein (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse et al., (1989) Science 246, 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a NgR protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be "humanized" by techniques well known in the art. See e.g., U.S. Pat. No. 5,225,539. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. Antibody fragments that contain the idiotypes to a NgR protein may be produced by techniques known in the art including, but not limited to: (i) an F(ab)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
[0201] Additionally, recombinant anti-NgR antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No. 125,023; Better et al., (1988) Science 240, 1041-1043; Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84, 3439-3443; Liu et al., (1987) J. Immunol. 139, 3521-3526; Sun et al., (1987) Proc. Natl. Acad. Sci. USA 84, 214-218; Nishimura et al., (1987) Cancer Res. 47, 999-1005; Wood et al., (1985) Nature 314, 446-449; Shaw et al., (1988) J. Natl. Cancer Inst. 80, 1553-1559); Morrison (1985) Science 229, 1202-1207; Oi et al., (1986) Biolechniques 4, 214; U.S. Pat. No. 5,225,539, Jones et al., (1986) Nature 321, 552-525; Verhoeyan et al., (1988) Science 239, 1534; and Beidler et al., (1988) J. Immunol. 141, 4053-4060.
[0202] In a preferred embodiment of the invention a portion of a NgR is joined to an Fc portion of an antibody to form a NgR/Fc fusion protein. Preferably, the Ig fusion protein is soluble. The NgR/Fc fusion protein may be formed by recombinant techniques as described above. In one embodiment, a portion of a NgR including the entire amino acid sequence of NgR except the C-terminal hydrophobic region is fused to an Fc portion of an antibody. In preferred embodiments, the NgR is a human NgR and the Fc is also human. More preferably, the human Fc portion is derived from an IgG antibody. In other embodiments, the N-terminal signal sequence is omitted. Such antibodies are useful in binding Nogo to prevent Nogo signaling through the NgR.
[0203] In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of a NgR protein is facilitated by generation of hybridomas that bind to the fragment of a NgR protein possessing such a domain. Antibodies that are specific for one or more domains within a NgR protein, e.g., domains spanning the above-identified conserved regions of NgRs, or derivatives, fragments analogs or homologs thereof, are also provided herein.
[0204] Anti-NgR antibodies may be used in methods known within the art relating to the localization and/or quantitation of a NgR protein (e.g., for use in measuring levels of the NgR protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for NgR proteins, or derivatives, fragments analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds [hereinafter "Therapeutics"].
[0205] An anti-NgR antibody (e.g. monoclonal antibody) can be used to isolate NgR by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-NgR antibody can facilitate the purification of natural NgR from cells and of recombinantly produced NgR expressed in host cells. Moreover, an anti-NgR antibody can be used to detect NgR protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NgR protein. Anti-NgR antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
[0206] Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response. The amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.
[0207] Another aspect of the invention is directed to anti-idiotypic antibodies and anti-anti-idiotypic antibodies. An anti-idiotypic antibody is an antibody that recognizes determinants of another antibody (a target antibody). Generally, the anti-idiotypic antibody recognizes determinants of the antigen-binding site of the target antibody. Typically, the target antibody is a monoclonal antibody. An anti-idiotypic antibody is generally prepared by immunizing an animal (particularly, mice) of the same species and genetic type as the source of the target monoclonal antibody, with the target monoclonal antibody. The immunized animal mounts an immune response to the idiotypic determinants of the target monoclonal antibody and produces antibodies against the idiotypic determinants of the target monoclonal antibody. Antibody-producing cells, such as splenic cells, of the immunized animal may be used to generate anti-idiotypic monoclonal antibodies. Furthermore, an anti-idiotypic antibody may also be used to immunize animals to produce anti-anti-idiotypic antibodies. These immunized animals may be used to generate anti-anti-idiotypic monoclonal antibodies using standard techniques. The anti-anti-idiotypic antibodies may bind to the same epitope as the original, target monoclonal antibody used to prepare the anti-idiotypic antibody. The anti-anti-idiotypic antibodies represent other monoclonal antibodies with the same antigen specificity as the original target monoclonal antibody.
[0208] If the binding of the anti-idiotypic antibody with the target antibody is inhibited by the relevant antigen of the target antibody, and if the anti-idiotypic antibody induces an antibody response with the same specificity as the target antibody, it mimics the antigen of the target antibody. Such an anti-idiotypic antibody is an "internal image anti-idiotype" and is capable of inducing an antibody response as if it were the original antigen. (Bona and Kohler (1984) ANTI-IDIOTYPIC ANTIBODIES AND INTERNAL IMAGE, IN MONOCLONAL AND ANTI-IDIOTYPIC ANTIBODIES: PROBES FOR RECEPTOR STRUCTURE AND FUNCTION, Venter J. C. et al. (Eds), Alan R Liss, New York, N.Y., pp 141-149, 1984) Vaccines incorporating internal image anti-idiotype antibodies have been shown to induce protective responses against viruses, bacteria, and parasites (Kennedy et al., (1986) 232, 220-223; 1047; McNamara et al., (1985) Science 226, 1325-1326). Internal image anti-idiotypic antibodies have also been shown to induce immunity to tumor related antigens (Raychauhuri et al., (1986) J. Immunol. 137, 1743-1749; Raychauhuri et al., (1987) J. Immunol. 139, 3902-3910; Bhattacharya-Chatterjee et al., (1987) J. Immunol. 139, 1354-1360; Bhattacharya-Chatterjee et al., (1988) J. Immunol. 141, 1398-1403; Herlyn. et al. (1989) Intern. Rev. Immunol. 4, 347-357; Chen et al. (1990) Cell Imm. Immunother. Cancer 351-359; Herlyn et al., (1991) in vivo 5, 615-624; Furuya et al. (1992) AntiCancer Res. 12, 27-32; Mittelman, A. et al. (1992) Proc. Natl. Acad. Sci., USA 89, 466-470; Durrant. et al., (1994) Cancer Res. 54, 4837-4840; Mittelman. et al. (1994) Cancer Res. 54, 415-421; Schmitt et al. (1994) Hybridoma 13, 389-396; Chakrobarty. et al. (1995) J. Immunother. 18, 95-103; Chakrobarty. et al. (1995) Cancer Res. 55, 1525-1530; Foon, K. A. et al. (1995) Clin. Cancer Res. 1, 1205-1294; Herlyn et al. (1995) Hybridoma 14, 159-166; Sclebusch et al. (1995) Hybridoma 14, 167-174; Herlyn. et al. (1996) Cancer Immunol Immunother. 43, 65-76).
[0209] Anti-idiotypic antibodies for NgR may be prepared, for example, by immunizing an animal, such as a mouse, with a immunogenic amount of a composition comprising NgR2 (SEQ ID NO:2), NgR3 (SEQ ID NOs:4 or 14), or immunogenic portion thereof, containing at least one antigenic epitope of NgR. The composition may also contain a suitable adjuvant, and any carrier necessary to provide immunogenicity. Monoclonal antibodies recognizing NgR may be prepared from the cells of the immunized animal as described above. A monoclonal antibody recognizing an epitope of NgR is then selected and used to prepare a composition comprising an immunogenic amount of the anti-NgR monoclonal antibody. Typically, a 25 to 200 μg dose of purified anti-NgR monoclonal would be sufficient in a suitable adjuvant.
[0210] Animals may be immunized 2-6 times at 14 to 30 day intervals between doses. Typically, animals are immunized by any suitable route of administration, such as intraperitoneal, subcutaneous, intravenous or a combination of these. Anti-idiotypic antibody production may be monitored during the immunization period using standard immunoassay methods. Animals with suitable titers of antibodies reactive with the target monoclonal antibodies may be reimmunized with the monoclonal antibody used as the immunogen three days before harvesting the antibody producing cells. Preferably, spleen cells are used, although other antibody producing cells may be selected. Antibody-producing cells are harvested and fused with myeloma cells to produce Hybridomas, as described above, and suitable anti-idiotypic antibody-producing cells are selected.
[0211] Anti-anti-idiotypic antibodies are produced by another round of immunization and Hybridoma production by using the anti-idiotypic monoclonal antibody as the immunogen.
[0212] Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of NgR), diagnostic purposes to detect or quantitate NgR, and purification of NgR. Therefore, kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.
Kits
[0213] The present invention is also directed to kits, including pharmaceutical kits. The kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well appropriate controls, such as positive and/or negative controls. The kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like. For example, the kit can comprise: a labeled compound or agent capable of detecting NgR protein or mRNA in a biological sample; means for determining the amount of NgR in the sample; and means for comparing the amount of NgR in the sample with a standard. The compound or agent can be packaged in a suitable container.
Screening Assays
[0214] The DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a NgR polypeptide or polynucleotide will interact. Methods to identify binding partner compounds include solution assays, in vitro assays wherein NgR polypeptides are immobilized and cell-based assays. Identification of binding partner compounds of NgR polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with NgR normal and aberrant biological activity.
[0215] The invention also provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules (e.g., molecules of less than 1,000 Daltons) or other drugs) that bind to NgR proteins or have a stimulatory or inhibitory effect on, for example, NgR expression or NgR activity.
[0216] In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a NgR protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12, 145).
[0217] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., (1993) Proc. Natl. Acad. Sci. USA 90, 6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91, 11422; Zuckermann et al. (1994) J. Med. Chem. 37, 2678; Cho et al., (1993) Science 261, 1303; Carrell et al., (1994) Angew Chem. Int. Ed Engl. 33, 2059; Carell et al., (1994) Angew Chem. Int Ed Engl. 33, 2061; and Gallop et al., (1994) J. Med. Chem 37, 1233
[0218] Libraries of compounds may be presented in solution (e.g., Houghten (1992) BioTechniques. 13, 412-421), or on beads (Lam (1991) Nature 354, 82-84), on chips (Fodor (1993) Nature 364, 555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, above), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1865-1869) or on phage (Scott and Smith (1990) Science 249, 386-390; Devlin (1990) Science 249, 404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; Felici (1991) J. Mol. Biol. 222, 301-310; Ladner, above).
[0219] 1. Cell-Based Assays
[0220] The invention also provides cell-based assays to identify binding partner compounds of a NgR polypeptide. In one embodiment, the invention provides a method comprising the steps of contacting a NgR polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the NgR polypeptide. In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NgR protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NgR protein or biologically active portion thereof.
[0221] In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NgR protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NgR protein determined. The cell, for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NgR protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NgR protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NgR protein or a biologically active portion thereof, on the cell surface with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the test compound to preferentially bind to NgR or a biologically active portion thereof as compared to the known compound.
[0222] Determining the ability of the test compound to modulate the activity of NgR or a biologically active portion thereof can be accomplished, for example, by determining the ability of the NgR protein to bind to or interact with a NgR target molecule. As used herein, a "target molecule" is a molecule with which a NgR protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NgR protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A NgR target molecule can be a non-NgR molecule or a NgR protein or polypeptide of the present invention. In one embodiment, a NgR target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound NgR molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NgR. In a preferred embodiment, the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule.
[0223] Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant NgR products, NgR variants, or preferably, cells expressing such products. Binding partners are useful for purifying NgR products and detection or quantification of NgR products in fluid and tissue samples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of NgR, especially those activities involved in signal transduction.
[0224] 2. Cell-Free Assays
[0225] (a) Direct Binding:
[0226] The invention includes several assay systems for identifying NgR binding partners. In solution assays, methods of the invention comprise the steps of (a) contacting a NgR polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the NgR polypeptide. Identification of the compounds that bind the NgR polypeptide can be achieved by isolating the NgR polypeptide/binding partner complex and separating the binding partner compound from the NgR polypeptide. An additional step of characterizing the physical, biological and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention. In one aspect, the NgR polypeptide/binding partner complex is isolated using an antibody immunospecific for either the NgR polypeptide or the candidate binding partner compound.
[0227] In still other embodiments, either the NgR polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation, and methods of the invention to identify binding partner compounds include a step of isolating the NgR polypeptide/binding partner complex through interaction with the label or tag. An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.
[0228] (b) Immobilized NgR
[0229] In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized NgR polypeptide, or a biologically active fragment thereof with a candidate binding partner compound and (b) detecting binding of the candidate compound to the NgR polypeptide. In an alternative embodiment, the candidate binding partner compound is immobilized and binding of NgR is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety. Binding of a test compound to NgR, or interaction of NgR with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, and not by way of limitation, GST-NgR fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St Louis, Mo.) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NgR protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, and the complexes determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of NgR binding or activity determined using standard techniques.
[0230] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either NgR or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NgR or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NgR or target molecules, but which do not interfere with binding of the NgR protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NgR trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NgR or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NgR or target molecule.
[0231] Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, (v) determining the activity of the NgR, as well as other techniques well known and routinely practiced in the art.
[0232] Determining the activity of the target molecule, for example, may be accomplished by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NgR-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
[0233] (c) Competition Experiments
[0234] In yet another embodiment, the assay comprises contacting the NgR protein or biologically active portion thereof with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the test compound to preferentially bind to NgR or biologically active portion thereof as compared to the known compound.
[0235] In yet another embodiment, the cell-free assay comprises contacting the NgR protein or biologically active portion thereof with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the NgR protein to modulate the activity of a NgR target molecule.
[0236] The cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of NgR. In the case of cell-free assays comprising the membrane-bound form of NgR, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NgR is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.
Modulators
[0237] Agents that modulate (i.e., increase, decrease, or block) NgR activity or expression may be identified by incubating a putative modulator with a cell containing a NgR polypeptide or polynucleotide and determining the effect of the putative modulator on NgR activity or expression. The selectivity of a compound that modulates the activity of NgR can be evaluated by comparing its effects on NgR to its effect on other NgR compounds. Selective modulators may include, for example, antibodies and other proteins, peptides or organic molecules which specifically bind to a NgR polypeptide or a NgR-encoding nucleic acid. Modulators of NgR activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant NgR activity is involved. NgR polynucleotides, polypeptides and modulators may be used in the treatment of such diseases and conditions associated with demyelination. NgR polynucleotides and polypeptides, as well as NgR modulators, may also be used in diagnostic assays for such diseases or conditions.
[0238] Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator. A modulator is identified in those instances where binding between the NgR polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound. A modulator that increases binding between the NgR polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the NgR polypeptide and the binding partner compound is described as an inhibitor.
[0239] In another embodiment, modulators of NgR expression may be identified in a method wherein a cell is contacted with a candidate compound and the expression of NgR mRNA or protein in the cell is determined. The level of expression of NgR mRNA or protein in the presence of the candidate compound is compared to the level of expression of NgR mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NgR expression based on this comparison. For example, when expression of NgR mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NgR mRNA or protein expression. Alternatively, when expression of NgR mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NgR mRNA or protein expression. The level of NgR mRNA or protein expression in the cells can be determined by methods described herein for detecting NgR mRNA or protein.
High Throughput Screening
[0240] The invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.) of a NgR polypeptide. HTS assays permit screening of large numbers of compounds in an efficient manner. Cell-based HTS systems are contemplated to investigate NgR receptor-ligand interaction. HTS assays are designed to identify "hits" or "lead compounds" having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit" or "lead compound" is often based on an identifiable structure/activity relationship between the "hit" and the NgR polypeptide.
[0241] Another aspect of the present invention is directed to methods of identifying compounds that bind to either NgR or nucleic acid molecules encoding NgR, comprising contacting NgR, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds NgR or a nucleic acid molecule encoding the same. Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Ausubel et al. (Eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety. The NgR proteins, for example, can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., (1993) Cell 72, 223-232; Madura et al., (1993) J. Biol. Chem. 268, 12046-12054, Bartel et al., (1993) BioTechniques 14, 920-924; Iwabuchi et al., (1993) Oncogene 8, 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NgR ("NgR-binding proteins" or "NgR-bp") and modulate NgR activity. Such NgR-binding proteins are also likely to be involved in the propagation of signals by the NgR proteins as, for example, upstream or downstream elements of the NgR pathway.
[0242] Other assays may be used to identify specific ligands of a NgR receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., (1989) Nature 340, 245-246, and Fields et al., (1994) Trends Genet. 10, 286-292, both of which are incorporated herein by reference. The two-hybrid system is a genetic assay based on the modular nature of most transcription factors used for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs. The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast. The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein. The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene. The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene. For example, when the first protein is a NgR gene product, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction. Expression of the reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent results in lack of a reporter signal. The compounds to be screened include (which may include compounds that are suspected to bind NgR, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biological or chemical origin.
[0243] The function of the NgR gene product is unclear and no ligands have yet been found which bind the gene product. The yeast two-hybrid assay is useful to identify proteins that bind to the gene product. In an assay to identify proteins that bind to a NgR receptor, or fragment thereof, a fusion polynucleotide encoding both a NgR receptor (or fragment) and a UAS binding domain (i.e., a first protein) may be used in addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay. Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain. This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein. The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
[0244] Other assays may be used to search for agents that bind to the target protein. One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat. No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
[0245] Another method for identifying ligands of a target protein is described in Vieboldt et al. (1997) Anal. Chem. 69:1683-1691, incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by simple membrane washing. The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
[0246] The methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel (e.g., 125I, 35S, 32P, 33P, 3H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label. Modulators falling within the scope of the invention include, but are not limited to, non-peptide molecules such as non-peptide mimetics, non-peptide allosteric effectors, and peptides. The NgR polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell. One skilled in the art can, for example, measure the formation of complexes between NgR and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between NgR and its substrate caused by the compound being tested.
[0247] Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of NgR comprising contacting NgR with a compound, and determining whether the compound modifies activity of NgR The activity in the presence of the test compared is measured to the activity in the absence of the test compound. Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity. Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.
[0248] The present invention is particularly useful for screening compounds by using NgR in any of a variety of drug screening techniques. The compounds to be screened include (which may include compounds which are suspected to modulate NgR activity), but are not limited to, extracellular, intracellular, biologic or chemical origin. The NgR polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly One skilled in the art can, for example, measure the formation of complexes between NgR and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between Nogo-R and its substrate caused by the compound being tested.
[0249] The activity of NgR polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands. Alternatively, the activity of the NgR can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants and photons. Alternatively, the activity of the NgR can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels. Thus, modulators of NgR activity may alter a NgR receptor function, such as a binding property of a receptor or an activity. In various embodiments of the method, the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [35S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [3H]-arachidonic acid) and an assay for extracellular acidification rates, as well as other binding or function-based assays of NgR activity that are generally known in the art. NgR activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art. Biological activities of NgR receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of NgRs known in the art. Non-limiting examples of NgR activities include transmembrane signaling of various forms, which may involve phosphatidylinositol (PI) association and/or the exertion of an influence over PI; another exemplary activity of NgRs is the binding of accessory proteins or polypeptides that differ from known GPI proteins.
[0250] The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural NgR receptor ligands, peptide and non-peptide allosteric effectors of NgR receptors, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of NgR receptors. The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.
[0251] Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, ENZYM ASSAYS: A PRACTICAL APPROACH, Eisenthal and Danson (Eds.), 1992, Oxford University Press, which is incorporated herein by reference in its entirety.
[0252] The use of cDNAs in drug discovery programs is well-known; assays capable of testing thousands of unknown compounds per day in high-throughput screens (HTSs) are thoroughly documented. The literature is replete with examples of the use of radiolabelled ligands in HTS binding assays for drug discovery (see Williams (1991) Med. Res. Rev., 11, 147-184; Sweetnam et al., (1993) J. Nat. Prod 56, 441-455 for review). Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson (1992) Bio/Technology 10, 973-980; each of which is incorporated herein by reference in its entirety).
[0253] A variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art. Such systems include bacteria (Strosberg et al. (1992) Trends Pharmacol. Sci. 13, 95-98), yeast (Pausch (1997) Trends Biotechnol. 15, 487-494), several kinds of insect cells (Vanden Broeck (1996) Int. Rev. Cytol. 164, 189-268), amphibian cells (Jayawickreme et al. (1997) Curr. Opin. Biotechnol. 8, 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; see Gerhardt et al. (1997) Eur. J. Pharmacol. 334, 1-23). These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).
[0254] In preferred embodiments of the invention, methods of screening for compounds which modulate NgR activity comprise contacting test compounds with NgR and assaying for the presence of a complex between the compound and NgR. In such assays, the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to NgR.
[0255] In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to NgR is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test compounds are contacted with NgR and washed. Bound NgR is then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
[0256] Generally, an expressed NgR can be used for HTS binding assays in conjunction with its defined ligand. The identified peptide is labeled with a suitable radioisotope, including, but not limited to, 125I, 3H, 35S or 32P, by methods that are well known to those skilled in the art. Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al. (1994) Drug Dev. Res. 33, 373-398; Rogers (1997) Drug Discov. Today 2, 156-160). Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams (1991) Med. Res. Rev. II, 147-184; Sweetnam et al. (1993) J. Nat. Prod. 56, 441-455). Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama (1998) Curr. Opin. Drug Disc. Dev. 1, 85-91 Bosse et al. (1998) J. Biomol. Screening 3, 285-292). Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers (1997) Drug Discov. Today 2, 156-160; Hill (1998) Curr. Opin. Drug Disc. Dev. 1, 92-97).
[0257] Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch (1997) Trends in Biotechnol. 15, 487-494); changes in intracellular Ca2+ concentration as measured by fluorescent dyes (Murphy et al. (1998) Cur. Opin Drug Disc. Dev. 1, 192-199). Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder et al. (1996) J. Biomol. Screening 1, 75-80). Melanophores prepared from Xenopus laevis show a ligand-dependent change in pigment organization in response to heterologous NgR activation; this response is adaptable to HTS formats (Jayawickreme et al. (1997) Curr. Opin. Biotechnol. 8, 629-634). Assays are also available for the measurement of common second messengers, including cAMP, phosphoinositides and arachidonic acid, but these are not generally preferred for HTS.
[0258] Preferred methods of HTS employing these receptors include permanently transfected CHO cells, in which agonists and antagonists can be identified by the ability to transduce the signal for the binding of Nogo in membranes prepared from these cells through the putative GPI anchor. In another embodiment of the invention, permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabelled ligand specific for the particular receptor. Alternatively, a functional assay, such as fluorescent monitoring of ligand-induced changes in internal Ca2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be preferred for HTS. Equally preferred would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats. More preferred would be permanently transfected insect cell lines, such as Drosophila S2 cells. Even more preferred would be recombinant yeast cells expressing the Drosophila melanogaster receptors in HTS formats well known to those skilled in the art (e.g., Pausch (1997), above).
[0259] The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to NgR receptors. In one example, the NgR receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound. In another example, interaction between the NgR receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound. In either assay, an inhibitor is identified as a compound that decreases binding between the NgR receptor and its binding partner. Another contemplated assay involves a variation of the di-hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published Aug. 3, 1995.
[0260] Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as "hits" or "leads" in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms that are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Cane et al., Science (1998) 282, 63-68. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers (1997) Curr. Opin. Biotechnol. 8, 701-707. Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit" to modulate activity.
[0261] Still other candidate inhibitors contemplated by the invention can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins. A "binding partner" as used herein broadly encompasses non-peptide modulators, as well as such peptide modulators as neuropeptides other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified NgR gene.
[0262] Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with NgR. Radiolabeled competitive binding studies are described in Lin et al., (1997) Antimicrob. Agents Chemother. 41, 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
[0263] In other embodiments of the invention, the polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins. Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules. For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified. Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.
[0264] Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors. In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.
[0265] Alternatively, compounds may be identified which exhibit similar properties to the ligand for the NgR of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body. When an organic compound is designed, a molecule according to the invention is used as a "lead" compound. The design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such "lead" compounds. Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property. Furthermore, structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.
[0266] Comparison of the protein sequence of the present invention with the sequences present in all the available databases showed a significant homology with the transmembrane portion of G protein coupled receptors. Accordingly, computer modeling can be used to develop a putative tertiary structure of the proteins of the invention based on the available information of the transmembrane domain of other proteins. Thus, novel ligands based on the predicted structure of NgR can be designed.
[0267] This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
Compositions and Pharmaceutical Compositions
[0268] In a particular embodiment, the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral or parenteral administration. The compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's PHARMACEUTICAL SCIENCES, 16th ed., (1980) Osol, A (Ed.), which is incorporated herein by reference in its entirety. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[0269] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral and parenteral (e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal and rectal administration). Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0270] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0271] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NgR protein or anti-NgR antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0272] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.
[0273] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide or a nebulizer.
[0274] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams as generally known in the art.
[0275] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[0276] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
[0277] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by any of a number of routes, e.g., as described in U.S. Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91, 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
[0278] The pharmaceutical compositions can be included in a container, pack or dispenser together with instructions for administration.
[0279] The dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. For treating human or animals, between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
[0280] Another aspect of the present invention is the use of the NgR nucleotide sequences disclosed herein for identifying homologs of the Nogo-R, in other animals, including but not limited to humans and other mammals and invertebrates. Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs using screening procedures well known to those skilled in the art. Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with NgR sequences can be identified.
[0281] The present compounds and methods, including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth. In a particular embodiment, the present molecules are used in gene therapy. For a review of gene therapy procedures, see e.g. Anderson Science (1992) 256, 808-813, which is incorporated herein by reference in its entirety.
[0282] The present invention also encompasses a method of agonizing (stimulating) or antagonizing a NgR natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism. One embodiment of the present invention, then, is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize-NgR-associated functions.
[0283] Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996, and International patent publication number WO 96/22976, published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures or tables. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.
[0284] The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient. Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
[0285] Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out. For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI. Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.
[0286] Toxicity studies can also be carried out by measuring the blood cell composition. For example, toxicity studies can be carried out in a suitable animal model as follows: (1) the compound is administered to mice (an untreated control mouse should also be used); (2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and (3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present.
[0287] At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euthanasia, (1993) J. Am. Vet Med. Assoc. 202; 229-249). Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness or toxicity. Gross abnormalities in tissue are noted and tissues are examined histologically. Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs. In general, the greater the adverse effect the less preferred the compound.
[0288] For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness. Plasma levels should reflect the potency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.
[0289] NgR mRNA transcripts have been found in the brain and heart. SEQ ID NOs: 1 and/or, 3 will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize NgR and for compounds with potential utility in treating disorders including CNS disorders (e.g., stroke) and degenerative disorders such as those associated with demyelination.
[0290] For example, NgR receptor activation may mediate the prevention of neurite outgrowth. Inhibition would be beneficial in both chronic and acute brain injury. See, e.g., Donovan et al., (1997) J. Neurosci. 17, 5316-5326; Turgeon et al., (1998) J. Neurosci. 18, 6882-6891; Smith-Swintosky et al., (1997) J. Neurochem. 69, 1890-1896; Gill et al., (1998) Brain Res. 797, 321-327; Suidan et al., (1996) Semin. Thromb. Hemost. 22, 125-133.
Pharmacogenomics
[0291] Agents, or modulators that have a stimulatory or inhibitory effect on NgR activity (e.g., NgR gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., a disease condition such as a demyelination disorder) associated with aberrant NgR activity. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NgR protein, expression of NgR nucleic acid or mutation content of NgR genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
[0292] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum (1996) Clin. Exp. Pharmacol. Physiol. 23, 983-985 and Linder (1997) Clin. Chem. 43, 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
[0293] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
[0294] Thus, the activity of NgR protein, expression of NgR nucleic acid, or mutation content of NgR genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NgR modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring Clinical Efficacy
[0295] Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NgR (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NgR gene expression, protein levels or upregulate NgR activity, can be monitored in clinical trials of subjects exhibiting decreased NgR gene expression, protein levels, or downregulated NgR activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NgR gene expression, protein levels, or downregulate NgR activity, can be monitored in clinical trials of subjects exhibiting increased NgR gene expression, protein levels, or upregulated NgR activity. In such clinical trials, the expression or activity of NgR and, preferably, other genes that have been implicated in, for example, a disease or disorder, can be used as a "read out" or markers of the immune responsiveness of a particular cell.
[0296] For example, genes, including NgR, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NgR activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on demyelination disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NgR and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced by one of the methods as described herein or by measuring the levels of activity of NgR or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
[0297] In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NgR protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NgR protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NgR protein, mRNA or genomic DNA in the pre-administration sample with the NgR protein, mRNA or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NgR to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NgR to lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment
[0298] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NgR expression or activity.
[0299] Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, (i) a NgR polypeptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgR peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a NgR peptide) are utilized to "knockout" endogenous function of a NgR peptide by homologous recombination (see, e.g., Capecchi (1989) Science 244, 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a NgR peptide and its binding partner.
[0300] Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a NgR peptide, or analogs, derivatives, fragments or homologs thereof or an agonist that increases bioavailability.
[0301] Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a NgR peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
[0302] In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NgR expression or activity, by administering to the subject an agent that modulates NgR expression or at least one NgR activity. Subjects at risk for a disease that is caused or contributed to by aberrant NgR expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NgR aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of NgR aberrancy, for example, a NgR agonist or NgR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
[0303] Another aspect of the invention pertains to methods of modulating NgR expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NgR protein activity associated with the cell. An agent that modulates NgR protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NgR protein, a peptide, a NgR peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NgR protein activity. Examples of such stimulatory agents include active NgR protein and a nucleic acid molecule encoding NgR that has been introduced into the cell. In another embodiment, the agent inhibits one or more NgR protein activity. Examples of such inhibitory agents include antisense NgR nucleic acid molecules and anti-NgR antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NgR protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or down-regulates) NgR expression or activity. In another embodiment, the method involves administering a NgR protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NgR expression or activity.
Gene Therapy
[0304] Mutations in the NgR gene that result in loss of normal function of the NgR gene product underlie NgR human disease states. The invention comprehends gene therapy to restore NgR activity to treat those disease states. Delivery of a functional NgR gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson (1998) Nature, supplement to 392(6679):25-20. For additional reviews of gene therapy technology see Friedmann (1989) Science 244, 1275-1281; Verma (1990) Sci. Am. 68-84; and Miller (1992) Nature 357, 455-460. Alternatively, it is contemplated that in other human disease states, preventing the expression of, or inhibiting the activity of, NgR will be useful in treating disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of NgR.
[0305] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NgR expression or activity.
[0306] Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, (i) a NgR polypeptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgR peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a NgR peptide) are utilized to "knockout" endogenous function of a NgR peptide by homologous recombination (see, e.g., Capecchi (1989), above); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a NgR peptide and its binding partner.
[0307] Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a NgR peptide, or analogs, derivatives, fragments or homologs thereof, or an agonist that increases bioavailability.
[0308] Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a NgR peptide) Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
[0309] In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NgR expression or activity, by administering to the subject an agent that modulates NgR expression or at least one NgR activity. Subjects at risk for a disease that is caused or contributed to by aberrant NgR expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NgR aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of NgR aberrancy, for example, a NgR agonist or NgR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
[0310] Another aspect of the invention pertains to methods of modulating NgR expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NgR protein activity associated with the cell. An agent that modulates NgR protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NgR protein, a peptide, a NgR peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NgR protein activity. Examples of such stimulatory agents include active NgR protein and a nucleic acid molecule encoding NgR that has been introduced into the cell. In another embodiment, the agent inhibits one or more NgR protein activity. Examples of such inhibitory agents include antisense NgR nucleic acid molecules and anti-NgR antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NgR protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or down-regulates) NgR expression or activity. In another embodiment, the method involves administering a NgR protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NgR expression or activity.
[0311] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figure. Such modifications are intended to fall within the scope of the appended claims.
[0312] The following Table 5 contains the sequences of exemplary polynucleotides and polypeptides of the invention.
TABLE-US-00008 TABLE 5 The following DNA sequence NgR2 <SEQ ID NO. 1> was identified in humans: ATGCTGCCCGGGCTCAGGCGCCTGCTGCAAGCTCCCGCCTCGGCCTGC CTCCTGCTGATGCTCCTGGCCCTGCCCCTGGCGGCCCCCAGCTGCCCC ATGCTCTGCACCTGCTACTCATCCCCGCCCACCGTGAGCTGCCAGGCC AACAACTTCTCCTCTGTGCCGCTGTCCCTGCCACCCAGCACTCAGCGA CTCTTCCTGCAGAACAACCTCATCCGCACGCTGCGGCCAGGCACCTTT GGGTCCAACCTGCTCACCCTGTGGCTCTTCTCCAACAACCTCTCCACC ATCTACCCGGGCACTTTCCGCCACTTGCAAGCCCTGGAGGAGCTGGAC CTCGGTGACAACCGGCACCTGCGCTCGCTGGAGCCCGACACCTTCCAG GGCCTGGAGCGGCTGCAGTCGCTGCATTTGTACCGCTGCCAGCTCAGC AGCCTGCCCGGCAACATCTTCCGAGGCCTGGTCAGCCTGCAGTACCTC TACCTCCAGGAGAACAGCCTGCTCCACCTACAGGATGACTTGTTCGCG GACCTGGCCAACCTGAGCCACCTCTTCCTCCACGGGAACCGCCTGCGG CTGCTCACAGAGCACGTGTTTCGCGGCCTGGGCAGCCTGGACCGGCTG CTGCTGCACGGGAACCGGCTGCAGGGCGTGCACCGCGCGGCCTTCCGC GGCCTCAGCCGCCTCACCATCCTCTACCTGTTCAACAACAGCCTGGCC TCGCTGCCCGGCGAGGCGCTCGCCGACCTGCCCTCGCTCGAGTTCCTG CGGCTCAACGCTAACCCCTGGGCGTGCGACTGCCGCGCGCGGCCGCTC TGGGCCTGGTTCCAGCGCGCGCGCGTGTCCAGCTCCGACGTGACCTGC GCCACCCCCCCGGAGCGCCAGGGCCGAGACCTGCGCGCGCTCCGCGAG GCCGACTTCCAGGCGTGTCCGCCCGCGGCACCCACGCGGCCGGGCAGC CGCGCCCGCGGCAACAGCTCCTCCAACCACCTGTACGGGGTGGCCGAG GCCGGGGCGCCCCCAGCCGATCCCTCCACCCTCTACCGAGATCTGCCT GCCGAAGACTCGCGGGGGCGCCAGGGCGGGGACGCGCCTACTGAGGAC GACTACTGGGGGGGCTACGGGGGTGAGGACCAGCGAGGGGAGCAGATG TGCCCCGGCGCTGCCTGCCAGGCGCCCCCGGACTCCCGAGGCCCTGCG CTCTCGGCCGGGCTCCCCAGCCCTCTGCTTTGCCTCCTGCTCCTGGTG CCCCACCACCTC The following amino acid sequence <SEQ ID NO. 2> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 1: M L P G L R R L L Q A P A S A C L L L M L L A L P L A A P S C P M L C T C Y S S P P T V S C Q A N N F S S V P L S L P P S T Q R L F L Q N N L I R T L R P G T F G S N L L T L W L F S N N L S T I Y P G T F R H L Q A L E E L D L G D N R H L R S L E P D T F Q G L E R L Q S L H L Y R C Q L S S L P G N I F R G L V S L Q Y L Y L Q E N S L L H L Q D D L F A D L A N L S H L F L H G N R L R L L T E H V F R G L G S L D R L L L H G N R L Q G V H R A A F R G L S R L T I L Y L F N N S L A S L P G E A L A D L P S L E F L R L N A N P W A C D C R A R P L W A W F Q R A R V S S S D V T C A T P P E R Q G R D L R A L R E A D F Q A C P P A A P T R P G S R A R G N S S S N H L Y G V A E A G A P P A D P S T L Y R D L P A E D S R G R Q G G D A P T E D D Y W G G Y G G E D Q R G E Q M C P G A A C Q A P P D S R G P A L S A G L P S P L L C L L L L V P H H L The following DNA sequence NgR3 <SEQ ID NO. 3> was identified in mouse: ATGTCTTGGCAGTCTGGAACCACAGTGACACAATCTCCCGTGCAGGCT GCTCAGGTCTCAGGGTGCTGTGTGGAATTGCTGCTGTTGCTGCTCGCT GGAGAGCTACCTCTGGGTGGTGGTTGTCCTCGAGACTGTGTGTGCTAC CCTGCGCCCATGACTGTCAGCTGCCAGGCACACAACTTTGCTGCCATC CCGGAGGGCATCCCAGAGGACAGTGAGCGCATCTTCCTGCAGAACAAT CGCATCACCTTCCTCCAGCAGGGCCACTTCAGCCCCGCCATGGTCACC CTCTGGATCTACTCCAACAACATCACTTTCATTGCTCCCAACACCTTC GAGGGCTTTGTGCATCTGGAGGAGCTAGACCTTGGAGACAACCGACAG CTGCGAACGCTGGCACCCGAGACCTTCCAAGGCCTGGTGAAGCTTCAC GCCCTCTACCTCTATAAGTGTGGACTGAGCGCCCTGCCCGCAGGCATC TTTGGTGGCCTGCACAGCCTGCAGTATCTCTACTTGCAGGACAACCAT ATCGAGTACCTCCAAGATGACATCTTTGTGGACCTGGTCAATCTCAGT CACTTGTTTCTCCATGGTAACAAGCTATGGAGCCTGGGCCAAGGCATC TTCCGGGGCCTGGTGAACCTGGACCGGTTGCTGCTGCATGAGAACCAG CTACAGTGGGTTCACCACAAGGCTTTCCATGACCTCCACAGGCTAACC ACCCTCTTTCTCTTCAACAACAGCCTCACTGAGCTGCAGGGTGACTGT CTGGCCCCCCTGGTGGCCTTGGAGTTCCTTCGCCTCAATGGGAATGCT TGGGACTGTGGCTGCCGGGCACGTTCCCTGTGGGAATGGCTGCGAAGG TTCCGTGGCTCTAGCTCTGCTGTCCCCTGCGCGACCCCCGAGCTGCGG CAAGGCCAGGATCTGAAGCTGCTGAGGGTGGAGGACTTCCGGAACTGC ACAGGACCAGTGTCTCCTCACCAGATCAAGTCTCACACGCTTACCACC TCTGACAGGGCTGCCCGCAAGGAGCACCATCCGTCCCATGGGGCCTCC AGGGACAAAGGCCACCCACATGGCCATCCGCCTGGCTCCAGGTCAGGT TACAAGAAGGCAGGCAAGAACTGCACCAGCCACAGGAACCGGAACCAG ATCTCTAAGGTGAGCTCTGGGAAAGAGCTTACCGAACTGCAGGACTAT GCCCCCGACTATCAGCACAAGTTCAGCTTTGACATCATGCCCACCGCA CGACCCAAGAGGAAGGGCAAGTGTGCTCGCAGGACCCCCATCCGTGCC CCCAGTGGGGTGCAGCAGGCATCCTCAGGCACGGCCCTTGGGGCCCCA CTCCTGGCCTGGATACTGGGGCTGGCAGTCACTCTCCGC The following protein sequence <SEQ ID NO. 4> is deduced protein of SEQ ID NO: 3: M S W Q S G T T V T Q S P V Q A A Q V S G C C V E L L L L L L A G E L P L G G G C P R D C V C Y P A P M T V S C Q A H N F A A I P E G I P E D S E R I F L Q N N R I T F L Q Q G H F S P A M V T L W I Y S N N I T F I A P N T F E G F V H L E E L D L G D N R Q L R T L A P E T F Q G L V K L H A L Y L Y K C G L S A L P A G I F G G L H S L Q Y L Y L Q D N H I E Y L Q D D I F V D L V N L S H L F L H G N K L W S L G Q G I F R G L V N L D R L L L H E N Q L Q W V H H K A F H D L H R L T T L F L F N N S L T E L Q G D C L A P L V A L E F L R L N G N A W D C G C R A R S L W E W L R R F R G S S S A V P C A T P E L R Q G Q D L K L L R V E D F R N C T G P V S P H Q I K S H T L T T S D R A A R K E H H P S H G A S R D K G H P H G H P P G S R S G Y K K A G K N C T S H R N R N Q I S K V S S G K E L T E L Q D Y A P D Y Q H K F S F D I M P T A R P K R K G K C A R R T P I R A P S G V Q Q A S S G T A L G A P L L A W I L G L A V T L R The following protein sequence <SEQ ID NO. 5> is NgR1 from humans: M K R A S A G G S R L L A W V L W L Q A W Q V A A P C P G A C C Y N E P K V T T S C P Q Q G L Q A V P V G I P A A S Q R I F L H G N R I S H V P A A S F R A C R N L T I L W L H S N A T L A R I D A A A F T G L A L L E Q L D L S D N A Q L R S V D P A T F H G L G R L H T L H L D R C G L Q E L G P G L F R G L A A L Q Y L Y L Q D N A L Q A L P D D T F R D L G N L T H L F L H G N R I S S V P E R A F R G L H S L D R L L L H Q N R V A H V H P H A F R D L G R L M T L Y L F A N N L S A L P T E A L A P L R A L Q Y L R L N D N P W V C D C R A R P L W A W L Q K F R G S S S E V P C S L P Q R L A G R D L K R L A A N D L Q G C A V A T G P Y H P I W T G R A T D E E P L G L P K C C Q P D A A D K A S V L E P G R P A S A G N A L K G R V P P G D S P P G N G S G P R H I N D S P F G T L P G S A E P P L T A V R P E G S E P P G F P T S G P R R R P G C S R K N R T R S H C R L G Q A G S G G G G T G D S E G S G A L P S L T C S L T P L G L A L V L W T V L G P C The following amino acid sequence <SEQ ID NO: 6> is a Consensus Sequence of NgR based on homology with NgR1 C P X X C X C Y X X P X X T X S C X X X X X X X X P X X X P X X X X R X F L X X N X I X X X X X X X F X X X X X X X X L W X X S N X X X X I X X X X F X X X X X L E X L D L X D N X X L R X X X P X T F X G L X X L X L X L X X C X L X X L X X X X F X G L X X L Q Y L Y L Q X N X X X X L X D D X F X D L X N L X H L F L H G N X X X X X X X X X F R G L X X L D R L L L H X N X X X X V H X X A F X X L X R L X X L X L F X N X L X X L X X X X L A X L X X L X X L R L N X N X W X C X C R A R X L W X W X X X X R X S S S X V X C X X P X X X X G X D L X X L X X X D X X X C X X X X X P X X P X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X G X X X X X X X X X X X X P P X X X S X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X R X X X X X X X X X X X X X X X X X X X X X X X X X X X L X X X X X X X X X X L The following protein sequence <SEQ ID NO: 7> is the 66 amino acid active domain of Nogo: R I Y K G V I Q A I Q K S D E G H P F R A Y L E S E V A I S E E L V Q K Y S N S A L G H V N C T I K E L R R L F L V D D L V D S L K The following protein sequence <SEQ ID NO: 8> is the amino acid sequence of the mature NgR2: C P M L C T C Y S S P P T V S C Q A N N F S S V P L S L P P S T Q R L F L Q N N L I R T L R P G T F G S N L L T L W L F S N N L S T I Y P G T F R H L Q A L E E L D L G D N R H L R S L E P D T F Q G L E R L Q S L H L Y R C Q L S S L P G N I F R G L V S L Q Y L Y L Q E N S L L H L Q D D L F A D L A N L S H L F L H G N R L R L L T E H V F R G L G S L D R L L L H G N R L Q G V H R A A F R G L S R L T I L Y L F N N S L A S L P G E A L A D L P S L E F L R L N A N P W A C D C R A R P L W A W F Q R A R V S S S D V T C A T P P E R Q G R D L R A L R E A D F Q A C P P A A P T R P G S R A R G N S S S N H L Y G V A E A G A P P A D P S T L Y R D L P A E D S R G R Q G G D A P T E D D Y W G G Y G G E D Q R G E Q M C P G A A C Q A P P D S R G P A L S A G L P S P L L C L L L L V P H H L The following protein sequence <SEQ ID NO: 9> is the amino acid sequence of the mature NgR3: C P R D C V C Y P A P M T V S C Q A H N F A A I P E G I P E D S E R I F L Q N N R I T F L Q Q G H F S P A M V T L W I Y S N N I T F I A P N T F E G F V H L E E L D L G D N R Q L R T L A P E T F Q G L V K L H A L Y L Y K C G L S A L P A G I F G G L H S L Q Y L Y L Q D N H I E Y L Q D D I F V D L V N L S H L F L H G N K L W S L G Q G I F R G L V N L D R L L L H E N Q L Q W V H H K A F H D L H R L T T L F L F N N S L T E L Q G D C L A P L V A L E F L R L N G N A W D C G C R A R S L W E W L R R F R G S S S A V P C A T P E L R Q G Q D L K L L R V E D F R N C T G P V S P H Q I K S H T L T T S D R A A R K E H H P S H G A S R D K G H P H G H P P G S R S G Y K K A G K N C T S H R N R N Q I S K V S S G K E L T E L Q D Y A P D Y Q H K F S F D I M P T A R P K R K G K C A R R T P I R A P S G V Q Q A S S G T A L G A P L L A W I L G L A V T L R The following amino acid sequence <SEQ ID NO: 10> is a conserved cysteine motif (Cysteine domain 1) of the NgR and homologs based on the Consensus Sequence: C P X X C X C Y X X P X X T X S C The following amino acid sequence <SEQ ID NO: 11> is a conserved cysteine motif (Cysteine domain 2) of the NgR and homologs based on the Consensus Sequence: N X W X C X C R A R X L W X W X X X X R X S S S X V X C X X P X X X X G X D L X X L X X X D X X X C The following amino acid sequence <SEQ ID NO: 12> is a conserved Leucine-rich domain of the NgR and homologs based on the Consensus Sequence: R X F L X X N X I X X X X X X X F X X X X X X X X L W X X S N X X X X I X X X X F X X X X X L E X L D L X D N X X L R X X X P X T F X G L X X L X L X L X X C X L X X L X X X X F X G L X X L Q Y L Y L Q X N X X X X L X D D X F X D L X N L X H L F L H G N X X X X X X X X X F R G L X X L D R L L L H X N X X X X V H X X A F X X L X R L X X L X L F X N X L X X L X X X X L A X L X X L X X L R L
[0313] Unless otherwise indicated, X is any amino acid. For example, X where indicated may be no amino acid. Additional features of the invention will be apparent from the following Examples. Examples 1-5 are actual, while the remaining Examples are prophetic.
[0314] As shown by the following Examples, a gene encoding novel NgRs have been identified by computational analysis of DNA sequence data. The proteins encoded by NgR2 and NgR3 have a putative signal sequence, eight leucine-rich repeat domains in a conserved leucine-rich region (SEQ ID NO:12), a conserved cysteine-rich region (SEQ ID NO:10) N-terminal to the leucine-rich region, a second cysteine-rich domain (SEQ ID NO:11) C-terminal to the leucine-rich region, and a putative glycophosphatidylinositol-linkage (GPI-linkage) site. NgR2 and NgR3 differ from the previously identified NgR sequence. The NgR homologs, when compared to known NgRs, show a consensus sequence (SEQ ID NOs:6). The putative mature NgR2 and NgR3 are shown in Table 5 as SEQ ID NOs: 8 and 9, respectively.
Example 1
Tblastn Query of the HTG Database
[0315] The protein sequence for the human NgR (NgR1) (SEQ ID NO:5) was used to query the high throughput genomic (HTG) database the use of which is familiar to those skilled in the art. The HTG database is a part of GenBank, a comprehensive NIH genetic sequence database, which includes an annotated collection of all publicly available DNA sequences (Nucleic Acids Res. (2000) 28, 15-8). The HTG database includes sequences obtained from genomic DNA. Within genomic DNA, genes are typically encoded by multiple segments of DNA called exons. Thus when one aligns a cDNA sequence (or a protein sequence encoded by a cDNA sequence) to a genomic sequence, the sequence will be broken up into segments depending on the number of exons in the gene.
[0316] The BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., (1990) J. Mol. Biol. 215, 403-410, which is incorporated herein by reference in its entirety). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The basic BLAST algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
[0317] The BLAST algorithm (Karlin et al., (1993) Proc. Natl. Acad. Sci. USA 90, 5873-5787, which is incorporated herein by reference) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a NgR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a NgR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[0318] To query the HTG database with the NgR protein sequence, we used a variation of the BLAST algorithm known as the tblastn program, which compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames (J. Mol. Biol. (1990) 215, 403-410: Nucleic Acids Res. (1997) 25, 3389-3402). The results of the tblastn search indicated the presence of genes in the database with a significant identity to the NgR. In addition to finding hits to genomic clones which contain the human and mouse NgR genes, we found hits to clones where the identity was not as high, but still very significant. Three human clones were found (Accession numbers: AC068514, AC016869, AC013606) with an e-value of 4e-43 and one mouse clone was found (Accession No. AC021768) with an e-value of 1e-78. The three human clones all appeared to encode the same gene, so further analysis was confined to AC013606.
Example 2
Prediction of the Human NgR2 Protein Sequence (AC013606)
[0319] The human NgR protein sequence aligned with two regions of translated sequence from nucleotide sequence AC013606, indicating that the new gene was encoded by at least two exons. In order to define the complete gene, we used the computer program GENSCAN® (J. Mol. Biol. (1997) 268, 78-94) which can identify complete exon/intron structures of genes in genomic DNA. The gene prediction by GENESCAN® contained seven exons. By comparing these predicted exons to the NgR, it was concluded that the new human gene contains two of these exons and a part of another (containing the initiating methionine). The predicted cDNA (mRNA) encoded by these three exons was assembled from AC013606 (HTG11; deposited March 2000; length=143899; GenBank release 118.0; SEQ ID NO:15) by combining nucleotides from the three exons whose coordinates are: 123292-123322 (exon 1); 130035-130516 (exon 2); and 138589-139335 (exon 3). The sequence for this cDNA sequence is SEQ ID NO:1 (nucleotide sequence of human NgR2; AC013606). The translation of this cDNA provides the protein sequence of human NgR2 (SEQ ID NO:2).
[0320] We used the protein sequence of human NgR2 as a query sequence against the human EST database. A number of hits of high significance were found indicating that the NgR2 mRNA is expressed in a number of tissues including fetal brain. Furthermore, two of these ESTs provided support for the exon structure that we deduced. One EST (Accession No: GB_EST19:AI346757) contains 565 nucleotides corresponding to amino acids 84-271 of the human NgR2 (SEQ ID No:4). This spans the second intron located between amino acids 171 and 172, and provides positive evidence for the splicing of exons 2 and 3 at the mRNA level. Another EST (GB_EST26:AI929019) contains 545 nucleotides, part of which corresponds to amino acids 1-75 of the human NgR2 (SEQ ID NO:2). This spans the first intron located between amino acids 10 and 11, and provides positive evidence for the splicing of exons 1 and 2 at the mRNA level.
Example 3
Prediction of the Mouse NgR3 Protein Sequence (AC021768)
[0321] The human NgR protein sequence aligned with only one region of translated sequence from nucleotide sequence AC021768, indicating that most of the new mouse gene was encoded by one large exon. However, upon inspection, the protein encoded by this exon was missing an initiating methionine. In order to define the complete gene, we used the computer program GENSCAN as described above. The gene prediction by GENSCAN contained two exons; the large one found by visual inspection and a short one at the 5' end which provided an initiating methionine. The predicted cDNA (mRNA) encoded by these two exons was assembled from AC021768 (HTG14; deposited March 2000; length=215980; GenBank release 118.0; SEQ ID NO: 16) by combining nucleotides from the two exons whose coordinates are: the complement of 164265-164325 (exon 1); and the complement of 155671-156992 (exon 2). The sequence for this cDNA sequence is SEQ ID NO:3 (nucleotide sequence of mouse NgR3; AC021768). The translation of this cDNA provides the protein sequence of mouse NgR3 (SEQ ID NO:4).
[0322] We used the protein sequence of mouse NgR3 as a query sequence against the mouse EST database. One hit of high significance was found indicating that the NgR2 mRNA is expressed in the heart. This EST (GB_EST20:AI428334) contains 463 nucleotides, part of which correspond to amino acids 45-193 of mouse NgR3 (SEQ ID NO:4).
Example 4
Similarity Between the NgRs
[0323] An alignment between NgR1 and the two new receptors is shown in FIG. 1A-1B. The similarities between these proteins include:
[0324] (1) The SignalP program, which locates the signal sequence cleavage position, predicts a cleavage before the first conserved cysteine in all the proteins. Thus the mature protein in all cases will have a cysteine at the N-terminus.
[0325] (2) All proteins contain eight Leucine Rich Repeats (LRR). LRRs are short sequence motifs present in a number of proteins with diverse functions and cellular locations. These repeats are usually involved in protein-protein interactions. Each LRR is composed of a beta-alpha unit.
[0326] (3) All three proteins contain a leucine rich repeat N-terminal domain (LRRNT), in which four cysteines are conserved. LRRs are often flanked by cysteine rich domains at both their N and C termini.
[0327] (4) All three proteins contain a LRR C-terminal domain (LRRCT). The LRRCTs of the three NgR proteins can be distinguished from those of other LRR containing proteins, by the pattern of typtophans and cysteines which are completely conserved in this domain.
[0328] (5) All three proteins contain a conserved cysteine in the fourth LRR domain.
[0329] (6) All three proteins contain a conserved potential glycosylation site in the eighth LRR domain.
[0330] (7) NgR2 and NgR3 have a hydrophobic C-terminus, as does NGR1, an indication that they probably also undergo a modification similar to NgR1, where a GPI moiety is covalently linked to a C-terminal amino acid. This allows the protein to remain tethered to the cell.
Example 5
Preparation of Nogo Proteins
[0331] A Nogo binding assay was developed which utilizes a method widely used in examining semaphorin and ephrin axonal guidance function (Flanagan & Vanderhaeghen (1998) Annu. Rev. Neurosci. 21, 3 09-345, Takahashi et al., (1999) Cell 99, 59-69). It involves fusing a secreted placental alkaline phosphatase (AP) moiety to the ligand in question to provide a biologically active receptor binding agent which can be detected with an extremely sensitive calorimetric assay. For Nogo, an expression vector is created encoding a signal peptide, a His6 tag for purification, AP, and the 66 amino acid active domain of Nogo. The fusion protein can be purified from the conditioned medium of transfected cells in milligram amounts. This protein is biologically active as a growth cone collapsing agent with an EC50 of 1 nM.
[0332] Alternatively, a glutathione-S-transferase Nogo (GST-Nogo) fusion protein may be prepared. For GST-Nogo, an expression vector (e.g., a pGEX vector) is created encoding a signal peptide, GST, and the 66 amino acid active domain of Nogo. GST-Nogo may be purified from the culture medium and used as a GST fusion protein, or GST may be cleaved from the Nogo portion of the fusion protein with an enzyme that recognizes the specific amino acid cleavage sit engineered between the GST portion and the Nogo portion of the fusion protein. Such sites are part of the commercially available GST vectors. The specific cleavage sites and enzymes may be used in accordance with the Manufacturers specifications.
[0333] It has been found that AP-Nogo is actually slightly more potent than GST-Nogo, perhaps because the protein is synthesized in a eukaryotic rather than a prokaryotic cell.
[0334] Binding of Nogo to immobilized NgR homologs may be performed in an ELISA-type assay in which AP-Nogo is allowed to react with an immobilized receptor homolog. Specificity of binding may be demonstrated in a competitive binding assay using increasing amounts of GST-Nogo in the type of assay to show a decreasing amount of binding of AP-Nogo (as judged in the calorimetric assay).
Example 6
Transfected COS Cell Binding Assays
[0335] The homologs of the present invention may be used in transfection studies in COS cells to demonstrate binding of Nogo. Specifically, nucleotide sequences encoding NgR2 and NgR3 may be transfected into COS cells using a suitable vector. Non-transfected COS-7 cells do not bind AP-Nogo. However, transfection of COS cells with nucleic acid sequences encoding NgRs will make them capable of binding Nogo. AP alone does not bind with any stable affinity to these transfected cells, indicating that any affinity of Nogo for NgR2 or NgR3 would be due to the 66 amino acids derived from Nogo. Furthermore, specific affinity of Nogo for the NgR2 or NgR3 proteins may be tested in displacement of AP-Nogo assays using GST-Nogo. NgR2 and/or NgR3 may also bind homologs of Nogo, which may also be tested using this assay.
Example 7
Expression of NgR in Human Cell Lines using Northern Blot and a Random-Primed Probe
[0336] A Northern blot is purchased from a commercial source, or RNA samples from cells of interest are run on an agarose gel and blotted to a membrane using any of the well known techniques for Northern blotting. The blot is probed with a fragment of NgR2 (SEQ ID NO:1) or NgR3 (SEQ ID NO:3). The probe is prepared from 50 ng of cDNA labeled by a random-primed method (Feinberg and Vogelstein (1983) Anal. Biochem. 132, 6-13). Hybridization is carried out at 68° C. for 1 hour in ExpressHyb® solution (Clontech, Cat. No. 8015-1) followed by washing with 2×SSC/0.05% SDS at room temperature and two washes with 0.1×SSC/0.1% SDS at 50° C. Expression of NgR2 and/or NgR3 can be assessed by the presence of an appropriately sized band on the blot.
Example 8
Cloning of cDNA Corresponding to NgRs
[0337] To obtain the full-length clone corresponding to NgR2 from a cDNA library, the following method may be used. A cDNA library is generated using standard methods from a tissue known to contain NgR2. Such a tissue was identified in Example 2. 1×106 plaque forming units from the cDNA library may be screened in duplicate on OPTITRAN® filters. The filters are hybridized with 32P-labeled oligonucleotides that are generated from the ESTs corresponding to portions of NgR2. The hybridization reaction may consist of 400 mls plaque screen buffer (50 mM Tris pH 7.5, 1M NaCl, 0.1% Sodium pyrophosphate, 0.2% Polyvinylpryolidine and 0.2% Ficoll) containing 10% Dextran sulfate and 100 μg/ml tRNA and 80 μmol each 32P-labeled oligonucleotide at 65° C. overnight. The filters are washed twice with 2×SSC/1% SDS and twice with 1×SSC/1% SDS and exposed to film. Duplicate positives are purified. DNA from each of these clones is analyzed by restriction enzyme digest followed by agarose gel electrophoresis and Southern blotting. The filters are hybridized to the 32P-labeled oligonucleotides used for the original hybridization to confirm that inserts hybridize to the probe. The insert is then sequenced to confirm that it represents the cDNA for NgR2. Similar methods may be used to generate a full-length clone corresponding to NgR3.
[0338] Alternatively, a full-length clone of NgR2 or NgR3 can be obtained by a person of ordinary skill in the art employing conventional PCR techniques.
Example 9
Hybridization Analysis to Demonstrate NgR Expression in the Brain
[0339] The expression of NgR in mammals, such as the rat, may be investigated by in situ hybridization histochemistry. To investigate expression in the brain, for example, coronal and sagittal rat brain cryosections (20 μm thick) are prepared using a Reichert-Jung cryostat. Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.), and stored at -80° C. Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature. Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature. Microscope slides containing processed cryosections are allowed to air dry prior to hybridization. Other tissues may be assayed in a similar fashion.
[0340] A NgR-specific probe may be generated using PCR. Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes. For production of a probe specific for the sense strand of NgR, a cloned NgR fragment cloned in pBluescript II may be linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase. A probe specific for the antisense strand of NgR may also be readily prepared using the NgR clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase. The riboprobes may be labeled with [35S]-UTP to yield a specific activity of about 0.40×106 cpm/pmol for antisense riboprobes and about 0.65×106 cpm/pmol for sense-strand riboprobes. Each riboprobe may be subsequently denatured and added (2 pmol/ml) to hybridization buffer which contains 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1×Denhardt's Solution, and 10 mM dithiothreitol. Microscope slides containing sequential brain cryosections may be independently exposed to 45 μl of hybridization solution per slide and silanized cover slips may be placed over the sections being exposed to hybridization solution. Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur. Equivalent series of cryosections are then exposed to sense or antisense NgR-specific cRNA riboprobes.
[0341] Following the hybridization period, coverslips are washed off the slides in 1×SSC, followed by RNase A treatment involving the exposure of slides to 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections are then subjected to three high-stringency washes in 0.1×SSC at 52° C. for 20 minutes each. Following the series of washes, cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMax® MR-1 film. After 13 days of exposure, the film is developed, and any significant hybridization signal is detected. Based on these results, slides containing tissue that hybridized, as shown by film autoradiograms, are coated with Kodak NTB-2 nuclear track emulsion and the slides are stored in the dark for 32 days. The slides are then developed and counterstained with hematoxylin. Emulsion-coated sections are analyzed microscopically to determine the specificity of labeling. The signal is determined to be specific if autoradiographic grains (generated by antisense probe hybridization) are clearly associated with cresyl violate-stained cell bodies. Autoradiographic grains found between cell bodies indicate non-specific binding of the probe.
[0342] In some cases, such as using a probe to detect a NgR homolog in a heterologous species, in order to achieve optimal hybridization, it may be necessary to decrease the stringency conditions. Such conditions are well known to those of ordinary skill in the art and examples are provided above.
[0343] Expression of NgR in the brain provides an indication that modulators of NgR activity have utility for treating neurological disorders. Some other diseases for which modulators of NgR may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like. Use of NgR modulators, including NgR ligands and anti-NgR antibodies, to treat individuals having such disease states is intended as an aspect of the invention.
Example 10
Northern Blot Analysis of NgR-RNA with a PCR-Generated Probe
[0344] Northern blot hybridizations may be performed to examine the expression of NgR mRNA A clone containing at least a portion of the sequence of SEQ ID NO:1 may be used as a probe. Vector-specific primers are used in PCR to generate a hybridization probe fragment for 32P-labeling. The PCR is performed as follows:
TABLE-US-00009 Mix: 1 μl NgR-containing plasmid 2 μl fwd primer (10-50 pM) 2 μl rev primer (10-50 pM) 10 μl 10×PCR buffer (such as that provided with the enzyme, Amersham Pharmacia Biotech) 1 μl 10 mM dNTP (such as #1 969-064 from Boehringer Mannheim) 0.5 μl Taq polymerase (such as #27-0799-62, Amersham Pharmacia Biotech) 83.5 μl water
[0345] PCR is performed in a Thermocycler using the following program:
TABLE-US-00010 94° C. 5 min 94° C. 1 min 55° C. 1 min {close oversize brace} 30 cycles 72° C. 1 min 72° C. 10 min
[0346] The PCR product may be purified using QIAquick PCR Purification Kit (#28104) from Qiagen, and radioactively labeled with 32P-dCTP (#AA0005/250, Amersham Pharmacia Biotech)) may be done by random priming using "Ready-to-go DNA Labeling Beads" (#27-9240-01) from Amersham Pharmacia Biotech. Hybridization is carried out on Human Multiple Tissue Northern Blot from Clontech as described in manufacturer's protocol, or on a Northern Blot prepared by running RNA samples from cells of interest on an agarose gel and blotting to a membrane using any of the known Northern blotting protocols. After exposure overnight on Molecular Dynamics Phosphor Imager screen (#MD146-814) bands of an appropriate size are visualized.
Example 11
Recombinant Expression of NgR in Eukaryotic Host Cells
[0347] A. Expression of NgR in Mammalian Cells
[0348] To produce NgR protein, a NgR-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques. For example, a NgR-encoding sequence described in Table 4 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6® (Boehringer-Mannheim) and the transfection protocol provided in the product insert. Other eukaryotic cell lines, including human embryonic kidney (HEK 293) and COS cells, are suitable as well. Cells stably expressing NgR are selected by growth in the presence of 100 μg/ml zeocin (Stratagene, LaJolla, Calif.). As an alternative to FuGENE6®, the expression vector may carry the gene for dihydrofolate reductase (dhfr) and selection of clones with methotrexate (MTX) drug pressure allows for stable transformation of CHO cells. Optionally, NgR may be purified from the cells using standard chromatographic techniques. To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the NgR amino acid sequence, and the antisera is used to affinity purify Nogo-R. The NgR also may be expressed in-frame with a tag sequence (e.g. polyhistidine, hemaglutinin, FLAG) to facilitate purification. Moreover, it will be appreciated that many of the uses for NgR polypeptides, such as assays described below, do not require purification of NgR from the host cell:
[0349] B. Expression of NgR in CHO Cells
[0350] For expression of N in Chinese hamster ovary (CHO) cells, a plasmid bearing the relevant NgR coding sequence is prepared, using a vector which also bears the selectable marker dihydrofolate reductase (DHFR). The plasmid is transfected into CHO cells. Selection under MTX drug pressure allows for preparation of stable transformants of a NgR (NgR2 or NgR3) in an expression plasmid carrying a selectable marker such as DHFR.
[0351] C. Expression of NgR in 293 Cells
[0352] For expression of NgR in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant NgR coding sequence is prepared, using vector pSecTag2A (Invitrogen). Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants. The forward primer for amplification of this NgR cDNA is determined by routine procedures and preferably contains a 5' extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the NgR sequence. The reverse primer is also determined by routine procedures and preferably contains a 5' extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the NgR sequence. The PCR conditions are 55° C. as the annealing temperature. The PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector.
[0353] The DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP® transfection media (Boehringer Mannheim, Indianapolis, Ind.). Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-NgR peptide antibodies. Permanently transfected cells are selected with Zeocin and propagated Production of the recombinant protein is detected from both cells and media by Western blots probed with anti-His, anti-Myc or anti-NgR peptide antibodies.
[0354] D. Transient Expression of Nogo-R in COS Cells
[0355] For expression of the NgR in COS7 cells, a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:1, for example, can be cloned into vector p3-CI. This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.
[0356] The forward primer is determined by routine procedures and preferably contains a 5' extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to a nucleotide sequence of SEQ ID NO:1. The reverse primer is also determined by routine procedures and preferably contains 5'-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of a nucleotide sequence of SEQ ID NO:1
[0357] The PCR consists of an initial denaturation step of 5 min at 95° C., 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C. The PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI. This construct is transformed into E. coli cells for amplification and DNA purification. The DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine® reagent from BRL, following the manufacturer's protocols. Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.
[0358] NgR expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography. Purified NgR is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at -80° C. NgR3 may also be expressed using this method and the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:13.
[0359] E. Expression of NgR in Insect Cells
[0360] For expression of NgR in a baculovirus system, a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:1, 3 or 13 can be amplified by PCR. The forward primer is determined by routine procedures and preferably contains a 5' extension which adds the NdeI cloning site, followed by nucleotides which correspond to a nucleotide sequence of SEQ ID NO:1 (or SEQ. ID NO:3 or SEQ ID NO:13, respectively). The reverse primer is also determined by routine procedures and preferably contains a 5' extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of a nucleotide sequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13, respectively).
[0361] The PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.). The pAcHTL expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6×His tag upstream from the multiple cloning site. A protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present. Of course, many other baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitable vectors for the expression of NgR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al., Virology 170: 31-39, among others.
[0362] The virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al. (1987) A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555.
[0363] In a preferred embodiment, pAcHLT-A containing NgR gene is introduced into baculovirus using the "BaculoGold®" transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer. Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35S-methionine at 24 hours post infection Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE. Viruses exhibiting high expression levels can be isolated and used for scaled up expression.
[0364] For expression of a NgR polypeptide in a Sf9 cells, a polynucleotide molecule having the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO:3 or SEQ ID NO:13) can be amplified by PCR using the primers and methods described above for baculovirus expression. The NgR cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect. The insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus). DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells. Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the NgR-specific antibody. These results are confirmed after further purification and expression optimization in HiG5 cells.
[0365] F. Expression of Soluble Forms of NgR2 and NgR3 as NgR-Ig Fusion Proteins.
[0366] To generate a NgR2-Ig fusion protein, standard methods may be used as described in the literature (e.g. Sanicola et al. (1997) Proc. Natl. Acad. Sci. USA. 94, 6238-6243). For example, a DNA fragment encoding NgR2 without the sequence encoding the hydrophobic C-terminus (GPI anchor signal) may be ligated to a DNA fragment encoding the Fc domain of IgG1 (which may be human IgG1), and the chimeric fragment may be cloned into an expression vector to generate a plasmid. The plasmid may then be transfected into Chinese hamster ovary cells to generate a stable cell line producing the fusion protein. The fusion protein is then purified from conditioned media using standard methods. For example, clarified conditioned media from the cell line may be loaded by gravity directly onto Protein A Sepharose. The column may then be washed with five column volumes each of PBS, PBS containing 0.5 M NaCl, and 25 mM sodium phosphate, 100 mM NaCl (pH 5.0). The bound protein may then be eluted with 25 mM NaH2PO4, 100 mM NaCl (pH 2.8) and immediately neutralized with 1/10 fraction volume of 0.5 M Na2HPO4 (pH 8.6).
[0367] Similar methods may be used to generate a NgR3-Ig fusion protein.
Example 12
Interaction Trap/Two-Hybrid System
[0368] In order to assay for NgR-interacting proteins, the interaction trap/two-hybrid library screening method can be used. This assay was first described in Fields et al. (1989) Nature 340, 245, which is incorporated herein by reference in its entirety. A protocol is published in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1999, John Wiley & Sons, NY and Ausubel, F. M. et al. 1992, SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety. Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).
[0369] A fusion of the nucleotide sequences encoding all or partial NgR and the yeast transcription factor GAL4 DNA-binding domain (DNA-BD) is constructed in an appropriate plasmid (i.e., pGBKT7) using standard subcloning techniques. Similarly, a GAL4 active domain (AD) fusion library is constructed in a second plasmid (i.e. pGADT7) from cDNA of potential NgR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al. 1989, MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The DNA-BD/NgR fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis. Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity. Yeast cells are transformed (ca. 105 transformants/mg DNA) with both the NgR and library fusion plasmids according to standard procedure (Ausubel, et al., 1992, SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety). In vivo binding of DNA-BD/NgR with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2). Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes. Colonies are dually assayed for β-galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) supplemented media (filter assay for b-galactosidase activity is described in Breeden et al., (1985) Cold Spring Harb. Symp. Quant. Biol., 50, 643, which is incorporated herein by reference in its entirety). Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific NgR/library protein interactions. Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the NgR-binding protein.
Example 13
Antibodies to Nogo-R
[0370] Standard techniques are employed to generate polyclonal or monoclonal antibodies to the NgR receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including "humanized" variants. Such protocols can be found, for example, in Sambrook et al. (1989), above, and Harlow et al. (Eds.), ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). In one embodiment, recombinant NgR polypeptides (or cells or cell membranes containing such polypeptides) are used as antigen to generate the antibodies. In another embodiment, one or more peptides having amino acid sequences corresponding to an immunogenic portion of NgR (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used as antigen. Peptides corresponding to extracellular portions of Nogo-R, especially hydrophilic extracellular portions, are preferred. The antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production.
[0371] A. Polyclonal or Monoclonal Antibodies
[0372] As one exemplary protocol, recombinant NgR or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies). To increase antigenicity, peptides are conjugated to Keyhole Limpet Hemocyanin (Pierce), according to the manufacturer's recommendations. For an initial injection, the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously. At intervals of two to three weeks, additional aliquots of NgR antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously. Prior to the final booster injection, a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with NgR. Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize NgR. Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies.
[0373] To generate monoclonal antibodies, the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI. Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well.
[0374] To produce hybridoma fusions, spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated. The cell pellet is dislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI. Thereafter, the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×106 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning, N.Y.).
[0375] On days 2, 4, and 6 after the fusion, 100 μl of medium is removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusions are screened by ELISA, testing for the presence of mouse IgG that binds to NgR. Selected fusion wells are further cloned by dilution until monoclonal cultures producing anti-NgR antibodies are obtained.
[0376] B. Humanization of Anti-NgR Monoclonal Antibodies
[0377] The expression pattern of NgR as reported herein and the potential of NgRs as targets for therapeutic intervention suggest therapeutic indications for NgR inhibitors (antagonists). NgR-neutralizing antibodies comprise one class of therapeutics useful as NgR antagonists. Following are protocols to improve the utility of anti-NgR monoclonal antibodies as therapeutics in humans by "humanizing" the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-NgR antibodies).
[0378] The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins. To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred.
[0379] For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules. (See, e.g., Morrison et al., (1989) Adv. Immunol., 44, 65-92). The variable domains of NgR-neutralizing anti-NgR antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest. The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).
[0380] To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions ("CDR") of the non-human monoclonal antibody genes are cloned into human antibody sequences. (See, e.g., Jones et al., (1986) Nature 321, 522-525; Riechmann et al., (1988) Nature 332, 323-327; Verhoeyen et al., (1988) Science 239, 1534-1536; and Tempest et al., (1991) Bio/Technology 9, 266-271). If necessary, the β-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody. (See Kettleborough et al., (1991) Protein Engin. 4, 773-783; and Foote et al., (1992) J. Mol. Biol. 224, 487-499).
[0381] In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody. See Padlan (1991) Mol. Immunol. 28, 489-498.
[0382] The foregoing approaches are employed using NgR-neutralizing anti-NgR monoclonal antibodies and the hybridomas that produce them to generate humanized NgR-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein NgR expression or ligand-mediated NgR signaling is detrimental.
[0383] C. Human NgR-Neutralizing Antibodies from Phage Display
[0384] Human NgR-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al. (1997) Human Antibodies 8, 155-168; Hoogenboom (1997) TIBTECH 15, 62-70; and Rader et al. (1997), Curr. Opin. Biotechnol. 8, 503-508, all of which are incorporated by reference. For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII. Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody. A phage library comprising such constructs is expressed in bacteria, and the library is screened for NgR-specific phage-antibodies using labeled or immobilized NgR as antigen-probe.
[0385] D. Human NgR-Neutralizing Antibodies from Transgenic Mice
[0386] Human NgR-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann et al. (1996) Immunol. Today 17, 391-397 and Bruggemann et al. (1997) Curr. Opin. Biotechnol. 8, 455-458. Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with a NgR composition using conventional immunization protocols hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-NgR human antibodies (e.g., as described above).
Example 14
Assays to Identify Modulators of NgR Activity
[0387] Set forth below are several nonlimiting assays for identifying modulators (agonists and antagonists) of NgR activity. Among the modulators that can be identified by these assays are natural ligand compounds of the receptor; synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like. All modulators that bind NgR are useful for identifying NgR in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like). Agonist and antagonist modulators are useful for up-regulating and down-regulating NgR activity, respectively, to treat disease states characterized by abnormal levels of NgR activity. The assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa).
[0388] A. cAMP Assays
[0389] In one type of assay, levels of cyclic adenosine monophosphate (cAMP) are measured in NgR-transfected cells that have been exposed to candidate modulator compounds. Protocols for cAMP assays have been described in the literature. (See, e.g., Sutherland et al., (1968) Circulation 37, 279; Frandsen et al., (1976) Life Sciences 18, 529-541; Dooley et al., (1997) J. Pharmacol. Exp. Therap. 283, 735-41; and George et al., (1997) J. Biomol. Screening 2, 235-40). An exemplary protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate® Assay from NEN® Life Science Products, is set forth below.
[0390] Briefly, the NgR coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the FuGENE 6 transfection reagent. Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound. For a control, some wells are seeded with wild type (untransfected) CHO cells. Other wells in the plate receive various amounts of a cAMP standard solution for use in creating a standard curve.
[0391] One or more test compounds (i.e., candidate modulators) are added to the cells in each well, with water and/or compound-free medium/diluent serving as a control or controls. After treatment, cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature. The assay is terminated by the addition of lysis buffer containing [125I]-labeled cAMP, and the plate is counted using a Packard Topcount® 96-well microplate scintillation counter. Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of [125I]-cAMP compete for antibody bound to the plate. A standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation. Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of NgR modulating activity. Modulators that act as agonists of receptors which couple to the Gs subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels. Agonists of receptors which couple to the Gi/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%. Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists.
[0392] B. Aequorin Assays
[0393] In another assay, cells (e.g., CHO cells) are transiently co-transfected with both a NgR expression construct and a construct that encodes the photoprotein apoaquorin. In the presence of the cofactor coelenterazine, apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium. (See generally, Cobbold, et al. "Aequorin measurements of cytoplasmic free calcium," In: McCormack J. G. and Cobbold P. H., eds., CELLULAR CALCIUM: A PRACTICAL APPROACH. Oxford:IRL Press (1991); Stables et al., (1997) Anal. Biochem. 252, 115-26; and Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS. Sixth edition. Molecular Probes, Eugene, Oreg. (1996)).
[0394] In one exemplary assay, NgR is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.) into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert.
[0395] The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 μM coelenterazine (Molecular Probes, Eugene, Oreg.). Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM.
[0396] Dilutions of candidate NgR modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 μl/well. Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.). The instrument is programmed to dispense 50 μl cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds. Dose-response curves for the candidate modulators are constructed using the area under the curve for each light signal peak. Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC50 values are obtained. Changes in luminescence caused by the compounds are considered indicative of modulatory activity. Modulators that act as agonists at receptors which couple to the Gq subtype of G proteins give an increase in luminescence of up to 100 fold. Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists.
[0397] C. Luciferase Reporter Gene Assay
[0398] The photoprotein luciferase provides another useful tool for assaying for modulators of NgR activity. Cells (e.g., CHO cells or COS 7 cells) are transiently co-transfected with both a NgR expression construct (e.g., NgR in pzeoSV2) and a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B. Expression levels of luciferase reflect the activation status of the signaling events. (See generally, George et al. (1997) J. Biomol. Screening 2, 235-240; and Stratowa et al. (1995) Curr. Opin. Biotechnol. 6, 574-581). Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.).
[0399] In one exemplary assay, CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin. Cells are transiently co-transfected with both a NgR expression construct and a reporter construct containing the luciferase gene. The reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (Legally, Calif.). Transfections are performed using the FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions. Cells transfected with the reporter construct alone are used as a control. Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours. Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 μl of lysis buffer per well from the luciferase assay kit supplied by Promega. After incubation for 15 minutes at room temperature, 15 μl of the lysate is mixed with 50 μl of substrate solution (Promega) in an opaque-white, 96-well plate, and the luminescence is read immediately on a Wallace model 1450 MicroBeta scintillation and luminescence counter (Wallace Instruments, Gaithersburg, Md.).
[0400] Differences in luminescence in the presence versus the absence of a candidate modulator compound are indicative of modulatory activity. Receptors that are either constitutively active or activated by agonists typically give a 3-20-fold stimulation of luminescence compared to cells transfected with the reporter gene alone. Modulators that act as inverse agonists will reverse this effect.
[0401] D. Intracellular Calcium Measurement using FLIPR
[0402] Changes in intracellular calcium levels are another recognized indicator of receptor activity, and such assays can be employed to screen for modulators of NgR activity. For example, CHO cells stably transfected with a NgR expression vector are plated at a density of 4×104 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate. The cells are incubated for 60 minutes at 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3® AM, Fluo-4® AM, Calcium Green®-1 AM, or Oregon Green® 488 BAPTA-1 AM), each at a concentration of 4 μM. Plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane. In addition, a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.
[0403] A calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 μM; positive control), or ATP (4 μM; positive control). Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm). (See, e.g., Kuntzweiler et al. (1998) Drug Dev. Res. 44, 14-20). The F-stop for the detector camera is set at 2.5 and the length of exposure is 0.4 milliseconds. Basal fluorescence of cells is measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level is subtracted from the response signal. The calcium signal is measured for approximately 200 seconds, taking readings every two seconds. Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels. In general, activated NgRs increase the calcium signal at least about 10-15% above baseline signal.
[0404] E. [35S]GTPγS Binding Assay
[0405] It is also possible to evaluate whether NgR signals through a G protein-mediated pathway. Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog [35S]-GTPγS in the presence and absence of candidate modulators provides another assay for modulator activity. (See, e.g., Kowal et al., (1998) Neuropharmacology 37, 179-187.).
[0406] In one exemplary assay, cells stably transfected with a NgR expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca2+/Mg2+-free phosphate-buffered saline, and scraped into 5 ml of the same buffer. Cells are pelleted by centrifugation (500×g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen. After thawing, the cells are homogenized using a Dounce homogenizer (1 ml TEE per plate of cells), and centrifuged at 1,000×g for 5 minutes to remove nuclei and unbroken cells.
[0407] The homogenate supernatant is centrifuged at 20,000×g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM BEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA). The resuspended membranes can be frozen in liquid nitrogen and stored at -70° C. until use.
[0408] Aliquots of cell membranes prepared as described above and stored at -70° C. are thawed, homogenized, and diluted into buffer containing 20 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mM ascorbate, at a concentration of 10-50 μg/ml. In a final volume of 90 μl, homogenates are incubated with varying concentrations of candidate modulator compounds or 100 μM GTP for 30 minutes at 30° C. and then placed on ice. To each sample, 10 μl guanosine 5'-O-(3-[35S]thio) triphosphate (NEN, 1200 Cimmol; [35S]-GTPγS), was added to a final concentration of 100-200 μM. Samples are incubated at 30° C. for an additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl2, at 4° C. is added and the reaction is stopped by filtration.
[0409] Samples are filtered over Whatman GF/B filters and the filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl2. Filters are counted by liquid scintillation spectroscopy. Nonspecific binding of [35]-GTPγS is measured in the presence of 100 μM GTP and subtracted from the total. Compounds are selected that modulate the amount of [35S]-GTPγS binding in the cells, compared to untransfected control cells. Activation of receptors by agonists gives up to a five-fold increase in [35S]-GTPγS binding This response is blocked by antagonists.
[0410] F. [3H]Arachidonic Acid Release
[0411] The activation of NgRs may also potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of NgR activity. (See, e.g., Kanterman et al., (1991) Mol. Pharmacol. 39, 364-369.) For example, CHO cells that are stably transfected with a NgR expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin for 48 hours at 37° C. before use. Cells of each well are labeled by incubation with [3H]-arachidonic acid. (Amersham Corp., 210 Ci/mmol) at 0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer.
[0412] Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 μM ATP and the cells are incubated at 37° C. for 30 minutes. Buffer alone and mock-transfected cells are used as controls. Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy. Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of [3H]-arachidonic acid. This potentiation is blocked by antagonists.
[0413] G. Extracellular Acidification Rate
[0414] In yet another assay, the effects of candidate modulators of NgR activity are assayed by monitoring extracellular changes in pH induced by the test compounds (see, e.g., Dunlop et al. (1998) J. Pharmacol. Toxicol. Meth 40, 47-55). In one embodiment, CHO cells transfected with a NgR expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×105 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin. The cells are incubated in this medium at 37° C. in 5% CO2 for 24 hours.
[0415] Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.). The capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mM NaCl) at a flow rate of 100 μl/minute. Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path. During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds. The pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle. The rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program. Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate. The selected instrument detects 61 mV/pH unit. Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist. This response is blocked by modulators which act as antagonists of the receptor.
Example 15
mNgR3 does not Bind hNogo-A(1055-1120)
[0416] To functionally test the mouse NgR3 (hereinafter, mNgR3) for its ability to bind hNogo-A(1055-1120), a cDNA expression vector for a myc epitope-tagged mNgR3 protein was created. The mouse NgR3 cDNA was amplified by PCR from mouse adult brain cDNA, from the signal sequence to the stop codon, and ligated into the pSecTag2 vector such that the vector encodes a signal sequence followed by a myc tag followed by the mature mNgR3 sequence. This plasmid was transfected into COS07 cells, and expression of a myc-tagged protein of the predicted size was verified by immunoblot analysis. Alkaline phosphatase-hNogo-A(1055-1120) binding studies and myc immunohistology were conducted as described (Fournier et al., supra).
[0417] The cells expressing mNgR3 express the myc-tagged protein but binding to AP-hNogo-A(1055-1120) was not observed under the conditions employed (FIG. 8).
Example 16
Identification of Partial Human NgR3 cDNA and Protein Sequences
[0418] The tblastn program was used to search for the human homolog of mouse NgR3. The mouse NgR3 protein sequence (SEQ ID NO:4) was used to query a proprietary human expressed sequence tag (EST) database from Incyte yielding one highly significant hit: Incyte Template ID 190989.1. This sequence (937 nucleotides) contains an open reading frame of 312 amino acids in the second reverse frame that exhibits 88% identity with residues 66 to 381 of mouse NgR3 (SEQ ID NO:4), strongly indicating that it is part of the human NgR3 homolog.
[0419] A query of SEQ ID NO:4 against the public human EST database in Genbank also produced a hit with a 465-bp EST (Accession number: R35699; Version number: R35699.1; GI: 792600). There are a number of single nucleotide deletions and insertions within this sequence which cause frame shift errors. All of the reliable sequence contained in this public EST is present in the Incyte EST (Template ID 190989.1).
[0420] To obtain more nucleotide sequence that would extend the amino acid sequence at that carboxy terminal end, the I.M.A.G.E. Consortium clone No. 38319, which corresponds to Genbank accession No. R35699, was purchased from Incyte Genomics Inc. and subjected to further DNA sequence analysis. This clone consists of a NotI/HinD III fragment containing the sequence of interest, cloned into the Not/HinD III sites of the vector Lafinid BA (http://image.llnl.gov/image/html/libs/lafmidBA.shtml). The clone was received as an agar stab, which was streaked out on LB agar plates containing 50 ug/ml ampicillin to isolate individual colonies. Six colonies were grown in LB medium with antibiotic, and plasmid DNA was prepared using the Promega Wizard Plus Miniprep DNA Purification System (Promega #A7500). These DNAs were subsequently digested with NotI and HinD III restriction enzymes to confirm that the clones contained an insert. The insert of one isolate was sequenced using a combination of vector specific and gene specific primers yielding a partial nucleotide sequence of human NgR3 of 1176 nucleotides (SEQ ID NO:13). A translation of this sequence provides a partial sequence for human NgR3 of 392 amino acids (SEQ ID NO:14).
[0421] The nucleotide sequence of SEQ ID NO:13 differs from the Incyte EST sequence at three positions. Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereas the corresponding nucleotides in the Incyte Template ID 190989.1 are GT (i.e., positions 12-13 of the complement of Incyte Template ID 190989.1). In addition, position 641 in SEQ ID NO:13 is a C, whereas the corresponding nucleotide in the Incyte Template ID 190989.1 sequence is an A (i.e., position 641 of the complement of Incyte Template ID 190989.1). This results in two changes in amino acids when comparing SEQ ID NO:14 to the ORF encoded by Incyte Template 190989.1: SEQ ID NO:14 contains a valine at position 5, whereas the ORF encoded by Incyte Template ID 190989.1 contains a leucine; SEQ ID NO:14 contains an alanine at position 214, whereas the ORF encoded by Incyte Template ID 190989.1 contains a glutamic acid.
[0422] The nucleotide sequence of SEQ ID NO:13 differs from the public EST (Accession number: R35699; Version number: R35699.1; GI: 792600) sequence at two positions (within the first 200 nucleotides of reliable sequence). Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereas the corresponding nucleotides in the public EST are GT (i.e., positions 12-13 of the public EST; Accession no: R135699; Version no: R35699.1; GI: 792600) This leads to a single amino acid change when comparing SEQ ID NO:14 to the ORF encoded by the public EST: SEQ ID NO:14 contains a valine at position 5, while the ORF encoded by the public EST contains a leucine.
[0423] A Bestfit analysis of the partial human amino acid sequence with the full-length mouse amino acid sequence indicates that the human NgR3 amino acid sequence is complete at the carboxy terminal end and that they share 89.54% identity. An alignment of all the NgR proteins is shown in FIG. 9. Although the human NgR3 amino acid sequence is missing the first 25 amino acids, it can be determined that the human NgR3 protein contains the following features in common with the other NgR sequences: (1) eight Leucine Rich Repeat (LRR) domains; (2) an LRR carboxy-terminal (LRR-CT) domain; (3) a conserved cysteine in the fourth LRR domain; (4) a conserved potential glycosylation site in the eighth LRR domain; and (5) a hydrophobic carboxyl terminus.
[0424] As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention.
[0425] The entire disclosure of each publication cited herein is hereby incorporated by reference. This application claims benefit from U.S. provisional application 60/238,361, filed Oct. 6, 2000, which is incorporated by reference herein in its entirety.
KEY FOR SEQUENCE LISTING
[0426] SEQ ID NO:1 human NgR2 cDNA sequence derived from genomic sequence AC013606 [0427] SEQ ID NO:2 human NgR2 amino acid sequence [0428] SEQ ID NO:3 mouse NgR3 cDNA sequence derived from AC021768 [0429] SEQ ID NO:4 a mouse NgR3 amino acid sequence [0430] SEQ ID NO:5 a human NgR1 amino acid sequence [0431] SEQ ID NO:6 a consensus amino acid sequence for NgRs [0432] SEQ ID NO:7 #1055-1120 amino acid residues of hNogoA (Nogo-66) [0433] SEQ ID NO:8 a mature human NgR2 amino acid sequence [0434] SEQ ID NO:9 a mature mouse NgR3 amino acid sequence [0435] SEQ ID NO:10 a consensus NgR LLRNT amino acid sequence [0436] SEQ ID NO:11 a consensus NgR LRRCT domain amino acid sequence [0437] SEQ ID NO:12 a consensus NgR LRR domain amino acid sequence [0438] SEQ ID NO:13 a partial human NgR3 nucleotide sequence [0439] SEQ ID NO:14 a partial human NgR3 amino acid sequence [0440] SEQ ID NO:15 a genomic sequence encoding a human NgR2 sequence. [0441] SEQ ID NO:16 a genomic sequence (complementary strand) encoding a mouse NgR3 [0442] SEQ ID NO:17 a mouse NgR1 amino acid sequence [0443] SEQ ID NO:18 a consensus sequence for the NTLRRCT domain of NgR [0444] SEQ ID NO:19 an consensus NgR LRRCT domain amino acid sequence
Sequence CWU
1
1911260DNAHomo sapiens 1atgctgcccg ggctcaggcg cctgctgcaa gctcccgcct
cggcctgcct cctgctgatg 60ctcctggccc tgcccctggc ggcccccagc tgccccatgc
tctgcacctg ctactcatcc 120ccgcccaccg tgagctgcca ggccaacaac ttctcctctg
tgccgctgtc cctgccaccc 180agcactcagc gactcttcct gcagaacaac ctcatccgca
cgctgcggcc aggcaccttt 240gggtccaacc tgctcaccct gtggctcttc tccaacaacc
tctccaccat ctacccgggc 300actttccgcc acttgcaagc cctggaggag ctggacctcg
gtgacaaccg gcacctgcgc 360tcgctggagc ccgacacctt ccagggcctg gagcggctgc
agtcgctgca tttgtaccgc 420tgccagctca gcagcctgcc cggcaacatc ttccgaggcc
tggtcagcct gcagtacctc 480tacctccagg agaacagcct gctccaccta caggatgact
tgttcgcgga cctggccaac 540ctgagccacc tcttcctcca cgggaaccgc ctgcggctgc
tcacagagca cgtgtttcgc 600ggcctgggca gcctggaccg gctgctgctg cacgggaacc
ggctgcaggg cgtgcaccgc 660gcggccttcc gcggcctcag ccgcctcacc atcctctacc
tgttcaacaa cagcctggcc 720tcgctgcccg gcgaggcgct cgccgacctg ccctcgctcg
agttcctgcg gctcaacgct 780aacccctggg cgtgcgactg ccgcgcgcgg ccgctctggg
cctggttcca gcgcgcgcgc 840gtgtccagct ccgacgtgac ctgcgccacc cccccggagc
gccagggccg agacctgcgc 900gcgctccgcg aggccgactt ccaggcgtgt ccgcccgcgg
cacccacgcg gccgggcagc 960cgcgcccgcg gcaacagctc ctccaaccac ctgtacgggg
tggccgaggc cggggcgccc 1020ccagccgatc cctccaccct ctaccgagat ctgcctgccg
aagactcgcg ggggcgccag 1080ggcggggacg cgcctactga ggacgactac tgggggggct
acgggggtga ggaccagcga 1140ggggagcaga tgtgccccgg cgctgcctgc caggcgcccc
cggactcccg aggccctgcg 1200ctctcggccg ggctccccag ccctctgctt tgcctcctgc
tcctggtgcc ccaccacctc 12602420PRTHomo sapiens 2Met Leu Pro Gly Leu Arg
Arg Leu Leu Gln Ala Pro Ala Ser Ala Cys1 5
10 15Leu Leu Leu Met Leu Leu Ala Leu Pro Leu Ala Ala
Pro Ser Cys Pro 20 25 30Met
Leu Cys Thr Cys Tyr Ser Ser Pro Pro Thr Val Ser Cys Gln Ala 35
40 45Asn Asn Phe Ser Ser Val Pro Leu Ser
Leu Pro Pro Ser Thr Gln Arg 50 55
60Leu Phe Leu Gln Asn Asn Leu Ile Arg Thr Leu Arg Pro Gly Thr Phe65
70 75 80Gly Ser Asn Leu Leu
Thr Leu Trp Leu Phe Ser Asn Asn Leu Ser Thr 85
90 95Ile Tyr Pro Gly Thr Phe Arg His Leu Gln Ala
Leu Glu Glu Leu Asp 100 105
110Leu Gly Asp Asn Arg His Leu Arg Ser Leu Glu Pro Asp Thr Phe Gln
115 120 125Gly Leu Glu Arg Leu Gln Ser
Leu His Leu Tyr Arg Cys Gln Leu Ser 130 135
140Ser Leu Pro Gly Asn Ile Phe Arg Gly Leu Val Ser Leu Gln Tyr
Leu145 150 155 160Tyr Leu
Gln Glu Asn Ser Leu Leu His Leu Gln Asp Asp Leu Phe Ala
165 170 175Asp Leu Ala Asn Leu Ser His
Leu Phe Leu His Gly Asn Arg Leu Arg 180 185
190Leu Leu Thr Glu His Val Phe Arg Gly Leu Gly Ser Leu Asp
Arg Leu 195 200 205Leu Leu His Gly
Asn Arg Leu Gln Gly Val His Arg Ala Ala Phe Arg 210
215 220Gly Leu Ser Arg Leu Thr Ile Leu Tyr Leu Phe Asn
Asn Ser Leu Ala225 230 235
240Ser Leu Pro Gly Glu Ala Leu Ala Asp Leu Pro Ser Leu Glu Phe Leu
245 250 255Arg Leu Asn Ala Asn
Pro Trp Ala Cys Asp Cys Arg Ala Arg Pro Leu 260
265 270Trp Ala Trp Phe Gln Arg Ala Arg Val Ser Ser Ser
Asp Val Thr Cys 275 280 285Ala Thr
Pro Pro Glu Arg Gln Gly Arg Asp Leu Arg Ala Leu Arg Glu 290
295 300Ala Asp Phe Gln Ala Cys Pro Pro Ala Ala Pro
Thr Arg Pro Gly Ser305 310 315
320Arg Ala Arg Gly Asn Ser Ser Ser Asn His Leu Tyr Gly Val Ala Glu
325 330 335Ala Gly Ala Pro
Pro Ala Asp Pro Ser Thr Leu Tyr Arg Asp Leu Pro 340
345 350Ala Glu Asp Ser Arg Gly Arg Gln Gly Gly Asp
Ala Pro Thr Glu Asp 355 360 365Asp
Tyr Trp Gly Gly Tyr Gly Gly Glu Asp Gln Arg Gly Glu Gln Met 370
375 380Cys Pro Gly Ala Ala Cys Gln Ala Pro Pro
Asp Ser Arg Gly Pro Ala385 390 395
400Leu Ser Ala Gly Leu Pro Ser Pro Leu Leu Cys Leu Leu Leu Leu
Val 405 410 415Pro His His
Leu 42031383DNAMus sp. 3atgtcttggc agtctggaac cacagtgaca
caatctcccg tgcaggctgc tcaggtctca 60gggtgctgtg tggaattgct gctgttgctg
ctcgctggag agctacctct gggtggtggt 120tgtcctcgag actgtgtgtg ctaccctgcg
cccatgactg tcagctgcca ggcacacaac 180tttgctgcca tcccggaggg catcccagag
gacagtgagc gcatcttcct gcagaacaat 240cgcatcacct tcctccagca gggccacttc
agccccgcca tggtcaccct ctggatctac 300tccaacaaca tcactttcat tgctcccaac
accttcgagg gctttgtgca tctggaggag 360ctagaccttg gagacaaccg acagctgcga
acgctggcac ccgagacctt ccaaggcctg 420gtgaagcttc acgccctcta cctctataag
tgtggactga gcgccctgcc cgcaggcatc 480tttggtggcc tgcacagcct gcagtatctc
tacttgcagg acaaccatat cgagtacctc 540caagatgaca tctttgtgga cctggtcaat
ctcagtcact tgtttctcca tggtaacaag 600ctatggagcc tgggccaagg catcttccgg
ggcctggtga acctggaccg gttgctgctg 660catgagaacc agctacagtg ggttcaccac
aaggctttcc atgacctcca caggctaacc 720accctctttc tcttcaacaa cagcctcact
gagctgcagg gtgactgtct ggcccccctg 780gtggccttgg agttccttcg cctcaatggg
aatgcttggg actgtggctg ccgggcacgt 840tccctgtggg aatggctgcg aaggttccgt
ggctctagct ctgctgtccc ctgcgcgacc 900cccgagctgc ggcaaggcca ggatctgaag
ctgctgaggg tggaggactt ccggaactgc 960acaggaccag tgtctcctca ccagatcaag
tctcacacgc ttaccacctc tgacagggct 1020gcccgcaagg agcaccatcc gtcccatggg
gcctccaggg acaaaggcca cccacatggc 1080catccgcctg gctccaggtc aggttacaag
aaggcaggca agaactgcac cagccacagg 1140aaccggaacc agatctctaa ggtgagctct
gggaaagagc ttaccgaact gcaggactat 1200gcccccgact atcagcacaa gttcagcttt
gacatcatgc ccaccgcacg acccaagagg 1260aagggcaagt gtgctcgcag gacccccatc
cgtgccccca gtggggtgca gcaggcatcc 1320tcaggcacgg cccttggggc cccactcctg
gcctggatac tggggctggc agtcactctc 1380cgc
13834461PRTMus sp. 4Met Ser Trp Gln Ser
Gly Thr Thr Val Thr Gln Ser Pro Val Gln Ala1 5
10 15Ala Gln Val Ser Gly Cys Cys Val Glu Leu Leu
Leu Leu Leu Leu Ala 20 25
30Gly Glu Leu Pro Leu Gly Gly Gly Cys Pro Arg Asp Cys Val Cys Tyr
35 40 45Pro Ala Pro Met Thr Val Ser Cys
Gln Ala His Asn Phe Ala Ala Ile 50 55
60Pro Glu Gly Ile Pro Glu Asp Ser Glu Arg Ile Phe Leu Gln Asn Asn65
70 75 80Arg Ile Thr Phe Leu
Gln Gln Gly His Phe Ser Pro Ala Met Val Thr 85
90 95Leu Trp Ile Tyr Ser Asn Asn Ile Thr Phe Ile
Ala Pro Asn Thr Phe 100 105
110Glu Gly Phe Val His Leu Glu Glu Leu Asp Leu Gly Asp Asn Arg Gln
115 120 125Leu Arg Thr Leu Ala Pro Glu
Thr Phe Gln Gly Leu Val Lys Leu His 130 135
140Ala Leu Tyr Leu Tyr Lys Cys Gly Leu Ser Ala Leu Pro Ala Gly
Ile145 150 155 160Phe Gly
Gly Leu His Ser Leu Gln Tyr Leu Tyr Leu Gln Asp Asn His
165 170 175Ile Glu Tyr Leu Gln Asp Asp
Ile Phe Val Asp Leu Val Asn Leu Ser 180 185
190His Leu Phe Leu His Gly Asn Lys Leu Trp Ser Leu Gly Gln
Gly Ile 195 200 205Phe Arg Gly Leu
Val Asn Leu Asp Arg Leu Leu Leu His Glu Asn Gln 210
215 220Leu Gln Trp Val His His Lys Ala Phe His Asp Leu
His Arg Leu Thr225 230 235
240Thr Leu Phe Leu Phe Asn Asn Ser Leu Thr Glu Leu Gln Gly Asp Cys
245 250 255Leu Ala Pro Leu Val
Ala Leu Glu Phe Leu Arg Leu Asn Gly Asn Ala 260
265 270Trp Asp Cys Gly Cys Arg Ala Arg Ser Leu Trp Glu
Trp Leu Arg Arg 275 280 285Phe Arg
Gly Ser Ser Ser Ala Val Pro Cys Ala Thr Pro Glu Leu Arg 290
295 300Gln Gly Gln Asp Leu Lys Leu Leu Arg Val Glu
Asp Phe Arg Asn Cys305 310 315
320Thr Gly Pro Val Ser Pro His Gln Ile Lys Ser His Thr Leu Thr Thr
325 330 335Ser Asp Arg Ala
Ala Arg Lys Glu His His Pro Ser His Gly Ala Ser 340
345 350Arg Asp Lys Gly His Pro His Gly His Pro Pro
Gly Ser Arg Ser Gly 355 360 365Tyr
Lys Lys Ala Gly Lys Asn Cys Thr Ser His Arg Asn Arg Asn Gln 370
375 380Ile Ser Lys Val Ser Ser Gly Lys Glu Leu
Thr Glu Leu Gln Asp Tyr385 390 395
400Ala Pro Asp Tyr Gln His Lys Phe Ser Phe Asp Ile Met Pro Thr
Ala 405 410 415Arg Pro Lys
Arg Lys Gly Lys Cys Ala Arg Arg Thr Pro Ile Arg Ala 420
425 430Pro Ser Gly Val Gln Gln Ala Ser Ser Gly
Thr Ala Leu Gly Ala Pro 435 440
445Leu Leu Ala Trp Ile Leu Gly Leu Ala Val Thr Leu Arg 450
455 4605473PRTHomo sapiens 5Met Lys Arg Ala Ser Ala
Gly Gly Ser Arg Leu Leu Ala Trp Val Leu1 5
10 15Trp Leu Gln Ala Trp Gln Val Ala Ala Pro Cys Pro
Gly Ala Cys Val 20 25 30Cys
Tyr Asn Glu Pro Lys Val Thr Thr Ser Cys Pro Gln Gln Gly Leu 35
40 45Gln Ala Val Pro Val Gly Ile Pro Ala
Ala Ser Gln Arg Ile Phe Leu 50 55
60His Gly Asn Arg Ile Ser His Val Pro Ala Ala Ser Phe Arg Ala Cys65
70 75 80Arg Asn Leu Thr Ile
Leu Trp Leu His Ser Asn Val Leu Ala Arg Ile 85
90 95Asp Ala Ala Ala Phe Thr Gly Leu Ala Leu Leu
Glu Gln Leu Asp Leu 100 105
110Ser Asp Asn Ala Gln Leu Arg Ser Val Asp Pro Ala Thr Phe His Gly
115 120 125Leu Gly Arg Leu His Thr Leu
His Leu Asp Arg Cys Gly Leu Gln Glu 130 135
140Leu Gly Pro Gly Leu Phe Arg Gly Leu Ala Ala Leu Gln Tyr Leu
Tyr145 150 155 160Leu Gln
Asp Asn Ala Leu Gln Ala Leu Pro Asp Asp Thr Phe Arg Asp
165 170 175Leu Gly Asn Leu Thr His Leu
Phe Leu His Gly Asn Arg Ile Ser Ser 180 185
190Val Pro Glu Arg Ala Phe Arg Gly Leu His Ser Leu Asp Arg
Leu Leu 195 200 205Leu His Gln Asn
Arg Val Ala His Val His Pro His Ala Phe Arg Asp 210
215 220Leu Gly Arg Leu Met Thr Leu Tyr Leu Phe Ala Asn
Asn Leu Ser Ala225 230 235
240Leu Pro Thr Glu Ala Leu Ala Pro Leu Arg Ala Leu Gln Tyr Leu Arg
245 250 255Leu Asn Asp Asn Pro
Trp Val Cys Asp Cys Arg Ala Arg Pro Leu Trp 260
265 270Ala Trp Leu Gln Lys Phe Arg Gly Ser Ser Ser Glu
Val Pro Cys Ser 275 280 285Leu Pro
Gln Arg Leu Ala Gly Arg Asp Leu Lys Arg Leu Ala Ala Asn 290
295 300Asp Leu Gln Gly Cys Ala Val Ala Thr Gly Pro
Tyr His Pro Ile Trp305 310 315
320Thr Gly Arg Ala Thr Asp Glu Glu Pro Leu Gly Leu Pro Lys Cys Cys
325 330 335Gln Pro Asp Ala
Ala Asp Lys Ala Ser Val Leu Glu Pro Gly Arg Pro 340
345 350Ala Ser Ala Gly Asn Ala Leu Lys Gly Arg Val
Pro Pro Gly Asp Ser 355 360 365Pro
Pro Gly Asn Gly Ser Gly Pro Arg His Ile Asn Asp Ser Pro Phe 370
375 380Gly Thr Leu Pro Gly Ser Ala Glu Pro Pro
Leu Thr Ala Val Arg Pro385 390 395
400Glu Gly Ser Glu Pro Pro Gly Phe Pro Thr Ser Gly Pro Arg Arg
Arg 405 410 415Pro Gly Cys
Ser Arg Lys Asn Arg Thr Arg Ser His Cys Arg Leu Gly 420
425 430Gln Ala Gly Ser Gly Gly Gly Gly Thr Gly
Asp Ser Glu Gly Ser Gly 435 440
445Ala Leu Pro Ser Leu Thr Cys Ser Leu Thr Pro Leu Gly Leu Ala Leu 450
455 460Val Leu Trp Thr Val Leu Gly Pro
Cys465 4706440PRTArtificial SequenceConsensus sequence
6Cys Pro Xaa Xaa Cys Xaa Cys Tyr Xaa Xaa Pro Xaa Xaa Thr Xaa Ser1
5 10 15Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Pro Xaa Xaa Xaa Pro Xaa Xaa 20 25
30Xaa Xaa Arg Xaa Phe Leu Xaa Xaa Asn Xaa Ile Xaa Xaa
Xaa Xaa Xaa 35 40 45Xaa Xaa Phe
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Trp Xaa Xaa Ser 50
55 60Asn Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Phe Xaa
Xaa Xaa Xaa Xaa65 70 75
80Leu Glu Xaa Leu Asp Leu Xaa Asp Asn Xaa Xaa Leu Arg Xaa Xaa Xaa
85 90 95Pro Xaa Thr Phe Xaa Gly
Leu Xaa Xaa Leu Xaa Leu Xaa Leu Xaa Xaa 100
105 110Cys Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Phe Xaa
Gly Leu Xaa Xaa 115 120 125Leu Gln
Tyr Leu Tyr Leu Gln Xaa Asn Xaa Xaa Xaa Xaa Leu Xaa Asp 130
135 140Asp Xaa Phe Xaa Asp Leu Xaa Asn Leu Xaa His
Leu Phe Leu His Gly145 150 155
160Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Arg Gly Leu Xaa Xaa
165 170 175Leu Asp Arg Leu
Leu Leu His Xaa Asn Xaa Xaa Xaa Xaa Val His Xaa 180
185 190Xaa Ala Phe Xaa Xaa Leu Xaa Arg Leu Xaa Xaa
Leu Xaa Leu Phe Xaa 195 200 205Asn
Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Leu Ala Xaa Leu Xaa Xaa 210
215 220Leu Xaa Xaa Leu Arg Leu Asn Xaa Asn Xaa
Trp Xaa Cys Xaa Cys Arg225 230 235
240Ala Arg Xaa Leu Trp Xaa Trp Xaa Xaa Xaa Xaa Arg Xaa Ser Ser
Ser 245 250 255Xaa Val Xaa
Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Gly Xaa Asp Leu Xaa 260
265 270Xaa Leu Xaa Xaa Xaa Asp Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Pro 275 280
285Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290
295 300Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa305 310
315 320Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 325 330
335Xaa Xaa Xaa Xaa Xaa Pro Pro Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa
340 345 350Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360
365Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 370 375 380Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg385 390
395 400Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 405 410
415Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa
420 425 430Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Leu 435 440766PRTHomo sapiens 7Arg Ile Tyr
Lys Gly Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly1 5
10 15His Pro Phe Arg Ala Tyr Leu Glu Ser
Glu Val Ala Ile Ser Glu Glu 20 25
30Leu Val Gln Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Cys Thr
35 40 45Ile Lys Glu Leu Arg Arg Leu
Phe Leu Val Asp Asp Leu Val Asp Ser 50 55
60Leu Lys658390PRTHomo sapiens 8Cys Pro Met Leu Cys Thr Cys Tyr Ser
Ser Pro Pro Thr Val Ser Cys1 5 10
15Gln Ala Asn Asn Phe Ser Ser Val Pro Leu Ser Leu Pro Pro Ser
Thr 20 25 30Gln Arg Leu Phe
Leu Gln Asn Asn Leu Ile Arg Thr Leu Arg Pro Gly 35
40 45Thr Phe Gly Ser Asn Leu Leu Thr Leu Trp Leu Phe
Ser Asn Asn Leu 50 55 60Ser Thr Ile
Tyr Pro Gly Thr Phe Arg His Leu Gln Ala Leu Glu Glu65 70
75 80Leu Asp Leu Gly Asp Asn Arg His
Leu Arg Ser Leu Glu Pro Asp Thr 85 90
95Phe Gln Gly Leu Glu Arg Leu Gln Ser Leu His Leu Tyr Arg
Cys Gln 100 105 110Leu Ser Ser
Leu Pro Gly Asn Ile Phe Arg Gly Leu Val Ser Leu Gln 115
120 125Tyr Leu Tyr Leu Gln Glu Asn Ser Leu Leu His
Leu Gln Asp Asp Leu 130 135 140Phe Ala
Asp Leu Ala Asn Leu Ser His Leu Phe Leu His Gly Asn Arg145
150 155 160Leu Arg Leu Leu Thr Glu His
Val Phe Arg Gly Leu Gly Ser Leu Asp 165
170 175Arg Leu Leu Leu His Gly Asn Arg Leu Gln Gly Val
His Arg Ala Ala 180 185 190Phe
Arg Gly Leu Ser Arg Leu Thr Ile Leu Tyr Leu Phe Asn Asn Ser 195
200 205Leu Ala Ser Leu Pro Gly Glu Ala Leu
Ala Asp Leu Pro Ser Leu Glu 210 215
220Phe Leu Arg Leu Asn Ala Asn Pro Trp Ala Cys Asp Cys Arg Ala Arg225
230 235 240Pro Leu Trp Ala
Trp Phe Gln Arg Ala Arg Val Ser Ser Ser Asp Val 245
250 255Thr Cys Ala Thr Pro Pro Glu Arg Gln Gly
Arg Asp Leu Arg Ala Leu 260 265
270Arg Glu Ala Asp Phe Gln Ala Cys Pro Pro Ala Ala Pro Thr Arg Pro
275 280 285Gly Ser Arg Ala Arg Gly Asn
Ser Ser Ser Asn His Leu Tyr Gly Val 290 295
300Ala Glu Ala Gly Ala Pro Pro Ala Asp Pro Ser Thr Leu Tyr Arg
Asp305 310 315 320Leu Pro
Ala Glu Asp Ser Arg Gly Arg Gln Gly Gly Asp Ala Pro Thr
325 330 335Glu Asp Asp Tyr Trp Gly Gly
Tyr Gly Gly Glu Asp Gln Arg Gly Glu 340 345
350Gln Met Cys Pro Gly Ala Ala Cys Gln Ala Pro Pro Asp Ser
Arg Gly 355 360 365Pro Ala Leu Ser
Ala Gly Leu Pro Ser Pro Leu Leu Cys Leu Leu Leu 370
375 380Leu Val Pro His His Leu385
3909421PRTMus sp. 9Cys Pro Arg Asp Cys Val Cys Tyr Pro Ala Pro Met Thr
Val Ser Cys1 5 10 15Gln
Ala His Asn Phe Ala Ala Ile Pro Glu Gly Ile Pro Glu Asp Ser 20
25 30Glu Arg Ile Phe Leu Gln Asn Asn
Arg Ile Thr Phe Leu Gln Gln Gly 35 40
45His Phe Ser Pro Ala Met Val Thr Leu Trp Ile Tyr Ser Asn Asn Ile
50 55 60Thr Phe Ile Ala Pro Asn Thr Phe
Glu Gly Phe Val His Leu Glu Glu65 70 75
80Leu Asp Leu Gly Asp Asn Arg Gln Leu Arg Thr Leu Ala
Pro Glu Thr 85 90 95Phe
Gln Gly Leu Val Lys Leu His Ala Leu Tyr Leu Tyr Lys Cys Gly
100 105 110Leu Ser Ala Leu Pro Ala Gly
Ile Phe Gly Gly Leu His Ser Leu Gln 115 120
125Tyr Leu Tyr Leu Gln Asp Asn His Ile Glu Tyr Leu Gln Asp Asp
Ile 130 135 140Phe Val Asp Leu Val Asn
Leu Ser His Leu Phe Leu His Gly Asn Lys145 150
155 160Leu Trp Ser Leu Gly Gln Gly Ile Phe Arg Gly
Leu Val Asn Leu Asp 165 170
175Arg Leu Leu Leu His Glu Asn Gln Leu Gln Trp Val His His Lys Ala
180 185 190Phe His Asp Leu His Arg
Leu Thr Thr Leu Phe Leu Phe Asn Asn Ser 195 200
205Leu Thr Glu Leu Gln Gly Asp Cys Leu Ala Pro Leu Val Ala
Leu Glu 210 215 220Phe Leu Arg Leu Asn
Gly Asn Ala Trp Asp Cys Gly Cys Arg Ala Arg225 230
235 240Ser Leu Trp Glu Trp Leu Arg Arg Phe Arg
Gly Ser Ser Ser Ala Val 245 250
255Pro Cys Ala Thr Pro Glu Leu Arg Gln Gly Gln Asp Leu Lys Leu Leu
260 265 270Arg Val Glu Asp Phe
Arg Asn Cys Thr Gly Pro Val Ser Pro His Gln 275
280 285Ile Lys Ser His Thr Leu Thr Thr Ser Asp Arg Ala
Ala Arg Lys Glu 290 295 300His His Pro
Ser His Gly Ala Ser Arg Asp Lys Gly His Pro His Gly305
310 315 320His Pro Pro Gly Ser Arg Ser
Gly Tyr Lys Lys Ala Gly Lys Asn Cys 325
330 335Thr Ser His Arg Asn Arg Asn Gln Ile Ser Lys Val
Ser Ser Gly Lys 340 345 350Glu
Leu Thr Glu Leu Gln Asp Tyr Ala Pro Asp Tyr Gln His Lys Phe 355
360 365Ser Phe Asp Ile Met Pro Thr Ala Arg
Pro Lys Arg Lys Gly Lys Cys 370 375
380Ala Arg Arg Thr Pro Ile Arg Ala Pro Ser Gly Val Gln Gln Ala Ser385
390 395 400Ser Gly Thr Ala
Leu Gly Ala Pro Leu Leu Ala Trp Ile Leu Gly Leu 405
410 415Ala Val Thr Leu Arg
4201017PRTArtificial SequenceConsensus sequence 10Cys Pro Xaa Xaa Cys Xaa
Cys Tyr Xaa Xaa Pro Xaa Xaa Thr Xaa Ser1 5
10 15Cys1150PRTArtificial SequenceConsensus sequence
11Asn Xaa Trp Xaa Cys Xaa Cys Arg Ala Arg Xaa Leu Trp Xaa Trp Xaa1
5 10 15Xaa Xaa Xaa Arg Xaa Ser
Ser Ser Xaa Val Xaa Cys Xaa Xaa Pro Xaa 20 25
30Xaa Xaa Xaa Gly Xaa Asp Leu Xaa Xaa Leu Xaa Xaa Xaa
Asp Xaa Xaa 35 40 45Xaa Cys
5012196PRTArtificial SequenceConsensus sequence 12Arg Xaa Phe Leu Xaa Xaa
Asn Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
10 15Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Trp Xaa
Xaa Ser Asn Xaa 20 25 30Xaa
Xaa Xaa Ile Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Leu Glu 35
40 45Xaa Leu Asp Leu Xaa Asp Asn Xaa Xaa
Leu Arg Xaa Xaa Xaa Pro Xaa 50 55
60Thr Phe Xaa Gly Leu Xaa Xaa Leu Xaa Leu Xaa Leu Xaa Xaa Cys Xaa65
70 75 80Leu Xaa Xaa Leu Xaa
Xaa Xaa Xaa Phe Xaa Gly Leu Xaa Xaa Leu Gln 85
90 95Tyr Leu Tyr Leu Gln Xaa Asn Xaa Xaa Xaa Xaa
Leu Xaa Asp Asp Xaa 100 105
110Phe Xaa Asp Leu Xaa Asn Leu Xaa His Leu Phe Leu His Gly Asn Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Phe Arg Gly Leu Xaa Xaa Leu Asp 130 135
140Arg Leu Leu Leu His Xaa Asn Xaa Xaa Xaa Xaa Val His Xaa Xaa
Ala145 150 155 160Phe Xaa
Xaa Leu Xaa Arg Leu Xaa Xaa Leu Xaa Leu Phe Xaa Asn Xaa
165 170 175Leu Xaa Xaa Leu Xaa Xaa Xaa
Xaa Leu Ala Xaa Leu Xaa Xaa Leu Xaa 180 185
190Xaa Leu Arg Leu 195131176DNAHomo sapiens
13gagggcatcc ccgtggacag cgagcgcgtc ttcctgcaga acaaccgcat cggcctcctc
60cagcccggcc acttcagccc cgccatggtc accctgtgga tctactcgaa caacatcacc
120tacatccacc ccagcacctt cgagggcttc gtgcacctgg aggagctgga cctcggcgac
180aaccggcagc tgcggacgct ggcacccgag accttccagg gcctggtgaa gcttcacgcc
240ctctacctct acaagtgtgg gctcagcgcc ttgccggccg gcgtctttgg cggcctgcac
300agcctgcagt acctctacct gcaggacaac cacatcgagt acctccagga cgacatcttc
360gtggacctgg tcaacctcag ccacctgttt ctccacggca acaagctgtg gagtctgggc
420ccgggcacct tccggggcct ggtgaacctg gaccgtcttt tgctgcacga gaaccagctg
480cagtgggtcc accacaaggc attccacgac ctccgcaggc tgaccaccct cttcctcttc
540aacaacagcc tctcggagct gcagggtgag tgcctggccc cgctgggggc cctggagttc
600ctccgcctca acggcaaccc ctgggactgt ggttgtcgcg cgcgctccct gtgggaatgg
660ctgcagaggt tccggggctc cagctccgct gtcccctgtg tgtcccctgg gctgcggcac
720ggccaggacc tgaagctgct gagggccgag gacttccgga actgcacggg accagcgtcc
780ccgcaccaga tcaagtcaca cacgctcacc accaccgaca gggccgcccg caaggaacac
840cactcacccc acggccccac caggagcaag ggccacccgc acggcccccg gcccggccac
900aggaagccgg ggaagaactg caccaacccc aggaaccgca atcagatctc taaggcgggc
960gccgggaaac aggcccccga gctgccagac tatgccccag actaccagca caagttcagt
1020tttgacatca tgcctacggc ccggcccaag aggaagggca agtgtgcccg caggaccccc
1080atccgtgccc ccagcggggt gcagcaggcc tcctcggcca gttccctggg ggcctccctc
1140ctggcctgga cactggggct ggcggtcact ctccgc
117614392PRTHomo sapiens 14Glu Gly Ile Pro Val Asp Ser Glu Arg Val Phe
Leu Gln Asn Asn Arg1 5 10
15Ile Gly Leu Leu Gln Pro Gly His Phe Ser Pro Ala Met Val Thr Leu
20 25 30Trp Ile Tyr Ser Asn Asn Ile
Thr Tyr Ile His Pro Ser Thr Phe Glu 35 40
45Gly Phe Val His Leu Glu Glu Leu Asp Leu Gly Asp Asn Arg Gln
Leu 50 55 60Arg Thr Leu Ala Pro Glu
Thr Phe Gln Gly Leu Val Lys Leu His Ala65 70
75 80Leu Tyr Leu Tyr Lys Cys Gly Leu Ser Ala Leu
Pro Ala Gly Val Phe 85 90
95Gly Gly Leu His Ser Leu Gln Tyr Leu Tyr Leu Gln Asp Asn His Ile
100 105 110Glu Tyr Leu Gln Asp Asp
Ile Phe Val Asp Leu Val Asn Leu Ser His 115 120
125Leu Phe Leu His Gly Asn Lys Leu Trp Ser Leu Gly Pro Gly
Thr Phe 130 135 140Arg Gly Leu Val Asn
Leu Asp Arg Leu Leu Leu His Glu Asn Gln Leu145 150
155 160Gln Trp Val His His Lys Ala Phe His Asp
Leu Arg Arg Leu Thr Thr 165 170
175Leu Phe Leu Phe Asn Asn Ser Leu Ser Glu Leu Gln Gly Glu Cys Leu
180 185 190Ala Pro Leu Gly Ala
Leu Glu Phe Leu Arg Leu Asn Gly Asn Pro Trp 195
200 205Asp Cys Gly Cys Arg Ala Arg Ser Leu Trp Glu Trp
Leu Gln Arg Phe 210 215 220Arg Gly Ser
Ser Ser Ala Val Pro Cys Val Ser Pro Gly Leu Arg His225
230 235 240Gly Gln Asp Leu Lys Leu Leu
Arg Ala Glu Asp Phe Arg Asn Cys Thr 245
250 255Gly Pro Ala Ser Pro His Gln Ile Lys Ser His Thr
Leu Thr Thr Thr 260 265 270Asp
Arg Ala Ala Arg Lys Glu His His Ser Pro His Gly Pro Thr Arg 275
280 285Ser Lys Gly His Pro His Gly Pro Arg
Pro Gly His Arg Lys Pro Gly 290 295
300Lys Asn Cys Thr Asn Pro Arg Asn Arg Asn Gln Ile Ser Lys Ala Gly305
310 315 320Ala Gly Lys Gln
Ala Pro Glu Leu Pro Asp Tyr Ala Pro Asp Tyr Gln 325
330 335His Lys Phe Ser Phe Asp Ile Met Pro Thr
Ala Arg Pro Lys Arg Lys 340 345
350Gly Lys Cys Ala Arg Arg Thr Pro Ile Arg Ala Pro Ser Gly Val Gln
355 360 365Gln Ala Ser Ser Ala Ser Ser
Leu Gly Ala Ser Leu Leu Ala Trp Thr 370 375
380Leu Gly Leu Ala Val Thr Leu Arg385
39015143899DNAHomo sapiensmisc_feature(2044)..(2143)n is a, c, g, or t
15aagcacatac aggtgacatt acagaactga cagttatgcc aggcactgta cttagcccct
60ataccatcct caaacagctg tatgatgtag attgggtatt aaccccatta ataacaaaag
120tacagggaac aaagtgactt tccaaaggtc atgccattca aaggagggtg aatcttaggt
180tggacgcagg ctgtctgact ctggagtctg aggtgttaat gctgcctcct ccatgggaac
240agcccaagtg aaaaacagct gatccactct tcatttactt ggcatctgtg ctaagctggt
300ccctgagcca agctctgagc aacagaaaca gaagctctgc attaggagct tgtgagcatg
360tcaatgccgg gtaaaggagt gctggaaacc gctgggatgg ccgccgagca ctaggccgtt
420gaaggtgggc tctgtgtgac tggttcctct acactctggc ctggctgcct gcaggaagaa
480gatcaagctg agtgggctgg ccctggacca caaggtgaca ggtgacctct tctacaccca
540tgtgaccacc atgggccaga ggctcagcca gaaggccccc agcctggagg acggttcgga
600tgccttcatg tcaccccagg atgttcgggg cacctcagaa aaccttcctg agagtgagtg
660tctggtcaag gtgccggcct tgggggatag tgatggtggg tcctcatatt cagtgagcac
720tcatggttga gtatttattc gcacccctct tcagtcctta caacacccca tgatgtaggt
780ggggcatgct cctcatttac agatgggcac atcaaagctc agctaacgct gggaagttca
840gattcagggt taccctgctg gattcctggg attggggagg gaggagcttc caaaatgggg
900acaaggtctc tgggcctgtc gggtagctgg tttcctcagg gccccttgca acctctgagc
960ttattgcatc aggtgcagcc aggcccgtga gcctcctggc aggggtcctc cacacctggc
1020tgtcttttgc cccctgctgg tcacaggagg agctgcagca cctgcctggg ctgcttctca
1080ggagggtaca tgaagatccc aggaccgcca gctccatgat aagtggaagg agctccttgg
1140agtcaggagc gggagttgag gagtttgagt cctgctctcc agttataggc tatgtgactt
1200gtgtagatca cctaaccttg ctcttgattt ccttacctct taaactagca ctaaaagcac
1260cccacaaact gtaaagttag ttgtgatgat tgaatgacac catgggtgtg gaagctcttt
1320gtaaagtgca aaacggtgtg cagtttgagg gtggttaccc ccagtgccga ttctcagagg
1380gcaacatggc taagggcacg agctggagtt aggctgacct gctgcttcca gccctgtgag
1440cttgagcaag tcatttaact tcctgagctg cagtttcctc atcagtaaaa tgtgataagg
1500atagggttgt tgtaagattt tattaaatgg ggtaataaat gtcaagtatg tagcccatag
1560tgagtgcttc agagtttttt tcttttgttt ctttcccccc cgccccgaga tggagcctta
1620ctctgttgcc caggctggag tgcagtggca tgatcttggc tcactgcaac ctccgcctcc
1680cgggttcaag caattctcct gcctcagcct cccaaatagc tgggactaca ggcgtgcacc
1740accatgctcg gctaattttt gtatctttag tagagacggg gtttcaccat gttggccagg
1800ctggtctcga actcctgacc tcatgatgct cctgcctcag cccccgaaag ttttgggatt
1860acaagtgtga gcccccgtgc cctgccaggt tttttttttt tttttttttt tgtaaaacac
1920ccacagggta ttgctgttgc ctgggctgga gtgcggtagt gcaatcatag ttcactgcag
1980ccttgacctc ctgggctcaa gtgatcctcc tgcctcagcc tcctgagtag ctgggaatac
2040aggnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnttttgta ttttaagtag
2160agacagggtt ttcccaatgt tggccaaggc tggtctaaaa ctcccaacct caggtgatcc
2220acccacctca gcctcccaaa gtactgggat tacaggcgtg agacaccgtg cccagccagg
2280aggcttattt tcttgataaa ttacccagtc tcaggtattt ctctacagcg atgcaagaac
2340agcctaatac atccaggctc agcatcagtg gacccaggtg ggagagctta agatgtcaag
2400gtctgaatgc cgcttccaca cacctttggg acctagggac tccctctctt tttctttttt
2460cagtagaaga tgttatcttc tcctttctct gaccagtagt tggtgatggt ttcagagata
2520gtttttcagt caagatatat ttcagtggct tcactgagcc caagttccct cgcctctcta
2580ggactttatt tccttgtttc tagaagaggg ataacacata ttttctaagg tggttgtgag
2640attaagggag ctggtaccgg gtggtgcata aggacaggat agagcaatgg tgagaccact
2700caaaaagcga aaagttgacc tgcgagggtg acacttatca aatcagcaca cagtgggagt
2760ggaaggaatg tccctcatca gttacaatat ttggagagtg caagttatag aaaacccagc
2820cctggccggg cgcggtgggt catgcctata atcccagcac tttgggaggc tgaggcaggt
2880ggatcacgag gtcaggagtt caagaccagc ctgaccaacg tggtgaaacc ccacctctac
2940taaaaataca aaattagctg ggcgtggtgg tgtgtgcctg taatctgagc tactcaggag
3000gctgaggcac gagaatcact tgaaaccggg aggtggagtt tgcagtgagc cgagatcgca
3060ccactgcact ccagcctggg caacagagcg agactccatc tcaaacgaaa aaaaaaaaag
3120aaagaaaacc cagctctaac tggcttaaac agtaagaaga tctattatat tatccatctc
3180aggcagcagc aagcccagag gtaggggact ccaaggttgg ttgatccagg gcttaacgat
3240gtcatcaaag acccaggttc tttctgtctc ggcacctctg tctgcagggc cagcttcatc
3300ctaagccaga ttgttcttgt cttgattaca agttggctgc tgggccagca gacgctgcct
3360gcctccctgt tcatcttcag aagtagaaag tggcccttcc ccagtcatgg aatgaaagag
3420tttcctttct gtctgggatt gcttaggtcc acccacctga agccaatgac tgtcaccagg
3480aaggtaatat acactgattg tcttaagtca gggttcctga gccagtcttg ggcaaggagt
3540gtgatactgt catgattgtc ttgggctcat cagggcagct ctgcagatga gatcaaactc
3600caagctacat tattctgaac agtgggaagt aggaaagaga cattttggga gatacaaaac
3660acaatgtcta tcccatatcc ctaggtccag gtcacagtgt cttggttgga catcaaatgt
3720agaaaaagaa agactgtcca tccatttatc tacctattca tctggttttt gatttttttt
3780aaattttatt ttaagacatt ctcactctgt cacccagact ggagtgcagt ggtttgatca
3840tggctcatgg cagcctcaac ctcccaggct caagtgaccc tcccatgctc aagtgatcct
3900cctacctcag cctcccaagt agctagaact aaaggtgcat gccaccacgc tcagttaatt
3960tttgcatttt ttgtagagat ggggtttcgt catgatgccc atgctagtct ggaattcctg
4020aactcaagca atatgcctgc ctttgcctcc caaaatgctg ggattgtagg catgagccac
4080tgctcctggc tcatctgttt aataatttat gaaacaacta ctgggtgctg agcacggggc
4140caggggctgg agatctagca gggaccaggc agatctctgc caagtcgttg gtttcttaaa
4200ggttttgctc ataattcccc ttttcttttc tctttcgttt tttttctttt ctttctttct
4260ttctttcttt tttttttttt gagacagagt ctcactctgt tacccaggct ggagtgcagt
4320ggtgcgatct cagctcactg caacctctgc ctcctgggtt caagcgattc tcctgcctca
4380gcctcccgag tagctgggac tacaggcgcc tgccaccatg cccggctaat ttttgtgttt
4440ttagtagaga ctgggtttca ccatattggc caggctggtc ttgaactcct gaccttgtga
4500tccgcccgct tcggcctccc acagtgctgg gattacaggc gtgagccacg gcgcccagcc
4560agtttccctt ttcaatgagg cctccctgac ctccatactc tactcctcca cctggcccac
4620tcagctctac tttttcttcc ccatagcact caagacctcc taacatacta cgtaagttat
4680ttatttacta ggcttactgt gtattgtctg tcttcctcta ctagaatgta aactccatga
4740gaatagaaat ttttgccttt ttatttagtg tggtgtctgc agcccctggc ttagtccctg
4800gcatacaaca gtcactccac ccacagttgc tgaataagtg actaaaggtc cctgccctca
4860tattgttatg agggagtgtg catgttgtta gagaaaaatc tgaggcacaa taaaatttta
4920tagagtttaa gttttctttt ttaagcaatc cacgaattgg ggtagtttca gaggtagttt
4980ttcagtcatg acgtatttca atggcttcac tgagcccaag ttctttcacc tctctaggac
5040tttatttcct tatttctaga acggggataa cacatagttc ataaggcagt tatgagagta
5100agggagctgg tatggggtga tgcataagga caggatagag cagtggtgag accgctcaga
5160tgacaaagcg tcagagacca gtatttacga cggaaatgtg gaagcatgat aaagaaatta
5220tttgggctgg gcacaatgac tcacaactaa taaaactttg ggaggccaag gtgggaggat
5280cacttgactt gcagaaggtc aaggctgcag tgagctgtga ttttgccact gcactccagc
5340ctggtcaaca gagtgagacc ctggctcgaa acgttatttg attggttaca gttatacagt
5400tgccttattt ggtctattcc atttgaaagt tcctagttct ataattttaa gtttgttggc
5460tgtttctgat tggttaagct taagttttgt tttcctttaa tacagttaag tgccccataa
5520tgacattttg gtcaaggaca gaccacatat acagtggtgg tcccataaga ttataatgga
5580gctgaaacat tcctattgtc tatggcgtag tggtcctgat gttgtagcgc aatgcattag
5640ttatatgttt gtggcaatgc tggtgtaaac acacctactg cactgccagt gatataaaag
5700aatagcacat acagttatat atagtacata atatctgata atgataatac ataactatat
5760tactggttta tatatttact atattattta tctttatttt atttttgaga cagagtctca
5820ttctgtcacc caggctggag tgcagtggcg cgatcttggc tcaccgcaac ctccgcttcc
5880tgggttcaag tgattctcct gcctcagtct cctgagtagc tgggattaca ggtgtgcacc
5940atgacaccct gctaatatgt tttgtatttt tagtagagat ggggtttcac catgttggcc
6000aggctggtct tgaactactg acctcaagtg atcaccccgc ctcggcttcc caaagtgctg
6060ggattacagg cgtgagccac cacgcatggc ctatttataa ttattttaga gtgtacgcct
6120tatacttata aaaaaaagct aactgtcaaa cagcctcggg caggtccttc aacagatatt
6180ccagaagaca ttgttatcat aggagatgac agctccgtgc atattattgt ccctgaaaac
6240cttctagtgt ggaagtggaa gacagtgata ttgatgatag gacccagtgt aggcctaggc
6300taatgtgtgt gtttgtgtct ttgcttttaa caagaaagtt taaaaagtta aaataaaata
6360caaaaatttt taaatagaaa aaagctgccc aggaacaatg gctcacacct gtaatcccac
6420cattcgggga ggccaaggtg ggtggattgc ttgagctcag gagttcaaga ccagcctggg
6480caacatggtg aaaccccatc tctacaaaaa atacaaaaat tagccgggtg tggtggcatg
6540cggctatagt tccagctaat cgaggggctg aggtgggagg atcactgggg gggaggtggt
6600tgaggctgna gtgagctgtg attgnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6720nnnnatattc ttaaaaaaat ttttttttat ttttgagaca gaatttctct cttgttgccc
6780aggctggagt gcaatggcgc tatctcagct cagggcaacc tccacctcct gggttcaagc
6840gattctcctg ccttagcctc ccaggtacag gcgcccgcca ccatgctcgg ctaatttttg
6900tatttttagt agagatgggg tttcaccatg ttgtccaggc tggtcttgaa atcctgcctc
6960aggtgatcca cccccctcgg cctcccaaag tgctggaatt tacaggcgtg agccactgtg
7020cctggcctcc tttacatttt tttaaattta attttaattt tttaattttt aatttctcat
7080atatatatat ttttaagact agccaagtga agcagtggga gtggaaaagg aactggtttt
7140gatcaatagg tgtaaacacc actgcactgg gaccagccta ttttacattc ctgttagcag
7200tgatgagggt tcactttctt tgtagcctca acaatatgtg tcgttgccca tctttttttt
7260tttttttttt tttttttttg agatggagtc tcactctgtt gcctaggctg gaatgcaatg
7320gcatgatctc agctcactgc aacctccgcc tcccaggttc aagtgattct tgtgtctcag
7380cctcctgagt agatgggatt acaggcgtcc accaccacgc ccggctaatt ttttgtattt
7440tcagtagaga tggggtttca ccatgttggc caggttggtt tcgaactcct gacctcaagt
7500gatccgccca cctcggcctc ccaaagtgct gggattacag gcatgagcca ccgcgcccgg
7560cctgcccatc ttttttttgt tatagccatc ctagtggatg taaagttttt ttgtgatttt
7620gatttgtgtt tccctactga tcaatgatgt tgagcatctt ttcctgtgct tattggcttt
7680tggtatatct ttggagaaag gtctattcag gtcctttgcc cactttaaaa ttaggttatc
7740tttctattac tgagatgtaa gagttcttta tgttctagat ataagtctcc tacatatgat
7800ttgtaaaaat tttccttcca ttattgggtt gtctttcact ttcttttggt gtcctttagt
7860gcacaacagt ttttaatatt gaagtccaat tttctatttt tctcttttgc cacttgtatc
7920ttggtgtcat gtttaaggaa ctattgccta atctcaggtc acaaagattt acacctgtgt
7980ttccttcttt ccttccttcc ttccttcctt ccttctttcc ctccctccct ctctccctcc
8040ctccctctct ccctccctcc ctccttccct tcctccctcc ctccctcctt ccttccttcc
8100ttccttcctt ccttccttcc ttccttcctt ccttcctttg tccttctgac ggaatcttgc
8160tctgtcaccc aggctggagt gtagtggcac gatcttggct cactgcaacc tctgcctcct
8220gggttcaagc aattctcctg cctcagcctc ctgagtagct gggactacag gcacacacca
8280ccatgcccag ctaatttttg tatttttagt agagacgggg tttcaccaca ttggccagga
8340tggtttcgat ctcctgacct cgtgatccac ccgccttggc ctcccaaagt gctgggattg
8400caggtgtgag ccaccatgcc cggcctgtgt tttcttagag ttttgtagtt ttagctctta
8460tagttagatc cttgatccat tttgagttga ttttgtatat agtgtgagat atccacctgg
8520tgttgtaaat tgcccagaag tgggtatgct tctaaatctg gctgttaggg attactagag
8580gtgaccaaag tgaatttttt ctttgtttct tttttttttt ggagacagag tctccgtcac
8640ccaggctgga gtgcaatggc ttcatcttgg ctcagtgcaa cctctgcctt ctggtttcaa
8700gcagttctcc tgcctcagac tcctgagtag ctggtattac aggcgtgtac caccatgctt
8760ggctaatttt tgtattttta gtaaagatgc agtttcacct gttggccagg cttttctgga
8820actcccggcc tcaagtgatc catctgcctc tacctcccaa agtgctggga ttacaggtgg
8880gagccaccgt gcccagtcct tttctcagaa tttatttgtt tttttttgtt ttgtttcatt
8940tttgagatag ggtctcactc tgtcagctag gcaggagttc agtggtgtga tcattgctgc
9000agccttgaac ttctggactc acgtgatctt cccacctcag cctcctgagt agctaggatt
9060acaggcatgt gcttccacac ctggctaatt ttttaatttt ctaggactta tttgtccatt
9120cttgcaaagc agggtacaac atgcctatct ctacctacct ctcttccctt caagggactc
9180cagccaaaat ccttgaggct ctcgggctga ctgtgggtgc tgttgcctga tctgcctcag
9240tcatgctgca tgatcaaaag tgtccgtttt ctgcttcttg gaactttatt cactttgggt
9300gtcagtcttc ctctgcagtg tcccaagaac acagaattag accaggaatc tgtgttgcca
9360tagtgtgtgg aaagaggcag acttccaact ccgctatgtg ctgttgggtg attgaagctt
9420aattttcttt ctatctttct ttcttttctt ttcttttttt ttttttggag atggaatctc
9480gctctgttgc ccaggctgga gtgcagtggt gcgatctcac ctcactgcaa cctccgcctc
9540ccaggttcaa gcgattctcc tgcctcagcc tcctgagtag ctgggattac aggtgcatgc
9600caccatgccc ggctaatttg tgtaatttta gtagaaacag tgtttcacca tattggtcag
9660gctggtctcg acctcctcac ctcaggtgat ccacccgcct tggcctccca aagtgtcggg
9720attacaggcg tgagccaccg tgcctggcac ttaattttct taatacctca attaccccat
9780atggtaaaat gggactagta atccatacct tatagcgctg ttgtgaaaat gaaatgaggg
9840taagcagata aaatttcaga ctacggatgg gattgttact acattctgaa cctggctttg
9900ctgttatttg ctatgtgacc ttatcttctc tggatctcca ttctttccaa gtctataaaa
9960caaagtggac aattgtcaac ctttcttcca aagagcaatg atttaaggat caaatgatgt
10020catttaacaa aaatatgaag agctcaacaa atgaggaact cattattatt attacaatta
10080ttattatttt agaaataggg tcttgttctc ttgcctaggc tggagtccag tggtataaac
10140acagctcaat gcatcttcag cctcctggat acaagtgatc ctcatgtctc atccccctaa
10200gtagctggga ccacaggcat gtaccaccac gcacggctaa ttttttattt tttattttta
10260ttttttgaga cagtcttgct ttgtcgccca gactggagtg cagcagcgca atcaccgctc
10320actgcaacct ccgcctcctg ggttcaagtg attctgctgc ctcaacctcc caagtagctg
10380ggattacagg cctgtgccac catgcccggc taattttttt gtatttttgg taaagacggg
10440gtttcaccat gttgcccagg ctgatctaga acccctggcc tcaagtgatc cccctttctt
10500ggcctcctaa agtgctagga ttacaggcgt gagcctctgc acctggcctc ggctaatttt
10560ttattttttg tagagacagg ttctcactat gttgccaggg ctggtcttga actcctgggc
10620tcaagtgatc ttcccacctc agcctcccaa agtgctgaga ttacagatgt gagccactgt
10680gcctggcctg gaactcatta ttgaagcatt cactagtatc aactttgggg ttacctggcc
10740acatcctctg acctacctat aagggtatca cagctaacgg agcctctgtt tctcagaatt
10800taggcagaag cagttcaatt tatcacaaac tactctatat ccagcataag tgcccaaata
10860aaacaattgc taaagttctt taggcattta ctgtttgtta gttagatatt tagtcctcac
10920tacaaatctg tgatacaggt attattttta ttaaccccat tttatagaag agaaacctga
10980agctcagaga tgctaagtaa cttgtgcaag gtcacacagc tagtaaataa agggcagagt
11040aaagatttag tttcacattg gactccagaa cctttctact gggactcatg ggaatagtgt
11100ggatgtccct gaccttcagt ggcccagggc tctcctgggg gaatccagcc atagacaaga
11160caccagcgag agcccaatcc taagattttg tttgtttgtt tttgagacaa ggtctcactc
11220tgtcaccaga ctggagtgca gtggcatgat caatgctcac tgcaaccttg atctcccagg
11280ctcaagcaat cctcccacct cagcctcctg agtagcttgg actacaggtg cacaccacca
11340cacctgacta attttaaaat tttatttaat taattactta ctattatttt ttgagacagg
11400gtatcacttt gtcacccaag ctggactgca atggtgtggt ctcagctcat tgcgtcctcc
11460acctcccagg ttcaagtgat cctcccacct cagcctctgg agttgcaggg actgcaggtg
11520tgcgccacta tgctcagcta atgtttttat tttttgtata gatggggtct cactatgttg
11580ccagggctag tctcaaactc ttggactcaa gcgatcctcc tgtcttggcc tcccaaagtg
11640ccgggattac aggcataaac caccacaccc aacccctaag gtgtttttgc tgaatgtgac
11700catgtcagag gcaggaaagg gaagcatcat ggggttagga aaggaacact gagcagggag
11760acaaagaaaa tgggatcatt ttgtgagtgt tcgctgtgtg tgtatgtgtg acaattctca
11820gagccagcct ctcaggtggt tgagaccaca gtccccattt cccagatgag ataatggagc
11880ctcagagagt ttctgcagca cagctagtgg aattagaatt tgaacccggc tcttccagac
11940tccaggtgct tcacaaccat cccaaaccta gtcatttgca gtttaccttc atgattttac
12000catttccctt tgccatagct agtgttattt acttaataat tccttttgaa tcagtctgct
12060taaaaaaaaa tagcttcatt ctaaagtgta atattcttgg aatatcgggt ttgctgttac
12120ccacccccac acgttataca tatacatgta tgtttctaat acatatatat gtacgtatat
12180acgtgtatcg ttttttgtta ttttttttgt tgttgttagt tttttttaga tggagtctct
12240ctctgtagcc caggctggag tgcagtggtg tgatttcggc tcactggaac ctctgcctcc
12300tgggttcaag cgattctcct gcctcagcct ctggagtagc tgggattaca ggcacccacc
12360actacacccg gctaatgttt gtatttttag tagagacagg gtttcaccat gttggccagg
12420tgggtcttga actcctgatc tcaagtgatc cacctgcttt ggcttcccaa agtgctggga
12480ttataggtgc gagctactgc ggctggccaa tgtatgtttt taatacacat tcaaataacg
12540aataactatg aaacctgaaa aactgctcca tgttacttcc tgaacccatc ttgagtgctc
12600acatgctgtg cataccacat attgggaaac actgctttcc ctggcttcca agcccagctt
12660aatcactgtc ccatcctatg cttcgcttta tttgtctata aatgttgggg ttgggggttg
12720atgccaaaga ccttttctgt tgtcattaac atggacacag ctctaagagg tcttggcatc
12780ttgggctggc tctcctttta gttcagaatt tggattttta tccaactact cagagtgatc
12840aagccttcct tatgaatgaa ctcgttggtc aaactcataa aaggctgatc gataaaacag
12900gaatgaatgt atgaattgac actaagtcat tagcatttca cgggaatgga ttctccgtta
12960gtggaagagc acatgtcctt tctggcactg atgtgtgctt gggaaactta ctgagctaac
13020tggcccatgt aacacagagg ccctttggtg cagtggaaaa ctgttgactt tggagattat
13080cttgagtttg aatctgagcc tgcctgtaag aagctggcta actgaattgc tttgcttctt
13140ggacccttac catttataaa atggggacca ttgtactcac cctttagggt tattgcatgg
13200attaaatggg attctctata gaaaatattg gcacaaagta ggtgtaaatt tgcacgctag
13260tgggattgtt tgtgagggaa attgtcattt gattatcaaa gacttaggag caggaacagt
13320gtctaattca gggactgcaa atggaaatgc cagctgaggc caggcatttg ctaataattg
13380ggtaaagcag ggcaggtgta gaatagcaat gtctgggaat taaaagagag gtgaggacgt
13440gtatgacctt gagaaggcaa gccctggcaa aaggggatgg cctccactca gctacagtca
13500tgcctagatc ttctaacttt ttatttttat ttttattttt tgagacggag tcttgctctg
13560tcacccaggc tggagtgcag tggcgcgatc tcggctcact gcaagctccg cctcccgggt
13620tcacgccatt ctcctgcctc agcctcccaa gtagctggga ctacgggcgc ccaccaccat
13680gcccggctaa tttttttttt gtatttttag tagagatggg gtttcaccgt gttagccagg
13740atggtctcga tctcctgact ttgtgattta ccctccttgg cctcccaaag tgctgggatt
13800acaggcttga gccaccgcac ctggccgatc ttctaacttt ttaaagagaa gcaagacatc
13860tggattttta tgtgataact cctgatttta aactggcacc caattataat ttacaacact
13920ataagggtca acattgccag cagagcaaaa catgggtggg ggcaactgct ggtcaccggt
13980gtgcagcctc tggtctaaaa tcatctttgt atttcttctt gctttacgca ttgtcccagc
14040acagtgctgt tgtatagtaa atatccagta agtgggtgta gaatgaataa accaatgcag
14100ataaacctgt agagaggccg ggcacagttg ctcatgtctg taatctcagc acnnnnnnnn
14160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
14220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntagtccca gcactttggg aggccaaggt
14280gggtagatca cctgaggtca ggagttcaag accagcctgg ccaatatagt gaaaccccgt
14340ctctacaaaa ataaaaaaat tatctgggca tgattgcagg tgcctctaat cccagctact
14400cgggaggctg aggccggaga attgcttgaa cctgggaggc ggaggttgta gtgagccgag
14460atcatgccat tgcactccag cctaggtgac ggagcaagat tctgtctcaa aaaaaaaaaa
14520aaaaaaaaag aaaaaagaaa agaaaaagaa acaatgaatg agtgtgaggc tcatggtagt
14580attggttcct gagagtagcc aaccttattg gtcatcccag ccacgaagtg aaatggtacc
14640cctggcttgg gccaatgaat gaggaagaat aatggcaaat gggggtctat gcctccaccc
14700tccaccacta gggaggtctc aagcttgaaa tccagtgacc aggtttttag gtcctggacc
14760tggccagtcc tcctacagtc aagtagataa gtggagggtt tggtccgttg ggctacggag
14820atagtgatca aggccgttac tctgcaatca gactcagaaa tggcctctca gttacttctc
14880catttgtggg tcttttggaa gagcagagaa gaggaaggaa tttaggtctt ctcaccctct
14940gggctgcctg tccctgctcc ctgagccatg gagggctggg gtggaatatg gggaataaat
15000ctgtactttt tttttttttt ttttttgaga cagagtctcg ctccgtcgcc caggctggag
15060tgccgtggcg tgatctctgc tcacagcagc atctgcctcc cgggttcaag ttattcttcc
15120acctcagtct cctgagtagc tgggattaca ggtgcccacc accacgcccg gctaattttt
15180gtatttttag tagagacagg gtttcactgt gttgggcagg ctggtctcaa atacctgacc
15240tcaggtgatc cacccgcaca tgcctcccaa agtgctggaa ttacaggcat gagccaccgt
15300gcccggtcct accaatctgc acattttaat tgacaagggt caccctccac tcatgtgcca
15360ggcatagttc tgagaagcat cccacaagga tgcctctgag ttcaccctga caagtccact
15420agctcttggc agagacatct ggcaaattca aggcttgaga catgctggcc tctctttaaa
15480gtgcagcaaa ttttgtctag agcttggtca gttaaaattt tgatgttttg ttttgcatta
15540atttcaattt ttaagaaatg ttgcattaaa atgttattta tcttgaatag taaatttctt
15600agtgtcccct taatttctta gtgtgtctga gttgagagcc tcccctgcct gattctagtc
15660cagaccctgg ggtgacagaa gactggtggg agatgggagg tgaggagggg agtgttggtt
15720ggagaggatg atctacagag tgctggagag actctgtatg gagcttttca tgctgcctgt
15780ttgccagccc tgaagctatg ccttgaggtt gggcaaggtg gcatatccta gatcagagat
15840cctcaactgg ggccattttt ctccccagag gacatttgga aacatgtgga gacatttttg
15900atcatctgcg ggggtgggga gaggggctac tgacatctgg tgagtagaga ccagagggac
15960cattaaactt tctacaacgc ccaggacagc ccctccacaa taaagagtta tttgacctca
16020catattaata gcacaaagtt gaggaacctt gatctagatc cacagcacag aagaaaggat
16080gtagattttt cacacattaa agatgagaaa gcttgtgcct gtaatccctg tgactcagga
16140ggctgtggca ggaggattac ttgagcccag gaattcaggg ttacagtgaa ctatcatcgc
16200agcactgcac tccagcctgg gtgacagagc aagattttgt ctcttaaaaa aaaaaaagat
16260gaggacaggc acagtggctc atgcctgtaa tcccagcatt ttgggaggcc gaagtgggtg
16320gatcacgagg tcaggagttc aagaccagcc tggccagcat agtgaaaccc catctctact
16380aaaaatacaa aaaattagcc agctacttgg gaggctgagg caggagaagc gcttgaaccc
16440gggaggtgga gcttgcagtg agccaaaatc ttgccattgc actccagcct gggcgacaga
16500gcaagactcc gtctcaaaaa gaaaaaaaaa aaagatgaga aagaggaagg gagagaaaaa
16560agagagagag gaaagaaaga gagaaggttt tggagtcaaa aagacttaga aattccagtt
16620cttccacttc ccatggaacc ttggcaagtt gccttctctc tttctctgaa tctcacattt
16680tgcctctgtg aagtaggggt ggtacctggt ggagatgatg cggagatgag ggtgaggggt
16740gtgttgcaca ctatgcccct aggatgggtg agagcttggg agcactgaac ctccctttcc
16800cctcttgttt cttcccccca ttgtctccca ccagctccct gggatctcca cttcactctc
16860tgggattcca ccagcaggag gctactcctg gagttaaggc gtgttgttca gactggggca
16920ttttaggggg cataaataat aattatgcct ggacaatgga cataacatct agggccttct
16980gaagcaaacc agggtgtggg gtacccaaac aaggcagtag gccccaggag gcaggtccct
17040gcagtcccag cagagagcag ggcacagggt tgagaagact gagcaaactt cattatcagc
17100tcctttgtcc cccactctgt cctggagcaa tcattctggc ctcttcccac ttccccaaaa
17160acccagtata aaggctgctt ctggcccctg aagccagagg cactgagagt ggaggtctca
17220gactcttgga aggtgagttc ttttctggct gcccaggcag gaccagtgta ggccctggga
17280agaagcagca cctcataggg caaacacgta ggaggcctgt ccttaggaac atcatagcta
17340agcagacctg tccccgcagg ggcaggagtc tgggctaagg gtgatactgg agagcagcaa
17400cggagactgg aagacaaatg aaatttggta cctgagttat ccctcccacc attccttttc
17460tagactctcc agctcagggt ctgttcatgg caagaggaga aagcaatctt gtttgctctt
17520taatcaaaca attaaacaaa tattccctct atactatgtg ccaggggcta tactagacac
17580acaaagacag ccccaagaag gacggtggag tagtgtcctc gctaaaagac agtagatatg
17640caatgcctct tgctcctgcc ctttctcctg ctgggaacag tttctgctct tcatctgggt
17700aagtctctcc cttccctcct catgcgtctt tccctttttt cctttttcct acactcccct
17760ccccccgctt ttatttgcac tcatgaggcc aggaccacag ccttccctct ttagctgata
17820cagctcatct ccggtaagat atcacttgga ctcagaactg taacctggaa ctttctcttt
17880tttgtttgat ttttttttgt tgttgttgtt tttgtttttt tttttgtttg ttttttgttt
17940tgttttgaga cggagtctcg ctctgttgcc caggctggag tgcagtggcg cgatctcggc
18000tcaccacaaa ctccgcctcc cgggttcaag caattcttct gcctcagcct cctgagtagc
18060tgggactaca ggcacatgcc accacgcctg gctaatcttt gtatttttag tagagatggg
18120gtttcaccat atttgccagg ctggtctcaa actcctaacc ttgtgattcg cccgccccgg
18180cctcccaaag tgctgggatt acaggcgtga gccaccgcac ccggcaaact gtaacctgaa
18240ctttcagaag gaaaaaccac ccacctgtta agatgaaggg ctggtgactg ccccaggctt
18300ctcacacgtg ctttctccca ccttcaaaac acacactcgt ggtgtcggcc agaagtcagg
18360ttcttgtcca tttgtgggtg tgacccgaga gatctctcct tacctaacac caaggaaatc
18420ctccagtctt gtcttcaggt ggaattccta ggaaagctcg agcgacgttg ctggagctgt
18480ccacggtgct ggaactagga agctcttgac ctgatggcag gttacctctt cttcccagag
18540aatgatgccc cccatctgga gagcctagag acacaggcag acctaggcca ggatctggat
18600agttcaaagg agcaggagag agacttggct ctgacggagg aggtgattca ggcagaggga
18660gaggaggtca aggcttctgc ctgtcaagac aactttgagg atgaggaagc catggagtcg
18720gacccagctg ccttagacaa ggacttccag tgccccaggg aagaagacat tgttgaagtg
18780cagggaagtc caaggtgcaa gatctgccgc tacctattgg tgcggactcc taaaactttt
18840gcagaagctc aggtaagtag tagggaggct actgcggagg acctggggga aaagagagta
18900cattcagtct tctgttccct attcatttag gctagtggtt ctcaaagcct cgcatgcatc
18960agaatcacct ggagttgttg ttaaaacaca gctttctggg cctcacctgc acgacttctg
19020atttaggagg gctgaggtga agcctgagaa tttgcattta caacaaatcc ccaggtgatg
19080atgatattgt tggtctgggg agaaccaccg atttaaacaa aaggctttgg tgttagaaac
19140gcctgtgtta aattctggtt ctgcctttta ttagctgtgt tacctgggca agttgctttg
19200cctttcaaag ctttagcacc ttcatttgta aaacgaagat atatagcacc aacttcttag
19260agttgtggtg agcattaaat gagataatac atgaaaagtg tttggaatag tcactgggct
19320gtaataaact ctcaataagc ggtggttata attattatga gtattatcat ttcctgtagg
19380attgtcctga cagctaatta agaagcaaaa gataggatta agggaggcaa gtaggtttat
19440ttttaacctg aaaagggatg ccgggctctt gcctggagac tcagaaactt gaaataaatg
19500agagggaatt cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
19560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngaattctct
19620gttagcacat agccagaaca tctagaaggg gtggtaggag tggggattag aggttccagc
19680tggaggcaat ggcacttgca aaggctttgt tgaagtggcg taagtgtgga ggtggagcat
19740tcaggaaagg agagcttcag cttcagtgtg gctggagtgc tgggtgtgaa gagaggtgaa
19800gatgaggctt ggaggctggg cagattttgc tccaaaagag cttggtgaac tgtgataagg
19860agtttggatt ttctcctact aaggacaaca gcaaactatt gaagagttta aatcgttcag
19920tgacaatgac acgtttgcgt tttggtggct cactcgagct gccagccagg tagacagtgg
19980cagaagatgg aagataaagc actaaagggt gatgaggcag gaagccagtg aggagagaaa
20040ggggacgatg tgagtgacag taaatcattt gttgggttgc tattgtgtgc taagctctgt
20100gctaaattct tcacgtgtat tatttcagct aatccatcta acaactctgt aaggcaggta
20160caatcgttcc cagctgaaga agctgaggct ctcaaaagct agtaacttgc ctaagttcat
20220gcagcatgca agttgtccag ccaggattct aacttagaca ccagaggcca cttttaacca
20280ctgctctagg actgggggaa atggtcccta gtgagatatg tgtcgagttt catatttcat
20340tcaacaatat tgttggcctg ctacatgtga agagctgtgg aaagcgccca aagtgagtta
20400gatccctatg agcaagtggg atgggggtgg agtggacagt aggagggctg gaacacacat
20460aaaagggtat aagaaataac aattaggccg gccaggggtg gtggctcacg cttttaatcc
20520cagcactttg ggaggccgag gagggtggat cacttgaggc caggagtttg agaccagccc
20580ggccaacatg gtaaaacccc atctctacta aaaatgcaaa aattagctgg gctggtggtg
20640cacgcctgta atcccagcta cttgggaggc tgaggcacga gaatcacttg aacccaggag
20700gcagaggtta cagtgaactg agattgcacc actctactcc agcctgggag acagagtgtg
20760accctgtctc aaaaaaagaa aacaaaacaa gtaggtactt tctgccatag ggaggattca
20820taaactgcta gtcctcaggt gcatttttgc ttatcagttt taaaaatcag agaatgtctc
20880aaagaattag gatgtcagct tcttttgaaa atttgggcca gaagcggtgg ctcacgcctg
20940taatcccagc actttgggag gctgaggtgg gtagatcacc cgaggtcagg agttggagac
21000cagcctgacc aacatggcga aaccccgtat ctactaaaaa tacaaaaatt agctgggctg
21060gtggtgcatg cctttagttc cagctactca ggatgctgag gcatgagaat cacttgaacc
21120cgggaggcag gggttacagt gaaatgagat tgcaccactg cactctagcc tgggagacag
21180agcaagaccc tgcctcgaaa aaaagaaaaa gaaaatttgg aagatctgac aacagttgac
21240ctgcattcct gctcggcaac agcctgatgg tggatgggca gaggctcagt tgtctgccaa
21300acctcccatc actgatgtct tccctcgctg tcatcatctg cttgacatgt aggcatttgg
21360tgtgtgcctt ctgctctggg tgcccagatg aattggatgc tatatgagaa aacattctgt
21420aaatgtcttg tggtaggcaa cctcaaagat cactggggcc tccaatgatc cctccttcct
21480ggtattcatg cctgtgtata atcctctccc ttgagtgtgt actacacctg gatacttgct
21540tctaataaac agaacacagc aagggtaatg ggatgctact tctaaggtta aattacaaga
21600gtgtaaagtc tgtcttgttt gtttccctct cttgatcttc ctctcattct ctctctctcc
21660ctctctctca ctttcttact gtcttgtcct tccctttgtt tactctgatg aagcaagcta
21720gcaagcatcc atgttgtgag ctgacctatg aagaggccca tgtggtggta aggaactgag
21780ggcagcctct acccagcaag gaactgagtc actcatcata tgggtgagct tggagacaaa
21840tccttcccca cttgagcttt cagatgacgg cagccctggc tgatgctttg caggcttgtg
21900agagaccctg agacagaaca ctcagctaag ctatacccta tctcctgaga tagagtataa
21960tacatgtagt tttaagctac tatgttttgg gataatttgt tactcagcaa tagataacca
22020atacatatac catgtacata actgtttcag ttgtctgaga ctatatttag tcattttaca
22080cctacatcaa gaatgtgtca ggcaccattc caggtacttg gaatacatca attaacagaa
22140taggtaaaga ggccaggcat agggctcaca tctataatcc cagcactttg ggaggcccag
22200gtgggaggac tgcttgagcc caggagttga gaccagcctg ggtaaaatag tgagacactg
22260tctcaactaa aaaaaaaaaa aattagttgg gcacagtggc acatgcctgt ggtgccagct
22320gctcaggagg ctgaggtggg aagatcgctt gagcccagga gtttgaagct ccagtgagcc
22380acggtcacaa aactgcactc tagcctgagc aacagaaaaa gaccctgtct caattaaaaa
22440aaaaaaaaaa aaaaggaaag aaagaaaaaa ataggtaaag atccttgatt cttgccctct
22500tggaacttct attctagagg gggatggttt ttcacagtag aagtctgtgt tgacagcgct
22560gtttaaagct ccttcagcat ctggggaaaa ggttnnnnnn nnnnnnnnnn nnnnnnnnnn
22620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
22680nnnnnnnnnn nnnnattttt tagagatagg gtcttgctat gttgcccacc aggctggtct
22740tgaactcctg ggctcaagca atcctcctgc ctcagcctcc tgagtagctg ggaatacagg
22800tgtgcaccac catgcctggc ttatttcata tatatatatt tttatatata tgtatattta
22860tatatataaa tatatatata atttctgtat ataaataaat aaatatatat atatatatat
22920ttttagagat agggtcttgc tatgttgacc accaggtctt gaactcctgg gctcaagtga
22980tcctcctacc tctgcctttc aaagtgttgg gattacaggc gtgagccatg gcacctaact
23040gagttatttt taccacacga agcataggac atacatccaa aaatgttctg agctgagcaa
23100gagcctggag gcaagtgaat ctgaactttc ccgtctttga agaaaccagt ctctctccaa
23160agtcacatag ttagtgtcac tccccccaag aactgcatga gctgggacaa tcagagggca
23220gtggaaggtc tggggctcag gggcgccccc tgctgtctcc ccagggtctg tccccttacg
23280caagagcctc tgctccccca ctttcctgtg gagcctcctc accatgggca tgacccagct
23340gcggatcatc ttctacatgg ctgctgtgaa caagatgctg gagtaccttg tgactggtgg
23400ccaggagcat ggtgaggcac cgctgaggcc cctgggggtt gggggcacag gcgggtcacc
23460ctggctgagc tcccctcacc atacgtttcc ctacccacag agacaaatga acagcaacaa
23520aaggtggcag agacaggtag ggctatgaaa gcagggccct ggctcacgcc caccccactg
23580caacccgctt ctcagggggc gggactcctc taggcctggg cccacccagg taaccctttt
23640gtgggatgta agagtctggg ttcagaggaa ggctattttg gtgctctctg gcctccgctg
23700gaaggggtga tagtgtccac tgagtgccag ttcctgaccc cactgccctt cccatcctgc
23760ccagttgggt tctactcctc cgtcttcggg gccatgcagc tgttgtgcct tctcacctgc
23820cccctcattg gctacatcat ggactggcgg atcaaggact gcgtggacgc cccaactcag
23880ggcactgtcc tcggagatgc caggtgacct gcctgtacag ggatggtgac agcaagtggt
23940caggcagtgc ttttcatttt ctctgtgcgt ttacatccag cagcttgttg ctttctccca
24000agaaccctag gagatcaggg gtacctcccc attttacaga tgaggaaact gaggctagga
24060agggacctgg cttgcttaat aataagaata gctaatgcag agtgctgact gtgcacttgg
24120caccttgcct tgtttagtcc tacaacacct ctttgaggta gatgcgttaa tatcttcatt
24180ttgcagttga ggaaaccgag gtacagggtt gcacagttag gtcattcacc caagatcaca
24240cagctttcag tggcagcctc cagaacctgt gttataaggg tacacgctaa agtcttgtta
24300gggctagaat aggtagagtt ggtatattag atatttattg ctgtataaca aatcacccca
24360aggcttggca ttttaaaaca acaaacactt ctcatctcat acagtttctg acagtcagaa
24420atcagggaga gactcagccg gctgattctg agtcacagtc tctcatgaag acatagtcag
24480gctgtcagcc agggctgcag tcatctgaag ggctgactgg ggttggagaa tctatgtcag
24540ttcaattacc cccatggcct ctccataggg ctgctcagga cacagcacct gctttccctt
24600gagcaagagg gctaagcgac agagaccccg tatcttctct cacataatct cagacgtagc
24660ataccatcac ttctgttacg ttctattata ggcacagagc aaccctgata tactgtggaa
24720ggagactgga caaagcaggg gaataccagg aggcaggatc cttgagggct gtcttgttgg
24780ctggagacca ccattgaggg tttttttttt tttttttatt gagacagtct tgctctgtcg
24840cccaggctgg agtgcagtgg cacgatctca gctcactgca acctctgcct cccaggttca
24900agcgattctc ctgcctcagc ctcccgagta gctgggattc accatggagt cttgaaccca
24960gattctgtga ctgcttttgc tctttttgtg ttcatccaaa cagtccctgt ttatcctaag
25020aggatgggag aaagagactg ggagagaagg aaatccagtg gcctccctcc ctgctagcag
25080agcctggccc tggcactgag ccttcctcct ctaccctctg ctcctaatgg tgagggtccc
25140ctagcagggc ccttctgtcc aggacacatg ggccgcctgt cctcacccca gcctactgac
25200ctctctcctg ggctggcctc agtgcccttg attgtgccgg agagaggaag cgctggacag
25260tcaggccaag ctgctgtccc caggagggca tctgcttatg tctagggcag ggacaccttc
25320ctgaggactt ctgatgagag acggtgtgag agcttcccac ttcccacctt ccttcccatc
25380cttggttctc aaaccttcaa gtgtgcatga gaatcactta gtgggggata tttgtccaaa
25440tgcagatttg cagatatccc cgctgagatt ctgagggccg agatgaggcc tgtgaatctg
25500catgttaaga aagcacccgc tttgatgcgt gtgtcattgg gtaggggagc aacactttga
25560gaaacatgga gctagagaac gtgggtttct atgggtttcc catagaaaca tggatttctg
25620tgttttctgc tgccctgaca tcgaaggcac atctgaaggg ggaggggcca ggccaagaac
25680cagggagtcc tgggaacgta gaggcagcag ccagtgactt cccgtactcc tcagggacgg
25740ggttgctacc aaatccatca gaccacgcta ctgcaagatc caaaagctca ccaatgccat
25800cagtgccttc accctgacca acctgctgct tgtgggtttt ggcatcacct gtctcatcaa
25860caacttacac ctccaggtac ccaccttcat ccttcccctc tccctgcctc ccgaggctcc
25920tccaaaggga tggtccatcc agcacctgcc ttccaggaag cgcagttctg gtcttctgat
25980ctggatctat tttccgggtt ctccaggaag tgtttctagt agattgggtt ggcgaggggg
26040tgggaattga ggcccagttg gcctcttcgc cctacccctc cttcctccag cctccacaca
26100ctctcctaac ctcttcactc tctctttttg gttttagttt gtgacctttg tcctgcacac
26160cattgttcga ggtttcttcc actcagcctg tgggagtctc tatgctgcag tgtgagtctg
26220ttgggctgaa atgccttcct gagctttgca accgtgatca gagaacccca gggaagggtt
26280gggagggccc caggcatccc ctaatgcacc tctctctgag accctctgat ggcagggagc
26340tcacttcctt aaaggcagcc tatcctgctg taattgactc cccctgttgg agtcttccct
26400tagaggaagc tgaaatacct ggcttgatga cactttggtt ctatgtctgc tgtttgaaac
26460ggcccccaga atggcctccc ctccatgccc accctgaaga aatttcccaa gggcagccat
26520ttgccttata attttcctct tcatgttgga cagtccccac ttgcatctct ctcctggttt
26580cccctgctgg gcgctgctga gggactctcc cctgtgtatg tgatggagta acaggacatt
26640acaataatga tgacaaaatg acaaccatta tcaagtgctc cgttggtgca ggcagcaggc
26700aggatccttg accatcactc cctgagttca gcctcactgc agcggtctcg gcagagggca
26760gctctctttc cttcatctgc tcaagccaga accctggagt ttccttgatg tttctctccc
26820tcacactcca tgttcactcc atcctcagta cagccagcag cagcttctac acaccccaaa
26880tctgaccctt cttgtcacct ccactgctgc ctctccagtc ctagccacca acatctctag
26940cctggattat tgtggcagcc tttagtctcc cacatctgcc ctggccccgc tgtctcagtc
27000tatttttaac acaggggctg cagtcacctg tcaggacata agtctcttca catcactctg
27060tggtgtcctg tctcatctgt ctcagagtaa aagccaaagg ctttactatg gcctaaaaag
27120ccctgcaagc tctggcccca gcacttcact cccctctagc tccccctcct ccattgttca
27180ctctgccaca gccacagtgc ttcctagtgc tccggaagtc tcaagtgtgt tccctgcttg
27240gcatctttgc atgtactagt ccctgtttct agaacattct tctccagata tctgcaaggt
27300gcccaatctt accttctctc cttcttcagg tctttccctg actgtcctct tctcagtgag
27360gcctcccttg gctgtcccat gtacaattgc aacctcccta ctgcccgctt ctctgcttgg
27420tttttctcag cgtttatcac taacactctg cctatctctt gcttattgtc tgaccgccac
27480ctgctccatg ggaatgccac ctcctcgatg gcaggaatct gttgacttgc ttgatcgtgg
27540tatctccagc acctagagca gtgcctggca catagtaggt tctcagctaa atgtttgttg
27600acagaataca gtggacagtc ctgcgaggtc aatgccatcc ctgttattag tggaggaagt
27660ggggctcagg gagtttgagc cacttgccaa tatcacacat acaggaggtg tgagaaccca
27720gctcagtggc cctgaagttg gagcatttgc cctcaaggct ggggaccaaa gagcccatgc
27780aaagagcccg aacgcttaag caccaccctg cctggccagc ggggnnnnnn nnnnnnnnnn
27840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
27900nnnnnnnnnn nnnnnnnnnn nnnncccact gcgcctggcc cattactttt aatggcaaaa
27960accacaatta cttttgcacc cacataaata gttaccatgg gctgagcatg gtggctcagg
28020cctgcaatcc cagcactttg ggaggctgag ccaggcggat cacttgaggc caggagttca
28080agaccagcct ggccaacatg gtgaaacccc gtctccacta aaaaatacaa aaattagctg
28140ggtgtggtgg cgcgtgcctg taatcccagc tattcaggag gcagaggttg cagttcactg
28200aaatcatgcc actgcactcc agcctgggcg acagaatgag actctgtctc aaaaataaat
28260aaataaataa ataaatattt accatgtttt gaccacctgt tatgtgccaa ctgtattact
28320taaaaacacc catgggaggc tgggcacagt ggctcacgcc tgtaatcgga cactttggaa
28380gggcaagcgg ggaggatccc ttaaggccag gagttcaaaa ccagcctagg taacacagta
28440agccctgtct ctacaaaaaa taaaaaaatt aactgggcat ggtggtgtgt gcctgtaacc
28500ccagctcctc gggaggcaga gggagaggtt cgcttgagcc cagcagtttt aggttgcagt
28560gagccaggac caagacacta cactccagcc tgagtgacag agcaagacac tgcctctaaa
28620caaacaaaca aacaaaagcg acctgtgggt aggtaggaac aggctcatag tacagatgag
28680aaagcagagc ttggagggct caagcgattt gccaagcaga ggtccaagcc gaggtctctc
28740tgaatccaaa gttaattccg tctatcatat caccacagcc ctctctgccc cagggagagt
28800ctctgcccac tccagccact cacgtgtaat tgacttcctc aggggcagga aaggcttcga
28860tgggccagtt gagggtgcag ttcagaaaga taaggcaggc caggccagac caggtgaaca
28920tgatgaccac gaaggccaca ccggcatcgt agatcagctg tgagaggagg gggcaggccc
28980gtgggggaga ctgcctggcc ccagacccca ccaaggtaga tcccaggcct cagaggcctt
29040aaagaagttc tcttctcccc ttgtccttgt gcccaatttg cagatgagga aaccaagacc
29100agaagtttag agtcagactc agaagaccca tcattccttt ttctttttca cttgaggccc
29160cctagagagc tatgaaatag tctccacaaa gcctgaagtt gctggccact ggctcaaaat
29220atctctgaaa tttccattat cttaaaaaaa tacatacatt tttgcctatg actccacaaa
29280cattcatgtt catgttcgca caaaaatgtc catttcatag tacgtacaaa ggaaacttag
29340tgctctaggt ttaccgggcc taatcgtgtt tatcctgccc cttcctggca cattccccag
29400gggaaaaggc aaacccagac tgctcatgct cagccttttc tcacctttcc caggtcctcc
29460cacgtgcaac aactgggggg gttggggaga gggaggtgca agtgctctgc ccaagggctc
29520tcaaccccag ggcaggtaag ttctcaattg aatgagattc tgtgcaaatg tgtcagccct
29580tcttatggaa gaagctgatg caccatctgt cctcttgtcc tccccatacc atctgaccag
29640gataattaat gtctgctctc ccctcaggct cctgctcaaa cctttttctc tgcagtcttg
29700gaccttggtg ccttttcctc cctaggggca ggacagagct tcaaagggcc acacccccaa
29760atgtgtggag gtaagatctg gctcttcaaa cactacttca gttgaaaaga agggagaact
29820gcccaccctc catgcctgcc caccagaaca actgatggcc cccccaccca tgcgctctct
29880caaactcctt tggagacact gagcaaaagt accttcttta gtactctttg taaagtgcaa
29940aacggtatgc agtttggtac tgcccaccgt ggaggttgag gagcatggca tggctcaaag
30000ggtcctttga tatttgacag aggaaattga ggcccccatc ttgcactgag ctaaaacttt
30060ggtcccctgg cttcgaggta caccaggttg acctgtccag gatccagcct ggcataaact
30120cactttgtga ccttggacca aaccacccat cctctctgga aggtgtggaa aaatgtggcc
30180ccaaaggctg aataaagcca gagagtcagg gaccttgaac gcatgtgaag gggctggact
30240tgattctgta ggtgaagcta aaccactgaa ggtttttcag cagtgtgtga gccagttccc
30300catctgagat ctttctggaa gtcacgtgag tgacagagta cagagaaaaa gaatcagagg
30360cagggagacc agctgagaaa gcttgctgtg gcccaggaga gagggggaag gcctgcattg
30420ggatgatgac agagaaagga gagcggagaa gtcagacccg tgggtcagca ctagctgctg
30480ctcactcggc cccacccggt tcttgtgtca agacaaaaag aaaacccagg tggcctcata
30540ccttgattcc tgggaacgta atggcagaag aggcgtaaga gccaatcatg agggccatta
30600acgtggagcg caggttccca aacatgttgg gcagctgagg agggaaagca gcacccatga
30660ggtggggaca ccgtgaccct tgcccagcat tcccagccct gctccataca atagctccag
30720gagacgcagc agaaaagccc caaggtaaaa caaacagaaa aatcaatgtg ggaaactgta
30780ctctgccccc tgcctacaca gtcacagtgc cctttagctt caaaaaggct cccagacacc
30840cctcagagag acattttgtt aattttgttt aattccaggt ttcccaagtt tgttacgtaa
30900cacctctgaa aaacacatgg aataggtgct taagaaacac tgatcttggc tgggcgcagt
30960ggctcatgcc tgtaatccca gcactttggg aagccgaagc tggtgggaag cttgaggtca
31020ggagttcaag accagcctgg acaacatggt gaaaccccat ctccaccaaa aatacaaaaa
31080ttagctaggc atggtggcat gcgcctgtaa tcccacctac tccagaggct gaggcaggag
31140aatcgcttga acctgggagg tggaggttgc agtgagccga gatcgcacca ctgcacttta
31200gcctgggtga cagagcgaga ctatgtcccc accccccaaa aaaaaagaaa agaaaagaaa
31260gaaacagtga tcttgtccaa cccatttgag atgagacaat tgagacccag ggaggaaaag
31320tgtactcaag ttcacagagc acattaatgg ctttctcccc attgtcgttg tcccagccct
31380aacccaaggc tgtgaccatg gctgtgtccc ggtaataggc agtgcctctt aaccctctcg
31440gttgacgtcc cagcccagtt tctgcctaat caggacaaat cacatcctgg gaggtgaggg
31500tggaaataag ggagggaact gagccagggc agacagtctc cagaggaggt ggctctgacg
31560cagagcaggg tcagaaccca caccaggaga gaatttaatt gatcatgtgt tccactcacc
31620tgcctcagcc aagccctcag ggcaggggaa ggcaaagtca ggatgccctt cgcacacacc
31680ctcctctggc cccaccatcc tccccaagtc actagatccc acagctgaga aggaccttag
31740gatccgtaca aagcctaaac acactccaca gagggggaaa ctgagactct gaagggaggc
31800ctcaacagct ctggtaaaaa aggcgtttag gccgggcgca gtggctcaca cctgtaatcc
31860cagcactttg ggaggccgag gcgggtggat tgcctgagct caggagttcg cgaccagcct
31920gagcaacacg gtgaaacccc gtctccacta aaatacgaaa aaattagccg ggcgtggagg
31980cgtgcacctg tagtcccagc tactcgggag gctgaggcag gagaattgct tgaacctagg
32040aggcagaggt tgcagtgagc cgagatcgcg ccactgcact ccagcctggg cgacactgcg
32100agactccgtc tcaacaacaa aaaaaaaaaa atggtgttta aacacatata actaaattat
32160ccttccccct tcccctgaag tggctggctc aggaaaaacc tctacccact caggcagagg
32220ttttcctgca ccctgcatcc gtgaggcacc actgccaagg acgccaggga aggctgccag
32280gcctggagag gggcagggcc ccctcccctc caaggggcca caaacgctgt ctgcgcccag
32340taccgtgggt aaggcgaggc cggccggcta accccgggct ggcggccttg cagcgtgcgt
32400ggcaacagca gctgggcccg caagactcag cacgggacgt cctcgtccaa gtctgggcca
32460agagcagcgg cccagggggc ggggccggcc agagggagcg gggagaggct gaggggcggt
32520gccagcgccg gaccctgcca ttggctggag attacaggag gcggggacat agcagggagg
32580agccgctgga caagccccac ccggccgcca gggagggtct gaggtcaaga gccggagaga
32640agggatttag ggccctgggc caagttgcac agcagggaga aggggctgcg cagaggggcg
32700gggagaaagg gatccgcttc cttcctttag agctgtgaaa tgtccccggt tggaattaaa
32760ggcggctgct ggggagaggt gaaattcagc caaaaccacc cagtcaggca gcccttctca
32820gagataaaca gtccgagcca gcccggccag gaaccttccc ctccaacctc cctaagcctt
32880taacactcct aagcctttaa cgcgtttaca cactcacata aataaacaca ctttgagcaa
32940cacacataca ccactcacca catgtaatag gtcaagccat gtgcacgacg aggtgtcgac
33000aatttcatat ggttcaacct agtacactca caaacacacc taccaactca tggctttcac
33060agggacgggg tcacacaccc actctcccac gacatggcaa gcgtgcacac gctatctcaa
33120gctgctccct ccccctcaag atcatgttac ccagttttat tttcttccca gcacctatga
33180cgactgacat aatttattag tttacttgtt tattgggtta tctgtgcccc tcacccccaa
33240aatgtaacct ccagcaggga ggatgactcg gtcagtcctg attgtgctgt agtccaggac
33300ctagaacaga gctccatgga cattcatggg ctctgtacac acaaacacac acattaacat
33360acaccccgac acacagcctc atccacacac acacagcctc acacctgctc tttgcagcca
33420cctgcacagt ttctcacaca ctcacttgat ctagtgatct gcgtccacag gcccctcccc
33480cagcccactc atactgccct caccccactc actctgccct caccccactc gggggaactc
33540tgctgccagg ccaggcctgt gacactcacc gtgagtgaag tgaacgttag gcagatgcca
33600ccaaagccat tcagggacag cgccaggaat atcaacggag acagagctgg aaaggggaaa
33660gcagcagatg agggcatttg gggagctgtg ggaagccaag ggcgggagct ggggtaaaca
33720tccgccttca tcccacctat tcttttcttg tggggccaca agaggacaga caactcacct
33780tccacgtccc gggaggccag ggccatgagg gtgcaggacg cagtgaagca ggcactgtgg
33840agacacaggg aagggcgagg ggttggcctg tgagcacccc ccctcccctc cccctgcagc
33900acggtccctg tcctcccgtt ccccatagcc cagccacctc acctgccaac cagccgcacg
33960ggtcgggggc caaagcggtc catgaggatc cccagtggca gggtggtggc gctgagcacg
34020aaggaaccaa tggtgaagcc caggttgagc atctcgtcct gctggtcaca gcctggccac
34080ctgcgctgct catcctgggt ggtgttggtg ctgctctcag ctgaggaggg ggaagggagg
34140gctcagcaca tgacaccagg aacagctggg cacaggagac agcagcccac agtcaggcgg
34200cctgctttca aatcccatgc caagtgcctt tgggggtacc ctagagtcac atctcctctg
34260atggggctgc tccagaaatg gcagccatta gtacctgacc ctgggagagt cttgtgcaca
34320cacagcctga ggcttcaact agctcaaatg aaatactgga cataaaagta tttactaagt
34380tgtaatatgc actcagtgtc caagcttagg gggttgtgga cccccaacaa gaagtgcccc
34440catatctaga ggcaaaggca aaggcagtga gtggtactct aatggctata acaagaattc
34500attaaaatgg cccggcgtgg tagttcatgc ctgtaatccc accactgtgg gaggctgaga
34560caggcagatc gcttaagcct acaagtttga gaccagcctg ggcaacatgg taaaacccca
34620tctctaaata aaaaaaagaa atttagaaag aacactaaaa cttagaggaa gctttcccga
34680taaatgatag tctgataaaa taatagctaa tacttattga gcacttaact atgctccagg
34740cactgttata agtcagttaa taaagtatcc cgttccctag gtgatgaagc tgaggcacag
34800aatgagaacc aggcactgcc ctccagtccc ctctagaagt ccacttggag gacttgtcct
34860taacggtaaa ctgccaactt ggagttgtga caagttaagg agaaaagcta gtgataggag
34920acaaagggct gcttcgcttt actcaatgct cannnnnnnn nnnnnnnnnn nnnnnnnnnn
34980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
35040nnnnnnnnnn nntccctccc tccctccctc cctcccttcc ttccttcctt ccttccttcc
35100ttccttcctt ccctccatct tctccacacc tggtatcatc atacagaagc agagaggact
35160gcacttggtg aaagtttcaa ttctcctgtg tggagaggtg agcactgagg aaggggtggg
35220ggctgtcaaa ggagacttac ccaatctttc cagcccacca atcccttgcc cagtgtttct
35280ataaaataag ggccttttgc atctgattta agtaggaagc tgattcctga gcccctcaga
35340tctgctgaat cagatagcta agggggccct ggaatctgca ttttagcaag cggaggtggg
35400ttatgaagca ctccaaagtt tgagacacgc ctcaaaggtg gagtggttct gtggggggca
35460gaaaggaaaa tgcaaagggg gaaggggtca cacttgggga aggtttcaga caataccgag
35520tggaaagggt gatgccaggt gtggggagta acagatagag gaggcaaagt gagtggagac
35580caagccagac cggggaggag ggggccacag ccaaggtgag acaggtcagc agccagaaac
35640cgaagcagac acttgcaggg tgcaccccgc cctctcttcg tggcaatctg agaccgagga
35700cgtggagacc ctggagagcc cccaaccttg tttctggggg gtgggtcaga gaggaagcct
35760ctcatccccg ggcaccagcg gccttcccgg gaggctcaac acgcagatac ctggataggc
35820gtccatcact cccccggcca gagcccacca acgctcctcg aggtccgacc ttgtccctcc
35880ttctacccca cagtccccag tcctagctca tctgcataaa gctccaatta acatgttttt
35940cctttgctat ttgcgatccc agaactcgtt ccccaccccg agcccgtttc ccgccgcttc
36000ctcgcccctg ggagggcggc cccattaacc ctcgcgaccc gggccgctcc tggcggtcct
36060gaccccgcca ccccgtcccg cggcgggggt ctgggggtga ggggcgcgcc ctggggcaga
36120ggattgcgcg gcagggtctg ccacagggca gaggccaggg ctctccggga aaaaggcagg
36180cgcatatatg cccccctttc tgggaaaaga cggggagggg ggcttctcct gggagactcc
36240aggcttcgaa attcctcgtt ccctatcctc cggcccccgc acccctcctc ctccccgcca
36300cgcaccctct cccctcccca gccatctgtt ccactccgca gcgccgcgac aaacacggct
36360ccagctcgct tccgcccctg cccagccccc tccccaagcc ccggggagtg ggggagtgag
36420cagacgccct tctcctagga ggccggaatt tctgcctcca tctcccaccg gggtccggct
36480ggccagaggc aagcttcgag accccccacc aaccaccacc accgttgcga gggccggtga
36540ggctgcagat aacgcttgca aggacgggag tcggggaggg tgtagggcga gtttaaagga
36600cgggcagagc aagccccggg aagaggcagg ggttttccct cccgggtcgc cgccccccgc
36660accctcggag ccagccgcag ccacgcagcg ccgcctgccg ggcacaccaa ggacctggcg
36720cgcacgtggc gcttaccccc acccccgggt ccgctcctgg ctcgcgctca gcctccccag
36780actattcgca aattgaggat cccggacaca gagtgcagag accccggcaa gcctactgaa
36840agccagccga acccgctggt gggtgctagc caattctgat tttgtacttt acaaaaacaa
36900aaaaagtcag tgttggaagt cgggagtctg ggctcagagc agcagggatc tgcgatgtga
36960ctttgccaag tctccagacc cctgaggaca ggttttccta tctgaaaacg gaggggacag
37020tctctcttat taacttctca agagaaacaa agacaaaggg agggaaaatg gcttagctgg
37080aatgctgtct tacagagcca acctttggag gtgggggaga tggccaaggc ctctgaggtc
37140actcttggcc ccaggagcag ctgagaaccg gaaagaagct tgggacctcc tttctgcaga
37200gctatccttt ccacagactg ccgaggttcc aaattgagct ccaccaccta acactgtgtg
37260cccttgggtg tgtgccttaa cctctctggg cttgtttcct acagcgacaa gaaagaatga
37320caacaccaac ctcttaggct atagtttgga taaaatgaga tagctgtgta gaacagacag
37380atcctaaacc aatgttagtt ttcccttcat ttggggactt gctctaacct ccagggctta
37440tgtcccagag gcacaagcag gtgcagggct ggataaataa ggtatgtctt tctgcaggat
37500ctcttgtcct cactgatggt gtcttctctt gatatagata attttaaagc ttcacgttat
37560ttatttattt actttaaagc ctcactttaa tgttaaaggt aaatgtaaat atagtataac
37620aaggaagctc aaaatttgca taaagtttta agataaaata ggagactcca aaaaagtgtt
37680actttcggca ggccctaggg atgctatggt gggaagtttg agtcatacct tagcattctt
37740tctaaagcat tctgtcctaa tcctctgtat ggagaaaagc cagcttcctg gatgtacccc
37800aaatcctggg aagtaggggg caggagctgg actccctcca agcactaagg gcagggcatg
37860gttgggaaca gggaggtgag ccagacagcc agaggcgaac gggctggcat gccaagcgtc
37920ctagttaatg cccagctgag cctgggtgaa gaaggatggg ggtgtgggga agacaccccc
37980caccaaccgc caaagacagg cgcacaccag ccagtctctc acttcccttt ttatttcctc
38040taagacttgc aagcagcagc accagagagg gaacctgccc tcctggccct ggaaggggcc
38100gacccccaac ccctaaccca ggacacagct ggcacctcag gcccctttcc ttctgaaagg
38160agggctgtgt ctctctcaca ttcacacata cacagacaca tgcatgtgtg cacactcatg
38220gcacatggga cctcaggggt agcctgtttg ccgatccccc caagaggtac caggaggcag
38280accgctagaa ggagataaga ggcaccctgg tctcctccaa cccaaggagg aagaaagctc
38340aacccctcta ggatagggac tgtcttcagt caatggagcg ttgacttagg gggcgttttt
38400gaaggttttt tttcctcctt tttgcagtct ttacaaaaat agaacttctc ttggtattta
38460taaatctacg gccatggctc tatgtgcatg ttacaggtag aaaagccata tggggcactc
38520cttttggttg ctcaggcctt gattgcctgt catccaggtc ccttggtctg agaagtctat
38580gcggtcacct cagagccgct aagcaccttc agtgggccca tcccattggc ggcgtactcc
38640tgctggagcc gggcacggta atagaagagg taggaaggca acaggaatcc caggagtgag
38700aatagcagga ggcccagatt cacctttagg gcaaggagag agaaacagag tcaagtaggt
38760agtcatctgc ccttagcctc ccacagggag gagaaggcgg ccatttttct ccaggtcctg
38820agccagaata aatacagcta gtacttatta tgtgtagtca ttgttccacc agtatctcac
38880ttaatgttca gcaattctgc aaagtggctg agatgagact tctcaggtat aacaagtggc
38940agggcctggt gggtgcccac accatatggc actcactagg taggtatgag gaaggcacag
39000cactgtagga gtctgggctg gtcaggctgc tcccgaaatg gggccttctg ggctcacccc
39060tctgaccttt ggagatgtta accaatggga tcccgttcag ggtggcgaga ggaggctctc
39120agacacagtt caaggaactg ggatgcacag cctggtggac agaaggcttg gaaggcccag
39180gacacgcggg ctctgactcg gttcacatcc cactctgcat tactcactgt gtgactttgg
39240gcaaataatg gcaattctta ctgagtgcct ccttctcagg gctgttgtgg cgaagatgta
39300agttaaaaaa aagtatgcat catgcttagc acatagtgag tgcttggtaa atagaagcag
39360ttatttcatc acaattcttt gggaggaggg tttacgtgtg ggtggcccca cagggcagat
39420gaaagatcag cgtcagggag gcagatgagt tcaatgtaag gaaaagactt actaacagca
39480gcagggctgc ctcgtgcagg agtgggtgcc ctaccactga gggtatctaa gctaagaggg
39540aagggtcccc tttcaggggt gctggagaca ggatcccaca ctaggtagaa ctggattgga
39600ccaatggtgc ctgaacacag gcccaagagt caggactggc cacttcacaa agcacctgga
39660gtttactaaa aacagactcc taggaggtca ggcactgtgg ctcacgcctg taaccccagc
39720actctgggag gccaaggtga gaagatcatt tgaggccagg agtttaagac tagcctgtgc
39780aacatggcaa gaccctgttt atctgtacaa aatttttttt taaaaaatta gccaggtatg
39840gtagccatca cctgtggttg cagctactca gaaggctggg gccggaggat cgcttgagcc
39900caggaatcag aggctgcagt gagctgtgat tttaccaccg cactccagac tgggcaacag
39960aacaagacac cttctctaca aaaaaaaaaa aacaataggg ccgggcgcgg tggctaaggc
40020atgtaatccc agcactttgg gaggctgagg agggcagatc acgaggtcgg gagatcgagg
40080ccatcctggc tagcacggtg aaaccccgtc tctactaaaa atccaaaaaa aaaaaaaaaa
40140ttagctgggc gtggtggtgg gcgcctgtgg tcccagctac ttgagaggct gaggcaggag
40200aatggcatga acccgggagg cggagcttgc agtgagccga gatcgcacca ctgcactcca
40260gcctgggcaa cagaatgaga ctccgtctca aaaaataaaa ataaaaataa ataaataaat
40320aaaataacaa taaattaaaa acaaaaacag actcctacgg tcaggctgag atatcctgat
40380tcaggggact ggggaatctg tatttttaac actccgtgag gggttctaaa aggcagacaa
40440cttggaaacc tgcagattag agacctctga ggtgcctctg gctgagatga gtgagggatg
40500gcaccacata caaggcccta cccctgcccc caggagagtg gctcctgctc cccccacacc
40560aaccctcgct ctcacccaga agggctctcc tttcaggggt cccaccatcc ccatgaaaag
40620tggctgctga agcaaggcga acacagcact ggtgagggac tgcaggcctg tcagcgtccc
40680aaaaggggtt ggatgggaac ctgtccccaa aacgggagat caaagggtgg tgggggcctt
40740tcagcccagg caagaacttt ttcttttcct tcccaacatg ggnnnnnnnn nnnnnnnnnn
40800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
40860nnnnnnnnnn nnnnnnnnnn nncactccag cttgggtgac agagtgaaac cctgtctcaa
40920aagaaaaaaa aatcttaaag aataaggata taaagaaaga aaatattttt gtgtagctgt
40980tcaatgtttg tatttcaagc caagtgttat tacaaaacag tcaaaagttt ttaaaaattt
41040aaaagtttat aaagtaaaaa agctaagtaa gctagggtta atttttttat cgaacaaaga
41100aaaatatctt tgtataaact tagtgtagtc taagtgtaca ttgtttttat tttatttatt
41160ttttattttt ttgaaatgga gtttcactct tgttgcccag gctggagtgc aatggcatga
41220tcttggctca cggcaagctc tgtctcctgg gttcaagcga ttctcctgcc tcagcctccc
41280aagtagctgg gattataggc acccgccacc atgcatggct agtttctttg catttttttt
41340ttgaaatgga attttgctct ttgacccagg ctggagtgca atggtgcaat ctgggctaaa
41400tgcaacctcc acctcccagg ttcaagagat tctcctgcct cagcctcctg agtagctggg
41460attacaggca tgcaccacca cactcggcta atttttgtat ttttagtaga gacagggttc
41520tcaactaaag agaaccatgt tggccaggct ggtctagaat tcctgacctc aggtgatcca
41580cccacctcgg cctcccaaag tgctgggatt gcaggcatga gccaccatgc ccagccagta
41640tacagtgttt ataaagcctc cagtagtgta cagcaatgtc ctagaccttc acattcactt
41700actactcact cactcactca cccagagcaa ctgccagtcc tgcaagctgc atgcatgata
41760agtgccctat ataggtgaac cattttttaa tattttatac tatattttta ctgcaccttt
41820tctatgatta gctacacaaa tgcttaccat tgtgttacaa ctgcctacag taatcagtac
41880agtactatgt atgggtttgt agcctaggct ataccatgtt gcctacgtgt gtagtcgtct
41940atactgtcta gtttgtacac tctatcatgt ttgcataaag ataaaatcac ctaatgacac
42000atttctctga gtgtattcct gttgttaagc aacacatgta taaacattta caagaaatag
42060ctcaaatttt tttttctttt gatacagggt cttgctttgt cacccaggct ggagtgcagt
42120ggcgcaatct cggcgcactg cgacatctac ctccccggtt caatcgattc tccggcctta
42180gcctcctgag tagttaggac tacaggcacg caccaccacg cctggctaat ttttttgtat
42240ttttattaag agatggggtt ttgccatgtt ggctaggctg gtctcgaact cctgacctca
42300ggtgatctgc ccgccttggc ctcccaacat gctgggatta caggcatgag ccaccatgcc
42360cagccattac gtttttttgg ttgtttaatt tttttttttt taagagacag attctcactc
42420tgtcatcaag gctggagtgc aatggcacaa ccatagctca ctgcagcctc caactcctgg
42480gctcaaggga ccctcctgcc tcagccttcc cagtaactga gactacaggt gtgagccacc
42540atgctcagct aattattttt tatcttttat tttttgtaga gggggggtct ttctatgttg
42600ctcaggtttg tctcaaactc ctgggctcaa tcaattctcc tgctttggcc tcccaaaggg
42660ctgggattac aggtgtgagc ctgaaaacct tctagtgtgg aagtggaaga taggcccagg
42720ccacttatgt tttcaagtta agcaaggttt aggtcactta tgaagcctga ctagttttgt
42780ttgcttaagg gatctgcagg cctgacctcg gttttcattt gttttaacag tgtctatgtg
42840tatgtgtgtg tttatgtacg tgcatgatgg ggggaaagct cagaaatcaa gtaagccaaa
42900cacaaacatg taattataag cagggataaa ttctatgatg aagaagtatg ggccacggga
42960gagtacttgt gccagtctgg tgatcaggaa caatgtcctt tgggaagtga catttgagcc
43020atgccctgaa gtacggtagg agttggttag gggtgaggca gtaagaccca gagctggggc
43080ttcctgcaca agctcagctg ggcactgagg acccagtgga ctctgctaca gggcagtgag
43140gagcagaaag gctgaggaag gctgggtgtg gtggctcaca cttgtaatcc cagggctttg
43200agaggctgat gggggaaaat cggtagagct caggagtttg agaccagcct gagcaacata
43260gcaagactcc atccctgtaa aaagctttta aaaattagct gggtgtggtg gtatgcatct
43320gcagtctcag ctactcaaga ggctggggta aggattgctt gagcctagga ggtggacgct
43380gcagtgcgcc acgattgtgc cactgtactc caacctagga gacaaagcga gatcctgtct
43440caaaactgaa tgaataggct gtgtgcggtg gctcactcct gtaatcccag cacttttgga
43500ggctgaggtg ggtggatcac ctgtgattgg gagtttgaga ccagcctggc caatatggtg
43560aaacccgata caaaaattaa ctgggcatgg tggctcacat ctgtaattcc agctactcgg
43620gaggctgagg catgagaatg tcttgaaccc ggggggcaga gggtgcagtg agctgagatc
43680gcaccactgc actccagcct gggagacagc gagactccat ctcaaaaaaa aaataataat
43740aataacaatt aaaaaaaaat taaaaggcca gggagcactg gcagcctgtc caaggtttca
43800ggtcacttta gtaaagggag aacaatggct cctcccagga cctctgggat ctcagcattg
43860atacgacagt catggaaatg ctagggccca ggcagaccat ctcagggaaa acaagtggct
43920ctgccctgcc ttggccactt cctggccctc tgcatgcccc agggtctcag caccaagctg
43980ttctcagtga gtagctctca tttagtgcca gggctctcgg gcttacatcc tacgatgacg
44040atggaatgca taaaagatgg ggctgtgata gcccagagct aggggtttga atctcatgag
44100atgttcatgg agccctggga gggagctcag tgcaagttca tttctctttt ttggttgaga
44160tggggctcag aggaggaagg acttgttcaa agacacacag ggagtgtttc agtgtgggac
44220ggaggtttat ggagaaaggg tgaccatcca aggcttggac aaagatcatg acttcgacca
44280gcaagcctca actctgtaga cttggtgggg gccaggccct cccaaacaca cctgacaggt
44340gtctgtggtc ttggggacat tgtcgctccc cttcctgctg atgctctgct gtccctctcc
44400catgaagcgt atctcttcgc cgtcccccat ccttgctgag agaggatggg ttctcttctg
44460accaatactg aagatcttta gtaaagttct cttttttttc attttctgaa agtccctctc
44520ttgagaaatc aggacaagtg agtcagggcc aggacaaaaa acagtgtggg acgagtgtgg
44580tggctcacgc ctgtaatccc agcactttgg gaggccaagg tggcggatca cttgaggtca
44640tgagtttgag actagcctgg ccaacatggt gaaacctcgt ctctacaaaa tacaaaaatt
44700agccaggcgt ggtggtgcat gcctgtaatc ccagctattc gggaggctga ggcaggagaa
44760tcacatgaac ccaggaggcg gaggttgcag cgagctgaaa ttgggccact gcactctggc
44820ctcttggcaa cagagccaga ctacctctca aaacaaaaac aaaaacaaac gacaaacagt
44880gtagactttg tgtttttctc aaaagcactg tcaagccagt gcccgcagca gtgggcctag
44940acacctccag tcttgcctca gggtcagttt ccagcctccc tggacacttc ccccaggtat
45000gtgtactttt tgattgtcct aaatccagag tctgtggcct gacctggttt gtcacagctc
45060tcagtccctc cccatcccga atcccaggga gccgcaggtg tgtgcagaag aggcacacca
45120cactcaatac atcttgcatc ctcgctggac ccaatccatt ggcttggtga tgtacagact
45180gagcctcatt atagccgttc gttcctgttg acctttccag atcaatctgc cagcttggct
45240tctccgagtt tcgcttgtca gcatttctcc aatcccatca tgtactttgg acctctttgt
45300tgggtggctt gctttatctg aaattttcag atttgacttc aggtctctcc tttgtccctt
45360aatatggctt aatggtggac cctgtcaggg gtagagaaaa tattgaggag ccctgacttt
45420gaggtgcaca agttagaggg ttagacaagt ccagccacaa ccagcccaag ctgcagtgta
45480gggaggcctg tccagctgct ccacggttga gggtggagca tacaggaagg cttccttctt
45540gctgcagccc aggtgttctg gctgccctag ctgcctggct ttggtagaag aaagaaaggc
45600tctgtctctg acttgtcaac taatggcact atgagattgc acataattaa cctgggtctg
45660ctcttccaaa agccttgggc ctctgactgc aacatggagt ctgggtatca ctccccatcc
45720ctgcgccact cacctgctct ggcgctaggc gtgtgcctaa tcacttaatt tctctgtgct
45780gcctcttagg tatcacttcc cctgatccca aatacttacc aggtgtggga tgacacctga
45840ctagttactc cttggaggta tctgcttctc accggggact ccgaaaccaa acgaaaagca
45900aggccaagcc cagcctaaag gacgcttcct acatgacttc aggcttgcgg gggctggagc
45960gtgggggtgg caatggagtt ggggggggct cagggagggg atgtggaagt gctttgcttt
46020gcaaactcta gagaaccgtg taaataggag tgattattct gtcccttccc tttctttcca
46080acaggaatca gcatcccaca gcccatgttc agctatgaag aatggaaact gaggctccgg
46140gaggggtata gggaggagcc agcagggtct tgagttcata ttagtgccct ttcctccata
46200ggcacatctg tgttttcttt tattttattt tgaatttaat tttttttttt tttggcagag
46260tcttgctctg tcgcccaggc tggagtgcag tggcgcggtc tcagttcact gcaatctccg
46320cctcctgggt tcaagtgatt ctcctgcctc agcctcccga gtagctggga ttacaggtgt
46380acaccaccac acccagctga tttttgcaat tttagtagag acagggtttc acagtgttgg
46440ccaggcttgt cttgaaatcc tgacctcaag tgatctgcta gcctcggcct cccaaagtgc
46500tggtattata ggtgtgagcc actgcgctcg gccacatctg tgttttaaat gagaggaaag
46560gggataatgt gcattttgtg gaagcttggg ccgtttgtgt ctaggactct tatgatcttc
46620ataagttttc ccccagggag gacactgttc cacttaggga gtcaggaccc ccagtcctta
46680caagattcag cctctcaaaa tggagacagc agttccaggc ctgggctggg ttctgttcac
46740actaggagag ggcaagtgag tggtgtttgg gatgtgggga agtattatga aaacagagat
46800gctccaattc ctagtgatag gaaaccatta agctacttgg catcttaaaa ccaagagcgg
46860ttcaagttct gagattgtta acacacctta caacaccgcc gccgttatta ggaagaagct
46920ctgtttgatg acgtcccaca ctgtgggtac ctttatgaac aggaatttgc tttttcaaat
46980cccagagaag taagattaaa gttggctgtt ctccatcctt gaaaaatttg gttttagggt
47040gaattcaaga atgactgacc atacagaatg gggagcaaac ttgggaagaa agaaggcaca
47100gttcagagct ctcccaatag tcacccctga actgcacccg gaccatcagt tatctctgtg
47160ggtagagctc aggaatctaa aatccatttt aaaattaaag tatatcgggg ctgggcgcgg
47220tggctcatgc ctgtaatccc agcactttgg gaggccgagg tgggaggatc acgaggtcag
47280gagtttgaga ccagcctggc cacatggtga aaccccgtct ctactaacaa tacaaaaatt
47340agccaggcat ggtggcagac acctgtagtc ccagctattc ggaaggctga gtcagaagaa
47400ttgcttgaac ctgggaggca gaggttgcag taagccaaga ttgtgccact gcactccagc
47460ctgggcaaca gagggagact ctgtctcaaa aaaaaaaaaa aaaaaattaa agtatgtcat
47520acatactgtt acaggcacag accttaagtg tacagcccaa tgaaatttta cacatctata
47580cagctatata actaccacct atatcaagac acattccagg aactcagact ccatcatacc
47640cctcctcagc agaggtaaca gacccacacc tctcctgctc cggtggtaat taaccactat
47700tctaactttt ctatcaatta gttttgccca ttcttgagct tcacacagat atacattgtc
47760aggcatgatg actcatgcct gtaatctcag cactttggga ggccgagacg ggagtatcac
47820ttgagcccag gagttggaga ctactctgga caacatagtg agacccccga ctctacaaaa
47880aaaataaatt agctggtcat ggtggtgcgt gcctgtagtc ttagctattt gagacgctga
47940gagaggagaa tctcttgagc ctgggaggtt gaggctgaag tgagccgtga ttgcaccact
48000gcactgcagc ctaggtgaca gagtgagatt ctgcctcaaa aaagaaaaaa tatggccggg
48060cgcggtggct caagcctgta atcccagcac tttgggaggc caaggcgggc ggatcacgag
48120gtcaggagat ggagaccatc ctggctaaca cggtgaaacc ctgtctctac taaaaataca
48180aaaaaagaaa gaaaaaaaaa ttagccaggc atggtggcgg gctcttgtag tcccagttac
48240ttgggaggct gaggcaagag aatggtgtga acccgggagg cagagcttgc agtgagccga
48300gatcgcacca ttgcactcca gcctgggcga cagagtaaga ctctgtctca aaaaaaaaaa
48360ggaaaaagaa aaaatatata tacattgtgt actttttggc atctggttta ttttgctcaa
48420tatcacatct gcgaaattaa tctacactgt gtgtatgaaa ggttggttct ttttgttgtg
48480atgcagtatt ccgtcgtgtg actacgggac aatttgctta tccgtattcc tatcggtggg
48540catttgggct gttaccaggt tctggctgtt atgaataaag ttgctatgga tattcttgta
48600cactacttct ggtgagcgta tgcactcatt tcgcttatgt aaatatcttg ggtggaatta
48660cctgatcata aggtaggtgt gttggctttg taatgtgctg acttggttat gctgaattcc
48720cttttttgtg tatttctggt tagagcggaa catgagggtg tctcttcagg gaatctggag
48780ggtggaaggg aagcaggagt cggtttctgg ctcacacatg ttgtgactga actgctggta
48840cacctggttg gcatggagct ggcttctcct ttggcgttgc ctactgttgg ggcaggtgtg
48900tatgtggtta gctccatgca atgaacccgg gcttctgcaa aatacattaa caacgacaga
48960gacaacaaaa gctgatgtgg atttaaaggc ttcagttcan nnnnnnnnnn nnnnnnnnnn
49020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
49080nnnnnnnnnn nnnnnnnnnc cagcagtggt tctcaactga gcatagtttt gcctcagagg
49140ggacatttgg taatgtctgc agacattttt tgattgtcac agcccagccg agaaggtact
49200actagtatct ttttggtaga ggctagagag gctgctaaac atctaacaat gcacaggaca
49260ggcctctgta acaaaaaagt atccagtcaa aaatgtccac agtgttgaga ggtttaggta
49320agtaggcgct aaaacataag gagactgtgc ctgagagcaa gaaggagtaa ttggaaagtg
49380ctggtgtgat tagctctggg ttttagaaag ctcattttgg ctgcttgtag acagtgcatc
49440agaggtggag gagggtggta agactggagg cagggaaagt aatttgggag ccactgaaat
49500gatccaggtg aaaaacggtc agcaggtgac taggaaagtg gcagaggcaa tggggatggg
49560tggctggatg agatggtgaa gaaagcacta taactaacta atgtgtggat gatgggcagg
49620aggggtgaag gatgaccaga gtcctgcctt gcaggtctag ttggaaggtg atggtttctc
49680ctgagaaagt gaccacaaaa agtgaagcag gtttgtgcgt gtgtgtgtgt gtgtgtgtgt
49740gtgtgtgttg agttcagtct gagatgtgtt ggactcacaa tgtccatggg acatccaagt
49800ggagaagcat cttgggtgac catatgtgtg agtctgcagc tcagaaacag gcctggggct
49860ggagatgaag acttgggaat gatctgcgta tatatttggt agcttgagcc acaagagtag
49920atgacataac ccgtggtggg tgtgcagaat taggagagac gtgcaccaag aagccaggtg
49980atccccaata tttaaccatc tggaagaata agaggagcct gccaacagaa attgggaggg
50040aatggccaca aaggctactg agaagggaag cagttcttaa gaagggggaa gtgaagaggt
50100atcactactg cagaggtcaa gtaggataag aactgaagaa tgtctgttgg gtttggcaat
50160ggggtagtca gtgggcacct gggcaaaagc agttttggtg gagcaatagg gataacagaa
50220acaagactgc tatggtaaga ggaggaagag ggtgttgagg aagtggccag cgagtctaca
50280ccacttgctg gaggagcttg gctttggtgc aaagcagaga agccagctca ctcattgact
50340taacctccaa gaaacacaaa atcatccata tcctggctca aattccagca ctaccaggag
50400atggttggcc cctagaaatg ccatcccact tctcctctgc ttatcctatc ctatctgtca
50460gtctgttgag cccaggctaa gcgctacctc ctcaagcaag ccttctctgc ctgccgtcac
50520actttaagtg atcctgacaa cactgaaaat gtgtgtctct tccattcatg ttagttctac
50580acttctgagt atctcctcaa tatattgcct tgttttacta atatgctcgt tctgtttgcc
50640ttatttatca gctaccttaa acctccctgc aactagagat tctctttaag tatttgttga
50700ataaatgaat gaatcaatcg atgatccaga gcctggtaga ggcttgtgtc catggtggat
50760gaggctcaga aaatacctgt agaatcgaaa taaatgcatg tgtgctctga tctaaactca
50820gctaaacttt ctccaggggg taaagttcaa gttgattagt caattgatta attaattcat
50880tatgtaatgg aaaaactcct tctatgacct gggcagagtt ataggcagtg aacaagacag
50940acaaggtcct tgttgtcatg aagtttgctt tctgaaggag agagataata aacaagaaac
51000cagtaagaaa gcaagattat atcattttgg taaatgttct tgtggaaata aatgtgatga
51060tgtgtaacaa aagtaccaaa taggagagtg gggtgggtgg gcttctttta gaaagagttc
51120tcggagaagg cttatctgag gaggtggcct tttaaccagt acaaatgctt tagcttggcc
51180agtggagctg ggaccaggat gacaagggtc acttgtcatg ccagtgagtt tgagcttgta
51240gacaagagcc tgatcatgaa agactttgca gatggtggta atgggtttgg gttaattgct
51300actatgtggg aagactttga atgggaagca tggggacaat ggcctgtgat acatgttatc
51360aaatatggtc gcaggggcta gtgaggtggc agcagagata gggagaagta gacggactgg
51420ggaaggtaga agatggggca ggggaggcaa ttactgcaaa gacatattcc ttctaagctc
51480actgagtgtt catggtctct gggagcagag gttcctggag gggaaagagg ataatgtcac
51540ttcctgagga agcgggaaga acccatctga gacgtgggga ctgtgctggt tcgtttctaa
51600ggggccttcc agatctcaca tgccaatcgt cttggtctat gtcaattgtt ggggcatcca
51660aatggggaac tgttgtccag gccgatttca cagaacaacc gcccagtcca tatctcccga
51720gccattcacc cttgcagtgg cgttagctct ttcaccagct tttatctgcc ccgtggggat
51780gttggccaag cccagttaac aagcagttga tcagccccag agatcaggtc cctggagtct
51840gtcacttttc tgagggtggg gagagaatcc tggagcagaa catgtaacta gaagggccac
51900ctggcttcct atggtctgag ggagagaatg gtgggatctc tggcctgaat caaacctccc
51960tttctcagtg tccatcttac ctctctgctg taccttcgtt attttccagc agctcctcag
52020cccgttcctg tgggaccctt ctctgccaat ccctacaccc actgtaaatt tcaccgtggg
52080agggagatgg gccttgaggg ctgtattagt cttctattct gcataacaaa ttgcctcaaa
52140tttagcagct tcaaacaact catgtttatt agctcatcgt gagttcatca gcagtgtggg
52200cccagcatgg ctaggttttc tgctcagggt ctcacaaggc taaaatcaag atgttgtctg
52260ggctgtgtgc tcatctggag tttagggttc tcttccaggc tcacgtggtt gtggcagaat
52320tctgttccct ggagttgcag ggctgaggtc ctgttttctt gctgactgtc agatgagggc
52380tgctctcagg tcctcgaggc tgcccacatt gcttgccacg tgcgtggtct tttccatcct
52440tgaagccagt gatggagaat ttcccttgga ttgaatcacc cacatggttg gactctctga
52500cttcaggaag agagccctgt ctcttttatg ggatcacctg attagatcat acccatagag
52560ggcagttcct tttccttaaa gtcaactgtg gcatgtaaca tcacacaacc acaggagtaa
52620aatccatcat atttacagtc ccagggatta tgcacagtgc accaggggac aactgaattc
52680tgcctgtcaa aagggccaag caggacttta ttggtgaaga acagtggaat gtcattcttg
52740gttcttccag aaaaaaatca ctcagtaaag ttagaggttc tcttgccttt tgggaagtca
52800tcaaagaatc tcatggaggg tttggacctt caccctagaa acatcacacc atgttttcta
52860taattgcagg gttcatggtc ccttgaagcc tattcatagt ttccaggttg aaaagctctg
52920ctgcagggtg tggggaggga tgcaggtgga ggtgagggct gaatagtgtg agctgcatat
52980ctggagctgt ggtggttttt ttagtcttta agctgtcatg tgttgggggt tgggcatggg
53040aggggcatcc caagagctcc ttggtattga caccatctcc aaggtgatct ctgctctgcc
53100tggtgcacac atgtttttct cctgttgcaa cagcccactc ttgtagaaga gcagacccct
53160cagtaccagg tctgaccctg gacagcttgt accaggagct acagcacact cccccacaag
53220cctaaagttg ggatgagccc cccgagaatt agatcagaaa agattaaatg cagaggtgat
53280ctgtcaggtc ccctttggaa gtgctggtat ggagaggatt gactgagtct gtttaggaac
53340ctccaagctc tgtagtaact ttagggctag aaaggaggat gcctaagatt caggatcctg
53400cagtgatgag tcaacatttc ttggggaagg aggcagggct gaggattaaa cggagatgat
53460gggtatcgtt ctcttgctca aaggcactgg accccaaggc ctccagctct tcgctcccat
53520ttgaaattca agtcctgagc acaccacagt tgtgatgcag ggaaagaatg tgcttatcag
53580agagcctggg caagtgggcc ccttgtgagt accgttcaac ctcatttatg tcattggcac
53640caaaagtaga catcagtctc ttgaaagttt gattaatgct ggtcacactc aaagaccctg
53700ggtagcattc atttactaag caattactaa ataccagttt ctgtgctaaa tgctgcatca
53760gtcagggctc ttaatggcag gcagcagaaa ctctccttgg ctgatctaag tagaaaaatc
53820caggactgaa aggaaacgga gtagctcatg aaattgcagg aagggccgga aaaccagaca
53880tggagccaaa gtcaggctgc agaacaggtc tagggaggat cccactgctg ctgagaccta
53940gaccttgtgt ctggcaccca ggatgttgta gggctcagac cctggatcaa tgtatcctgc
54000agtgcctctg tgggtactgc aactccagga actcaatctt gtcaacgcca ccgccagaga
54060gaggccttct tggcctccat ctttttggtc actagctcca gattcaaaat cttgaataga
54120tgcttcttct ctttgataga gcccagtcat atgcgttagc tgcaaaggaa gctgaaaatc
54180tattaggaac ttttgtcttc aaaaatgaga ggcctgtcct ccaccaagat ccataggaaa
54240tggaatccaa gaaaccacag gaaggggtga ggtgactggg cagctcacag catgcatgct
54300acatgtgaat tatctcattc atttctcaca ctacccagtg aggtaggtat tgtcatccct
54360acttcataaa tgatgatatg aggtacagaa agtttaagga acttgcccag gacacgacac
54420gcagctatta agtgctagac ccagtcaatt tgagtctgac ttggactgtc tgactccaga
54480agccaccctc tcagacactg ctgtatactt ccagtgaatg ttgatgaaat tttcagggtt
54540gctaagctgt ggatttcaga tcctggattg tatgacctaa aagagagact tccctaggag
54600tgagggtccc tgaacagtca actggtttcc aagaatgggc tccctctcat caccttatga
54660cagtaatcct ctgtccaaca gccaaagagg tcctgtgggg agggcttgca gatgggagtg
54720cgcagagccc agctcaaagc tcctgactag gctcttgttg agtattcctt tgattcctgc
54780ttctgtcttt ttaaatcaat ggagacaggg gagggttatc tccatcctcg gctcaagatg
54840aaatgcatcg ttcctcgttt ttctcattcc ttcccaatgt gtgtactgtt aactttagtt
54900atgaaggaaa ttacagtgtc ctgtgcatat accaaggctg tccaacctcc acacctttgc
54960tcaagctgtt ccttctactt gaaatgcctg tttccttccc ttctaattgc atctttccat
55020ccaggtagga atcagctcct tggttcatgg agccttttct gctctgtttt actatgcatg
55080gacttccttc tgaattagca gaggatgttt cctagcttgg tcttaaccct tctccttttg
55140tttgacctca atttactcat cttacaaatt aggttgtaag ctaattgaat acaggatcta
55200tgcttcactc tgattttatc tccacctgga tagcatcatt tttgacacac aagcaggcat
55260atgggagggg agagaagttt ggtgccagaa agaactggat ttgaattcta accctgttgt
55320ttacgtgagt acgttactta accattaatt acttcaatgt atatttatta agtacctact
55380atgtgccggg cactgtacta agcaccaagg atacaatggt gagtaaagag atgcagcctt
55440caccatcacg aaggaagaca gatgttaatc cattaaccaa gtaatctcac aagaaaagta
55500aaatgactaa ctgataagga caagcccctg gagctacaag agggtgtata cagggcatcg
55560atccaataag ggcagtgttg cggggagatc aggagccaca cagagcctgg gttgtctcac
55620ttggaaaatg gggtatcaac cacctacctc actaggtttt taaaatcagg ttaaatgagg
55680taatacttgc catgaacagt attttgttga ttgatgattg attgaaacgg agtctcactc
55740tctcgcccaa gctggagtgc agtggtgcaa tctcagctca ctgcaacctc tacttcctgg
55800gttcaagtga ttctcctgcc tcagactccc aagtagctgg gattacaggc agccacccct
55860atgcctgact aatttttgta tttttagtag agacaaggct ttgcaatgtt gaccaggctg
55920gtctcaacct cctgacctca aaagatccac ccacctcagc ctcccaaagt gctgggatca
55980caggcatgag ccactgcatc cagccacttg ccatgcatgg catttaaaaa tgttcagtaa
56040atgttaccat aatgaaggct ggtaggttgg ccaactgagt ggtctgattc agaaggaaag
56100aagttagaca tacgtgaaca tttcctgtac ttgaagatcc tcaggacagt gactcctaga
56160cccatcttcc atcacagtca gctgggaagc ttttaaaaaa atgcagacat ctgaccttca
56220cgctagacct attagccaag cagaagtttc tgggcagggc atctgcatat ttttaaaaat
56280ctttaataag gcagcctcaa aattacagat tcagcacgca tttaccataa ccactgaaga
56340aatgcaaagt tataaaaaga agataaacaa caatctgtct cctgctttct tccctctcct
56400cccctgcttc tggaggcaac aaggtcaact atttggtgtg attcctttta gcattccctc
56460catcaatggt cacataagga tgctcacaga taagcaccta tgcgggggtt ttttttttcc
56520ttgtaaaact attcacatac taaatacttt cctcagtatc ttgccttttt tcacttcatg
56580tcacagaaac atctcttcag gtttatagat acaggtccag ctcttctttt catagccata
56640taacattctg tagaatagag aggacacatt ttactcagtg tccgattgat ggatatcaat
56700attgttttca tttctacaaa tagtcaagga ataacataac tctgtaaaag ttttattact
56760tataggcgca tttatgccta aaggatagtc tcaaaagagt gaaactgatc aaatgtgcat
56820ttttttattt taataggtat ggacagattt gttctcaaaa tgtttgtggc agttcaaaac
56880accagtaaaa caggggagat atgtattttg gaaaagcacc caaggcgatt ctgaagtgta
56940gcccaggata agaaccattg cccagagctg ttccagatgg cccctgggtt cctgaagtgg
57000gtatcgggag agaaatcttc actgaatgaa tgagtgggct ccccagggaa gtgatgaaat
57060ggtccttatc agccttgcta tctccctctg acagaggcaa actctctctc cctgggggaa
57120gttcctccaa ggcctctata taagaagtct ttgtgagagg aagcaaagaa ggacctgggc
57180tttgggaaga tctaaagacc caggaaggtc tctgggtggg tgagtgcttt ctctgctgtg
57240gtggagctgg tgacagttta ttctcccagg aggtccctgg ctgtggctga cagtttctgg
57300agggctggca ggcgtctacc tgtggctttc aggttatgag gatgtcagca ggggcagcct
57360tcatcctctg ccttgcacat tccttctgcg ggatgtgaaa gtgctccttg gctggggaaa
57420ggagatggtg gagacatgga ggagggtgtg ggtggcttct tgaactctga ggaggggaca
57480taccttctaa gtcctatgtg ttcctaggaa agccaataat cattgcttct cccgcctttt
57540ttatgtcata gactctgagg gacccattaa gtacaaacaa ataagcgtaa tagtcccttc
57600tttacttccg ggcctgaagg aaagccagcc tcagccaccc ctcagggttt gctgcgttct
57660gtttagaaag aggtccttgc gtcctggatc ctggagcatc aggagctggg cttggcatga
57720gcttttctgg cccatcctga tttctattca ggccttcttt ttctccacct cactcccacg
57780gtcccctaat ggtgtgattg tgatgtgtgt gcatgtgtgt ctgtgtgtgt caatgacaaa
57840ctgtgttctc cgttgcagga taaagccaag atgaaactcc ccttacttct ggctcttcta
57900tttggggcag tttctgctct tcatctaagt aagtgttttt tgccttcagt ctttctttct
57960ctgttttttc cctttctatg gtagatgggg tcagagttac acacccaccc ccttctttga
58020tcgtcttcta tttctgaatt tctgtgtgct taaagggatg gggactctat ggccaggagt
58080tgaaaggatt tctcaaggcg tctgttatgt ctgtggtctt ggttctactg tgacattccc
58140aattttgtcc tttctccatt atgcttactt tgagcttact gagtgccttc tctcctttaa
58200ctctcttagc atcgccatga agtaggtggt attgtatacc catttcacag aaatacagct
58260ggtggatgat ggaaccagta cccaagccca tgactgcccg actctaagtc catgctctta
58320accaccttga ccttgtcagg cagcttgggt tcccctcata gagactgggt tccaggttcc
58380ccttcccagg cagagttgag cactctgatg cccagggcaa ggtgtgagct gtctgtggtt
58440ctggggagga acaaggggag atgtgaagga aggacactta gctatcctcc ctgccagggt
58500ctgagacttc cacctttgag acccctttgg gtgctaagac gctgcctgag gatgaggaga
58560caccagagca ggagatggag gagacccctt gcagggagct ggaggaagag gaggagtggg
58620gctctggaag tgaagatgcc tccaagaaag atggggctgt tgagtctatc tcagtgccag
58680atatggtgga caaaaacctt acgtgtcctg aggaagagga cacagtaaaa gtggtgggca
58740tccctgggtg ccagacctgc cgctacctcc tggtgagaag tcttcagacg tttagtcaag
58800cttgggtgag tggcctatgg ctgaggctga ggtgggagca tggaacgggt gtgggatatg
58860cccccagcat tgctatcact ggctcttttt cccattgagg gccctggggg tgtcagtaga
58920acctgagcct cagagaggtg ttggggtaag aggggagggc cacctacaaa cagaagttgc
58980attttggtct ccaaccttca aatggttgtg gcaggggagg gagggaatga attgtgggga
59040ctcaagaccc atgtgaattc atgtaggaag gatgctccat tctttgtctt ttatcctgcc
59100ctgtagttta cttgccggag gtgctacagg ggcaacctgg tttccatcca caacttcaat
59160attaattatc gaatccagtg ttctgtcagc gcgctcaacc agggtcaagt ctggattgga
59220ggcaggatca caggctcggt aagagaagtg tgaacactaa atggggtgca cctgctgatc
59280tcagccagca ctcagcttgc atcagatttg tctgtttttc tcctgtataa tctccagaag
59340aaccagggat agatggacac ccacagacaa cactgagggg gctgcctggg cattcaggga
59400agagctaagg atttagaatc aggaggtttg ggtccaagtt cctttccatc tctcactatc
59460tatgtaactt aagttagctg ggcatggtgg tgcatgtctg taatcctagc tacttgggag
59520gctgaggcag gagagtcact ggaacctggg agacagaggt tgcggtgagc cgagatggag
59580ccattgcact ccagcctggg caacaagagc gaaactccgc ctcaaaaata aataaataaa
59640taaataaaat aaaaaaaaaa ttaaaacaag accatgagtt tgtttcctca tctctaggat
59700gagttggcaa cccttgttct accttttgtt agggctggaa ggacaagcct gtcactggga
59760tgcatagaat ctgatggtga taattgccgt ggatcagcat ttcagatgac taggacagtt
59820cccatcatgg tccagcaggg aagggcccat tgcccggtgg gcagcagaaa gagctggcag
59880atacggggcc aggtctgctt ctctgccttc cctctgcccc atcccttctt cccctcttgc
59940tttctccagg gtcgctgcag acgctttcag tgggttgacg gcagccgctg gaactttgca
60000tactgggctg ctcaccagcc ctggtcccgc ggtggtcact gcgtggccct gtgtacccga
60060ggtgaggtgg ggctggggat gaacgatgga aaggtctggg agatgggaag tgccccaagg
60120aggagatgct acaaagagcc tgaccctttg tgggagaggc ttcctgggtc ttttatatac
60180tctgactcca cagcagtgtg tgggtgggaa aagaggccct cctgtgggtt gagttgggat
60240ggacaagagg ctgaaagtcc ctttctgttc tgccttcaca ggaggccact ggcgtcgagc
60300ccactgcctc agaagacttc ctttcatctg ttcctactga gctggtccca gccagcagtt
60360cagagctgcc ctctcctggg cagctgcctc ccctcctctg cttgccatcc ctccctccac
60420ctccctgcaa taaaatgggt tttactgaaa tggatttatt ttctcctctg atcgcggatc
60480cactctgctt agccctcatt gaaacttctt ccttatcatc tctccccaca ccacaacttt
60540catagaagtg tcagaagcta ctactccttg aggaggagga tggagggtgg agttgggtct
60600atggagcctt ttggagatgg aggaatgggc tcagctagtt ctcttcatag aacacctgat
60660tactgggcac ctgcatagtg ctgccaggac ctttcaaggt tgtaggtaga ctcccaatgg
60720cccagtttgc atctctgtaa ccaaaggcct tttctctctc tctctccaac cccagaactg
60780tggttggttt tatatgtaag gaagttaaca tgtccctggg aacagtccac aacattcagg
60840aatgaatgta taagtaccgc aatccccggc ccctcaagtg gaataaatct aacatgtatt
60900gggcaccatt tcccagtggc ctgctgtggt agttggcctt attccatgca tttttatggg
60960ctgccttccc ttcctcaact gcattctctg ctccttccta ctctctgcaa ctcccaaata
61020aacacttgta cgcaactccc tctctcagga tctccttctg gggaaacctg atataagaca
61080gcttgccatg cgtcagactc tgaatgaggc ctgggaatac aagacatagt cctctggcac
61140ttgggatata tggttatttg taacataggc acaaaaacat ctactagttg ttatcgctta
61200ttgagcaccc acaacatacc ccctgctgtg gcaggcacct tgcctagatg acctcatgtg
61260atcaataatt atgagcccta ttttacagaa ccaggctcag agaagttagg atctgtcaaa
61320agacttgccc aagactgaac ctctaaatgc aactcatatt gaaattcaac tctgctccaa
61380agcatgttac tttaaccctt gtgcttttac agctggctac tctcccctta tggtcacacg
61440gggatgaagc acggggggag gaaagccaga ctgtctcact cttgggttca tcttgggaca
61500caggacacca gcccagctgg aggtgaggga gctttaatca gaggggaggg aggaaggcat
61560tctcaacccc ttctgtacta gggaggtcag cagaagaaaa taattcaatg ttctaaagcc
61620atttttttct ccagcattcc tccaattcat agatcttcat atgggattag gggctcagag
61680aggggtgaaa caagaactct atttttttgg agtgtggtat agagaaggga tgctacttct
61740ctaaggtcac atagtaagtt gagaaagaga gagaaatcaa actcaggttc atttcaacta
61800ttgttccaca agaatctgtt gatttcaaag atggtggact atgggttcat ccctgtggtg
61860agtgctgtga ggatgcagct gaggtggaac tttcactcct tgccctcttg gactttatat
61920tctggtgtgg aaaggcattg cttcccttat ttcaatatta acaacaaagg gtaataatat
61980ttcccattta ttaagcattt actaggtgtc aggtactgtg ctaaatgtta ggtgaacttt
62040gtcttgttcc tcataaatct ctgccgctgt gggtgtgtac tttgacagaa gtttgacttc
62100cagtccacag agatcttctt tgggggagta atatcaagaa ggggcacgaa ggaagctgca
62160gggctcctag tcccatcctg tatctcgacc taggcatgtt tacattggtg cattcactgt
62220gaagtttccc tgagcagtcc actctatagt gtgctttata ggagcacatt gtacatccat
62280tgaaaaattt ttcttggccg ggcacggtgg ctcatgtctg taatcccagc actttgggag
62340gccgagacag gcggatcacc tgaggtcggg agtttgagac ctgcctgacc aacatggaga
62400aaccccgtct ctactaaaaa tacaaaaaaa ttagccgggt gtggtggcac atgcctgtaa
62460tcccagctac tcaggaggtt gaggctggag aatcgcttga acctgggagg cgaaggttgc
62520agtgagccga gatcgtgcca ttgcactcca gcctgggcaa caagagcgaa actccgtctc
62580aaaagaaaga aagagatttt ttctttttct taaaaagtaa aaatcatgaa ataaggggac
62640tgggctaata ttccaaaata tgggtttgtg tgtgaatttt cctctccagt aagatactaa
62700ctaagctctg tgaaactgtt tatctatggt tctttatcat tgaatccttg gagttcctta
62760cactgtgcag agcacagagt aggggctcaa tcaacagtgc actcattgct ttttcataga
62820caagggccac cctcactcaa ctcatgtgcc aggcatagtt ctgagagctt tgcttaagct
62880gatnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
62940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnggtcact aatttggtat
63000agagttatgt tcattgattt cattttattt tgtttctgat ttttaaagat tgttttactt
63060gttttcttcc tattattatt ttattttatt tgtaaaacat ttacatatca gacatttaca
63120ttttcccaaa ggtaaaactg tgaaacaaga tatattcaaa gaagtttact ttccctctct
63180gtttcttgta ccccttttcc tcttctttag gtaaccattt ttattttttt aaatataaac
63240attgtgtagg tgtatataca tgtattagtc tgttttcatg ctgctgataa agacctatct
63300gagactggga agaaaaagag gtttaattgg acttacagtt ccacatggct ggcaaggcct
63360cagaatcatg gcaggaggtg aaaggcactt cttacacggt ggtggcaaga gaaaaatgag
63420gaagaatcaa aagtggaaac ccctgataaa cccatcagat ctcgtgagat ttattcacta
63480tcacaagaat agcgtgggaa agactggccc ccatgattca gttaccctcc cccactgggt
63540cccacccaca atacgtggga attctgggag atataattca acgtgagatt tgggtgggga
63600cacagccaaa ccatatcaat acatttccct ctcttttaga taaaaggtag tatactgtat
63660acactattct gcagagtttt tttttttttt gatgtaactc tatcctgagg gtgctctgta
63720gcagggacct ctcatgcctt ttaaccactg cctgggtctc cattacatgg ctgcagcata
63780gttgccacag cattcctgta ctgatgacta tttggattgt ttccagtctt ttgctattac
63840cagtagtgtt acaaagagga tctggctaca tgttcagggt ggggaggggc agatgtgtag
63900cctgtcagga gggtattgca gtaatccatg actgagttaa tggtagttta aagctaggat
63960gagtcagtgg ggttggagag aagtgggcac atttgaatga tatgtaggag gtgaatgatc
64020agcattattg atgagtttga ggtggggcat gtggggaaag gattcgagga tgactcccag
64080gtttctgttg ggacagtgga tggatagtgg ctcctcccct ttttccaatc ttccttggcc
64140cttcgctgac ttctgttggg ttggcctaca gagagcttct ttttcctctc tgttcgccca
64200ggttcctcca ctttggcggt ggccctctgc tcgacggtgc cttcgctggc cctgacatcc
64260ctgctgtgcc tgggcttcgc cctctgtgcc tcagtcccca tcctccctct ccagtacctc
64320accttcatcc tgcaagtgat cagccgctcc ttcctctatg ggagcaacgc ggccttcctc
64380acccttgcgt aagtggcctt ggggcgggct ctgtggagac ggacacactg gggcaaagag
64440aagctggagg taaagaaatt gggaggcaag gcggggcctg gaggcagtca ggtgcgggag
64500actgggtttg ggggcaggtg tggagggggt gagaccagag gtggtgggaa ggatagaaca
64560ttcatgcact tgagccttta catctgcggt gccctctccc tctgttttct acctggtgaa
64620ctcgtattca tcctctgagg cccacttctg tttcagttct ccagggaaga aatggaaaag
64680tgtcttccct tctttgtgcc cttagtactc tagtcttact tcctttgcta gtgcgtgcat
64740tgtctggcat gccatccatt tacatgcctg tcttttcttt cctggtgcag cctgcatgag
64800ggtcctgtct gtttttccag ggccccgcat gtgccttctt ctgggttctg tgggtcaaat
64860gtctgagcag agctgaagag ggaaaggcca gacaggtgtg gttggagggc aggcctagga
64920caggggagct ggggacaagc ggccgacagc ccccagaggc caggcttctg cttggaggga
64980gggtccctga agctcactgg aacccctctg gtttctctcc ccagtttccc ttcagagcac
65040tttggcaagc tctttgggct ggtgatggcc ttgtcggctg tggtgtctct gctccagttc
65100cccatcttca ccctcatcaa aggctccctt cagaatgacc cattttacgt gagtactggg
65160aggatgggga tccctggcag gaggcctggg ccttaggcct tggctgcccc aaatctggct
65220gtgatggcct gggtatgtag catggtgcag cttcccaaag ggtctgtgtt attcaagtat
65280ttggggcaaa agtatttgtg tgtgtgggga aacagacatt ctggagtagg gtggggaatt
65340ctcacgaaac ttcaagcaaa atcctgagac ctcaaaggtg tttcctgctt gtggtgagtg
65400caggcccacc ctggcctctc ccctaggccc acacagggtt tccacagttg gccccaggga
65460caggacctct gtgctttcac ctctgtgtcc ttacacctgg agggatgctc tgaggtcctg
65520ctctaggagg tggtcgtgag tctcctgctc tttgcagaaa ctgaggctca aagaggttac
65580ttacgtgttc agaggcacca gctaaggagc aaaagtcaac tttgaattct gtgttttgac
65640tactgcacag ctctatttgc ctcatttttt atttttaaag cagcaaatct tagaatagga
65700gtttaaatcc atcacttgga gaaaagaaag actaaatgtt ttttgttttt gttttggaga
65760cacgatcttg ctttgtcacc caggctggag tgcagtggca caatctcggc tcactgcagc
65820ctcgatctcc tggactcaag cgatcctctc atctcagcct cctgagtagc tgacactaca
65880ggcatgtgcc accatgccaa gcttatttta ttttattttt ttgatagaca ctggggtttc
65940gctatgttgc ctgggctggt tttgaattcc tggcctcaag cgatccaccc gtctctgcct
66000tccaaaatgc tgtgattaca ggcgtgaacc actgtgcatg gccaaaagag taaacttgaa
66060atctgaggcg aatgacttga ttgtgacatc aggtgaccta gtaatcagct gtgtattcta
66120gctggtgcct ctaccagctt cccatgtgac cttgaacatg tcattgaatg ctcgctaggc
66180ctctgtttct ttatctgtga aatgggcttg atattcctcc tctaccccaa ccgatagtgc
66240agaatgaaaa gtaactgaaa gtccttcctc cagggcacca tagtgtctgg gtgaaaagta
66300gaatataaac tcggtagact tctggtccct tcattggtca tggaatggac cagtgcttgc
66360ttcattgagc aacagttctg ttgttcagaa ttcctggatt tcacctcact tctgctctcc
66420ctgcaggtga atgtgatgtt catgcttgcc attcttctga cattcttcca cccctttctg
66480gtatatcggg aatgccgtac ttggaaagaa agtccctctg caattgcata gttcagaagc
66540cctcactttt cagccccgag gatggttttg ttcatcttcc accacctttg aggacctcgt
66600gtcccaaaag actttgccta tcccagcaaa acacacacac acacacacac acacacaaaa
66660taaagacaca caaggacgtc tgcgcagcaa gaaaagaatc tcagttgcca agcagattga
66720tatcacacag actcaaagca aaggcatgtg gaacttcttt atttcaaaac agaagtgtct
66780ccttgcactt agccttggca gacccttgac tccaggggag atgacctggg ggaggaagtg
66840tgtcaactat ttctttaggc ctgtttggct ccgaagccta tatgtgcctg gatcctctgc
66900cacgggttaa attttcaggt gaagagtgag gttgtcatgg cctcagctat gcttcctggc
66960tctccctcaa gagtgcagcc ttggctagag aactcacagc tctgggaaaa agaggagcag
67020acagggttcc ctgggcccag tctcagccca gccactgatg ctggatgacc ttggcctgac
67080cctggtctgg tctcagaatc acttttccca tctgtaaaat tgagatgaat tttggtgttg
67140aaagttcttc ctggagcaga tgtcctagaa ggttttagga atagtgacag agtcaggcca
67200ccccaagggc catgggagcc agctgacctg cttgaccgaa ggatttctga cagactatct
67260ttggggatgt tttcaagaag ggatataagt tatttacttt gggcatttaa aagaaaattt
67320ctctcgggaa taattttata gaaaaataaa gcttctgtgt ctaaggcaac tactgtttcc
67380atctctctag gctttgggcc ggggctgtgt gtgtgtgtgt gtgtgtgttt gtgtgtatgt
67440gtatgtttct gaggaggccc taccctggca tgagagggta gggaatctgg ctacacatct
67500agtgtggcag ctggacccag aggtggggca ggaaccctga ctatgattca ccccgctggt
67560cctgggatgt gggcccagag acttcctccc ccaggaaccc ctctgcttcc tcttcctctc
67620cacatcctta actaacttta gcagaaccct actcctcact acacaccccc agctagaagc
67680gctggatgga atcagaaatt cctagtttga gtttcaattc tgcccctcag cagctgggca
67740agccccttaa ccactctgag tcactagttc cccacctgca aagtgcagtt aatcatttct
67800atctctgatg gcgattgtga gaatgtaaag tcattgcaac tgcctagcac atggtaggag
67860cacatgaggg tttgctcctg tgtttactca tgacccttgg ggaggacggg ggcaaagagg
67920gagaagttga gggtgcagga ggagagatgg caggtgggtg ggatgggaga atctggggca
67980cacctgctgt ctcattccca ccttgctagg agagggacta ggaaagaaca gtgggaggca
68040gggggatggg ggtggaaggc agggggtggc aggcaggttc atccatccat tcattcaaca
68100aatgtttatt gagcacctgc cacgtgtcag gccctgtcct gggtgctggg gctataaaga
68160tgcagaaggg tctgaaaccc agctcttcct tcttcctgtg gatgtcgggg tgtaatttcc
68220aggggccagg agcctgggtc tgagggcgga caccaaagtt ctagtggtgt ctattagcag
68280cgtttaaatc taatggatgg atttggtctt gttaccctgc tcaaaagctt tcagcagctc
68340cccactgtcc acaggacaaa aatccagatg ctagcctggc attcaaggct gtcactagtg
68400tgatctcaac ctctcccctt ccctctttac ctcctaccaa cagcggggca gagcccaccc
68460ctgtggacca agattcccag tctctgggtc tgtgtgtgca ccagttcctc tgcgtgggtg
68520gctcaccctg cctcagcttg tgaaatccat ctggtctgct gggatcctgc tcaaaatgtc
68580atcttctcca aaaatcatta ctcaggcttt ccagcatgtc tgagtccctg gcacttggtc
68640acacccttcc tggtgactgg catttgcctc cacatcatga ccctcccacc ccttgcctgg
68700gcagcatact ccaggaggca aggtctgttc tcgcctggct ctaattaatc tgtgcttacc
68760atccacatgg taccagctaa ttcttgttga atgaatgatc gttgaatgag tggattcttg
68820ttttggcctc agaaccaatt agaaggagcc agaaaaacac atgggggtgg gggaggtgca
68880gtgtggtgca gtggaaaaaa acccttctgg aaatctcagc tctgtcactt actttgtcag
68940ctctgtgact ttggatggac cacttctttg tcagtatggt gggagaaata gacatgcctc
69000tctgggctgt tgtaaggatt acaaattagg tcgagtgctt ggcatgtggt gggttgaaca
69060gatcacagct agcattacag atgatatatt aaagccaaaa aaagatgcct aatgtccacc
69120agttggtgaa cggacaaagg aaatgtacca tatttgggat attatttggc aatcaaaaaa
69180agtactgaca cctgctacaa cacggatgaa tcttgaaaac attagactaa gtgaaagaag
69240ccagacacaa gaaactgcta atgattccat ttaaatatga aatatcgggc cagggtgcag
69300tggctcatgc ctgtaatccc agcactttgg gatgccaagg tgggcagatc acttgaggcc
69360aggagttcgt gaccagcctg gccaacatgg cgaaaccccg tctctactaa aaattagccg
69420agtgtagtgg catgcacctg taatcccagc tacttggttg gctgaggcac aagaattggt
69480tgagcctggc aggtggaggt tgcagtgagc caagatcgtg ccactgcact ccagcctgga
69540tgacacagtg aggttccgtc tcaaaaaaaa aaaaaaaaaa ggaaaaagaa aaaaagaaat
69600ttccagaata ggccaatctg tagaggcaga aagtagattc atgattgggt aggcctgggt
69660gtggaggcca tgggtagtga tggctaatgg ggaaggggtt tcttttgggg tgatgaaaat
69720gggtggactt atggtatgtt aattatacct caataaaact gttatttaaa ggaagaaaag
69780atgcctggat tccccaggaa gtgtacagta gacttctgtg agaatcagaa atgatttctg
69840gggaagatgg gcgagaggag agtaagtggg agaagtgacc acgtgcgcaa ctctcatcgt
69900tctgccctga gagccttcct cctgcaactt tatttattta tttattttga aacaggttct
69960cactctgtta ccctggctgg agtgcagtgg tgtgatctca gctcactgca gcctcgacct
70020gccaggctca agcaatcctc ctgtttgagc tcctgagtag ctgggactac aggcgcatgc
70080caccacatct ggctaatctt ttatttattt atttatttat ttatagagat tggggagtct
70140cactctgttg ctcaggctgg tgtcaaacgc ctggactcaa gtgatcctcc caccttggcc
70200tcccaaagtg ttgggattat gggtgtgagc cactgtacct ggcacctcct gcaacttctt
70260cctcaagtgg aaccaatgag gaagcaagca actcagagct ttcacaagtt ttgatttcaa
70320tcagcaacgg gcttccaatg caacccttct ctcctgtaac cagcctcagt agagaggaac
70380tggaggtgaa ttggccccca tcacaccccc acagtgccaa gctgggccct tccatcaggg
70440ggagaacaca tgccgtgtaa gggacagcca acagcataaa ataggaattg tgtgatgatc
70500ccttttaagc ctattcagcc cagggaagtg catatgatca gccccatttc atagatgaag
70560aaagtcaggt tcacccatta gcacattgtg gggctggtat ttaaaccagg tctgtctggc
70620tcccaaggtc acattcattt agacattacc tttactttac atttcttctt cttttcttct
70680tcttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcttcttct
70740tcttcttctt cttcttcttc ttcttcttct tcctcttctt cctcttcttc ctcttcttcc
70800tcttcttcct cttcttttct tcttcctctt cttcctcttc ttcctcttct tcttcttctt
70860cttcttcttc ttcttcctct tcttctttct tcttcttctt cttttttttt tgaggtgggg
70920tcttgctcta ttgcccaggt tgaatgcagc atcatcatac ctaaatgcag ccttgaactc
70980ctggccttaa gcaatccccc tgcctcggcc tccaaaagtg ccaagatttc aggcatgagc
71040caccatgccc agcctgcatt tattctcttg taagaaagat atcatttaaa acagacgaga
71100aaataaagag ggacatgaaa aagacgcatc accattaatt ggaccactca gagataatca
71160tggttaacat gttggtatgt tccctcccgt catttgactg gatgtatgtg ataatttaaa
71220tgatctcata agcttttcct tatgtaatca aatagtagcc aaaaacatga ttttaaatgg
71280ctgctcacaa ccccatctcg tggttctgcc acgccttgtt tatccccatc caccccctac
71340tccctttccc cttccctgcc tgtgtggggg tcctagatga cggtgagcca gagggcagcc
71400ttggtcagca gattggagag tgcaaataat aaaaacactc agaaggcgag ctgttgtcaa
71460gtgggcttat cacaaaagag caccttggga tattccagag aatgacctca tacccgctaa
71520tcactatcca taatctggtg ctaactgtac tttagctgaa ggtgctggca ggtcctgccc
71580aggtgctgct aagaacactt ctattctgtg agaatcagag atgatttcta gggaaaatgg
71640gcgagaggga gtaagcagga gaaacaaccc acaggcacag ctctcatctt tctgccctga
71700gagccttcct cctgccacgt ggttttgttt gtttgtttgt ttgtttgttt cagatagggt
71760ctcactctgt cacccaggct ggagtgtagt ggcaagatca tggctcactg aagcctcgac
71820ctcccaggct caagcagtcc tccccaaatt caaagcttgg agtgatggtc ccagtggtta
71880tgtctaggag ccctttttcc tgccagcccc tcaggggatt gatgactctc aaatgcttca
71940ggtgtgacat gggcacagca gtgagtcatt cctctgacat tctttgggaa gaacattttc
72000catccaggct tccaggcata agatccagtc ctctggtgat aaggagttca cagacaggac
72060aatgtctgag tgtatcttaa acccaggacc atggcttgtg ttcacaccag accctccagg
72120gattttgagg tgttttgttt gtttgtttgt ttgtttgttt gttttttgag acagagtctc
72180tctctgtcgc caggctggag tgcagtggca cgatctcagc tcactgcaac cttcgcctcc
72240cggttcaagc gattctcctg tctcagcctc ctgagtagct gggactacag gtgtgcacca
72300ccacacccgg ctaatttttg tatttttaat agagactgtg tttcaccatg ttggacagga
72360tggtcttgat ctcttgacct cgtgatcctc ccgcctcggc ctcccaaaat actgggatta
72420caggcatgag ccaccgtggc ccgcccaatt ttgagttttt atgttctaat cccaaacatc
72480tgctcacagg cccctcagca tattctttcc tgggtccagt gtcacctccc aggcctgcag
72540gctggctaga gcagtagggt gtgtgggaaa gctctgggct ttgcaggcac tgatcagctg
72600tgtgacctta accaccctga acctcagttt cctcacctgt aatggaaata ggtaccacgg
72660cagtttgttg caaggactag agagtaacct tgggaataaa aggtagcagc agcttgggct
72720ctggagatgg actgtccaag accaacttcc agttcctccc cacacaagct ctggcactta
72780gattcctggt acctccgctg cttcatctgt aaaatggagt aacaatagga atactttata
72840gagttgtaag gattgagtgg ctggatgaac gtcaagcact tcaaagggga cctggcatgt
72900agtgagtgat caatataaac cacctggctt gtagcaggtg tgctgtgtgt ggctgcaggt
72960gttattagta acatctgtgt gcccttcaga gcgtgcacca cacttcacac cttgtggagt
73020ctggaatgcc actattatag ttcaggatag aaaacctccc tgcaagcact cgctttagct
73080tgtctccacc gaacaaaaca acacaagttc tttattactt ggaatgggaa aacttcaaag
73140gcaaaaaaaa aaaaagactt tcgagttacc ccaaatctta agccaaagtc aatgaaaaat
73200atcaatcttc atattcaatt tttgcgatac ttttgtctcc ccagcagtca atggagagaa
73260tccaagcaca cagaaatgtc aattaccagg ggcagggcta tgaattcctt tcagagccct
73320gggctgggga agagtgcagg cagacagatc tgggtcctgt tatcacgttc ttagattggg
73380tgtccttgta ggagtcatga agcatcttag tgcctttgtt tgctacctat aatgcctacc
73440tcagagagta ataaggataa gtaaggctct acgtgaaaag tgctcggccc tggcacatag
73500taggtccttc attaatggca gctactaatt tttattacat acgcaaaatc acattacagg
73560tcaagtacgc tacatgacag tgaaacagtt tttttgtttg tttgttttga gacagagtct
73620cgctctgtca cccaggctgg agtgcagtgg cacgatcttg gctcaccgca acttctgcct
73680tcaagcaatt ctcctgtctc agcctcccga gtagctggga ttacaggcat gtgccaccac
73740gccagctaat tttttttggt atttttagta gagacggggt ttcaccatat tggccagact
73800ggtctcaaac tcctgacctt gtgatctgcc caactcagac tcccaaagtg ctgggattac
73860tggcatgagc caccgcacct ggctgtgaaa cagttttatt gtgtttctgt ggaatgtgtc
73920ctacccaacc tatagctaac tcctatagtt ccctcagttc tcagctcaga tatcccttcc
73980tttctgtact gttacctagt actggttttc atagcaccag gtacctctct ggcatagagc
74040ttgtcacagt tgcagtttaa tgtaccatca taggatttta aaaatattca gttgtgtctt
74100ccattaggct ttcatttggg aactccacgc aggcagcagc tgtatatttt gtattgccta
74160ctgtatcctg agaactttgt accctactta gcacagaatg gaggctcagt aaatactgga
74220catgagagag agagagagag agagaggaga gggagagaga gagagagaga ttcaacctac
74280aatcccagct ctgagcttct agttccctga tggtgaggac tgtgatgtgt ctcacacggt
74340aatgagcact tatgcagaag aggctcagaa aatttctcct catggccaac ggaagactta
74400gagttctttt ccaagctcca ccgtttgctg gcatgcaaaa tttggactat cacttaagtt
74460ttccaagcct tgctttttct atccctaaca taggacaata ttcagcattg ttgtttgttt
74520gttgggggca ccatgtttca ggcacttagt agattattgt accaccacat ttcaattggt
74580cctcctcaag ccctgcaaca tctgtgaggt ggtcatcctt aacaactcac agatgagcaa
74640caggagactg gggggatgag ggaactgcca aggaggtcca gcttatgggc agcagagcca
74700agaatggaac cagggtcttt tattttttta tttttttatt tttatttttt aaccagggtc
74760ttttaacatc cgaggaccac attctttgtg ctttccaaat catcacctgc cccatgcaac
74820ttacagggta agttacatta aacaacgtat gtaaatggct ttgtgctagt tattcaccac
74880cacaggggaa gtgagtcacg gacaagagtg cagccgctcc attcggatcc tggctctgac
74940acttacctgg aaaatgactt aaccattccc aggatcagct gtttgtctgt aatttaggta
75000gtttaatggc acttgtgtcc tagagttgtt tagaaggttg aataatatgg agcacttaac
75060atacttagca cctagaaaca cttcctaaat attagttgct gctgttgtta tcgttattaa
75120aatttctgcc taagatctca tttcagggag cccaactcaa tctttgacaa gcttaaacaa
75180aaattgcttt tcttcattta ttcacttaca cagcaaacat gaattgagcc tgtactgtgt
75240ttccagaact gtgcaggacc agagaggcac aggtgaagga agcaaggctc tggctctact
75300ggggaaacag caagaagatt gctacaatga ggtgggaaga gggctggact agagagaagc
75360cctgattagt gtccttgcta cctttctctg ggagagccaa ggcaggcttc ctggaagagg
75420tgatccttgg ctgaaacttc gatgaagaaa aggaaagagc gcagtggtta gggaggaaag
75480ggcattctgg gcagatgaaa tgacatgtga caaaatatgg gtgatcannn nnnnnnnnnn
75540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
75600nnnnnnnnnn nnnnnnnnnn nnnnnnngca ctccagcctg ggtcactgag tgagagaccc
75660tgtctcaaaa aaattaaaaa aaaaaagtcc agaagaacat ttgggtctca ctctgtggcc
75720caggctggag tatagtggca caatcatagc tcactgcaca ttcaaactcc tggcctcaag
75780tgatcctcct gccttagcct tgaaataagc ttggattaca gatgagccac cacacccagc
75840cagaccatta ttcataatag ccaaaatgtg aaaacaaccc aaatctccat caactgacaa
75900atggataaat agaatggtgg ttgatccata caatggagta tttactcagc aataaaaaga
75960agtcctgata catgctacaa ggatgaacct cgaaaacatt atgctaagtg aaagcagcca
76020atcacaaaag gctacatatt acaagattcc atttaaatga aatgttcaga ataggtaaat
76080ctaactttta tcacaggcaa agctatgaca ggaaatagat gagtggttgc ctagtgcttg
76140ggggcagagg tgggggtgag gcgagtgagt actgctaatg gtacagagtt acttttgggg
76200ataaagaaac tgttctgaaa tggactctgg tgatggttgc actactctga acatactaaa
76260actgttaaat tatatacttg aaatgggtga cttgtgaggc atggaaatta tatcttaata
76320aagctgtttt acatatttta catatttaaa aatgcaggtg gagggatgag ccctctaaag
76380agaagcagga gtttgaggag gttctaaata ttgtgtggtg ggtactgagg catataaatt
76440tgtcagacct catcaaaatg tatgatgtaa tcttcaagaa agttgatttt aaagaaacac
76500caccagcacc aggtggagaa ggcaggaaga agttacacaa ggggtaggcc aagagtggtg
76560gctcatgtct ataatcccag cactgcggga ggccgagctg ggtgggtgac ttgaggtcag
76620gtgttcgaga ccagcctggc caacatggtg aaagcccgtc tctactaaaa atacaaaaat
76680tagccagacg tgctcgcgtg aacccagggg gagaaggttg cagtgagcga agatcatgcc
76740aatgcactcc agcctgggtg acagagtgag actctgtctc aaaaaaaaaa aaaaaagtta
76800cataggggac agtggcaggt gtcaagggca ggcagggtct ctcctatctc caggataaac
76860tcatagggga cttagatgcc atgtgggtcc ctaatagccc tccacttggt tcttgcagcc
76920actcttatgt gtatcatttc atgtcaggcc tcttcttccc aacccaccca gccatcccag
76980cctggctgcc aaccccacct cctccagccc ctgtcacccc ataattgggg ccaggaggca
77040tgggagagtc gccatctctc ggtgccatct gttgcatctt tacagataac catggctgga
77100tgcggcagat cctggggtgg agcagccgct gttcagagca gtgatcaaga cctccccatc
77160tccacccctc aaggaatcgg ttttcttcca tagccacatc aggtgctgtg caggaaggag
77220ttgaaacgag aagccaggag caacgagaag gacactaaca tttattaagc actgcagact
77280ctcacagcac tcccacggaa tcgatattat tatccccatt ctgaagacca ggcaactgaa
77340gctcaatgtt taaggaactc accgaagtca ccaactgata aaagtgatgg aagctgggat
77400tcaaatccaa gctaaacttc cttccaagct tactccacaa cacagaggtt ggggaaaggg
77460gataaaaaga gaggggagcc caattccatt tccacccagc tcctgaggcg gagcttgtca
77520gcacagctct ctccttccca gaataggaag atacccatca gaggcaagtc ctagacacca
77580gcagtggtaa ctccctgccc caaggcagct gcagacagcc tatggctgta gttactgctc
77640ccaaagagtg ttagaattcc cactcccagc ttcggggcca ctcacacaag gtgattgaag
77700tggaaaccag agactctcca caatgccctc ctagagtaaa tgaggctatg taactttgtc
77760caaatgagta atttgaaaac ctgggggctc ccagctcctg aaaagggaag gatgtggggc
77820cctttatatt catactccac tttgtgcagc tctcccttgt cttatgatag ccctattaag
77880aaattcctct cccagcacgt ctccttcaaa gagctctaga cctgaggctg tcagaggctt
77940aggactctgc ctattagtcc cagggtctgg atgaccagca ggacacctgg cattcagtga
78000ccactggatt agataaatga aacagtgggc agagtgccac ccaatctccc cctgaagttt
78060gaagaggtcg agaagtgagg ctgtccaact gctgaccctg ctttctgtcc acctggccac
78120ctaacctttt ctggcttcca cctgcccctt tgccatccct ccccccagcc cacccagccc
78180attttcaggc atacctgggc acgtgctgga atagaagccc tcgttcttca gaatgatcaa
78240cagggagccc cagcccagga gtacagcaga gaagaagagg ttctccagca cagccgtgca
78300ggccatccac cagcgcctcc ggtacgcctg ttgcagcgtg ggggccatgc tggccccgag
78360cctgcacaga aacagagcgc tgggtgaagg gccccccagt ggccccaggg aagggtcctg
78420catcatggtg gcacccgaga cctctcgggc cagcccgcga ggagcccctc atggaggccc
78480catagagccc tgggcttccc agccggtgcc aaggagctgg ctccgcgcgc actagcagtg
78540ccagaggtgc acgcggcacg gggctcccgc tgagccacta tcggaaacaa ggaaggtcct
78600gtctgcgcgc tgcagcttcc tagcaggctg ccgggttctc tcacccaggc cagggcgctc
78660agggccgggc tgctggggag aaagtccgca tctgcccagg tccccagagg acagcaaggg
78720gcagagcgcg ctctgaagca ccgcgggccc atgtccggac tctcgcgcca ggaaagaccc
78780ctagaagctg gcaggaagaa gggcaagttc aaggctaccc tacgacccca tcttccagtt
78840gcccctccaa gacctctcct tccctctggg gccgggcgac agcaagccct ccccctttcc
78900gtatcaggtg acccacgacc ctacagtctc tcgggccaag ccaacagctg ccacgtggag
78960ggagacccag gacgggctct cctcggttcc ctcctccccc gcgcgcccct cactcactcc
79020gcagggctcg gggcaccagg ctttgcacct cggaacccgc ttgcccccct ccagccccgg
79080gagggggctc ggacttcggc aggaagtctg gcggctgctg actttataag ggcagcggtg
79140gcggatgggc tggcgggcgg gtgtgtttac caaagggagg gaaagagccc cagctccccc
79200cgccgcggcc gctgcagcct cggcgggagg agagggaacg cgggcagcgc gggggcgggg
79260agcgacaact gggatgagac cgaggaaagc ggagaggaga agggcaagaa agacccagag
79320agaggggagg aagtaccagt cacttcttcc agggggactc ggtattctca tctgtgaaac
79380ggggctttgg gttcaagcgc tccaggaggt ccgctggaac tctggcaaac gcgcagctct
79440aagcagagga agtgcagcga gcggggaccc gggaggaaga gaagagtcgg aggggtcaga
79500gaaaagaaaa gggaaggacg cgcttggcga gatgggacac tgtgccgcgg gaccgcgggc
79560gcaagtaacg gtctttcctt gggaagcctg gcagtgtcgg cgggagccgg cctcggtgtc
79620tctcagccga cgcatagccg gagaccctac gcgcgccccc tccccgccca cgctgctcac
79680ctccggtcac cggcaaatga gcagccagca gctgcggacg cctccgggag cgcaacgctt
79740tcgcggcgcg tccggagtcc cgtgggccca gccctgagcc gcgccggcgc tggggtcttc
79800tctgcgtgca ggacccggcc gccacggagc ttcagcctga cagcccggtg gcctcgcctc
79860cgctgtctcc tcggaagaag cgggggaact gggaacccgc cgggcgccag aggtctgcga
79920agctgggctt ggatgaagtg gatctgcgga gttgatagtt gtatttacac gcgtccggag
79980ctgcgccccg aggtgggggc gggggctccc ttcttttccc ctccccttag gtcgagtttc
80040acgcgcacgt gactcgcccg ctggtcccgg acactctccc tctggcacag ccccagcacc
80100tacatttcca ccctggaccc ccatcttctc ccccaagccc ccagactaac atcaggcagc
80160gccctctgta tccttgttca aaacaaagtg cgattcggct gaagccgact gaccgcgatt
80220cagggccgcc ttgggtgggg ttttgaactg tgcagctgga agcagtgttt tccgagaggc
80280agagtggcac gggtttcttt ggagttagtc agatcgaggt ctgagtcttg actttttaac
80340tgactaccct gggttaccta gggcaagtta cctctctgag cctcagcttc ctcctcttta
80400aattcggtta aaatggaacc tacctaactg cccaaaggaa tcgcgattgt gatgcaggta
80460aaatgctaag catagcattt ggcatagtaa gcataatgtt aattgttgct gctgtcatta
80520tttcagaaga cctggtgatc ggatgcttcc agatcaacaa ttgattgact ccaggtaaat
80580ctctcagcct ccctgagcct cagtatcctc atctgtaaaa tagactacta tggtgtggag
80640taatgagaag taatctcatt acatgtgagt ttaattgtgt gttaagagtg ctgctaatgc
80700atgctgagct taatacctag gtgatgggtt gataggtgca ataaaccacc atggcataca
80760tttacctacg taacaaacct gcacattctg cacatgtacc ccagaactta aaataaaaat
80820aaaatttttt taaaaaaaga gtgatactgg tggccaggtg tggtggttca tgcctgtaat
80880cccagaactt tgggaggcca aggcaggagg atcgcttgag ctcaggagtt cgagaccaac
80940ctggacaaca tggtgaaacc ccgtctctac aaaaaagaaa aaaaaatagc caggcatggt
81000ggtgtgcacc tgcagtctca gctacccagc aggctgaagt gggaggatca ctgagctgga
81060gagatggagg ctgcagtgag ccaagatcat gccactacac tccagcctgg gtgacagagt
81120aagactctgt ctcaaaaaca aaacaagaat gactacagaa agctccaaga aggcctcaga
81180taaaagggaa cccctgaaca gatgagccac caagccaaga gaggaactaa tggctaccat
81240agacagggca ctttccaaaa taaaaatact gttattaatt cctcaagaca tcatggtccc
81300atttaaacct catagctttt cacagaggga gaaactgcag gcttgaagct ggagcaaggt
81360tagaggtagg atgcagagtc aggtcggcct ggcatttaag tacggctcct tccattcctc
81420ccagaaggag aatggcaaga gcaaaggctt agctgtggga atggcacaag gagttctcgg
81480tggccaaagc acatgtcagg ctctgatggt ttaacttctt aaaatgcaat actgcctccc
81540agaacttcca gatcaaggtc aaactcctca gctctacaca gggggaccta gagtcaactt
81600tctaagctag gagagtcatg gatccctttg agaatacaaa agacagtggg cgcggtggca
81660gtggctcatg cctgtaatcc caacattttg ggaggctgag gcaggaggat cacttgagcc
81720caggagttca agacctgctt ggtcaacata gtgagacccc tatttctaca aaaaattcag
81780ctgagcatgg tggcatgtgc ctgtagtctc agttactggg gaggctgaag taggatgatc
81840cctgagcctg ggaggtccag gaagctggag tgagccgaca tctcgccact gcactccagc
81900ctgggtgaca gagaccctgt ctcaaaaaaa aaaaaaaaaa gaagaaatat gttattgatc
81960tactcttgac aaaaatgctt gtgtgaacat ggacacacac actcatcaac attcacattt
82020caaggttttc atggaccctt tccatgaggc tctagtggtc catggacccc catggctgga
82080acacttgctc ttcctcatct caacccacat ttccatggag ttggactgtc tgctgcatga
82140ggacacaggc ctcatttggt gtgttcattc actgctgtgt atcccagcac ccagaacagc
82200acctcaccta aggggcactc agcacatgtg cagtgaagag tcagtcagct ggtttcacac
82260ctcccagtct ttgcacctgc tattccttct tgtgggaatg acagatttcc ttcatttctt
82320tttttttttt ttttgacaga ttccagctct gttgcccgag ttggagtaca gtggcacgat
82380ctcagctcac tgcaacctct gcctcccagg ttcaagcaat tctcatgcct cagcctccca
82440agtagctggg attacaggtg cacaccacca cctgtgagct gatatttttt tcttttcttt
82500tcttttttcc tgagacagag tctcactctg ttgcccaggc tggagtgcag tggcgtgatc
82560tcggctcact gcaagctcca cctcccgggt tcaagtgatt ctcctgcctc agcctcccaa
82620gtagctgaga ctacaggcgc gcaccaccat gcctggctaa tttttgtatt ttttagtaga
82680ggcggggttt caccatattg gacaggctgg tctcgaactc ctgacctcgt gatccgccca
82740cgttggcctc ccaaggtgct gagattacag gtgtgagcca ctgcactcgg ccattttttg
82800tattttttta gtagagatgg ggtttcacca tgttggccag gctggtcttg aactcttggc
82860ctcacgtgat ccacccacct tggccaccca aagtgttggg attacaggca tgaaccactg
82920cgctcagcct ccttcttcat ttctaatgta ctcatccttc acaactcagc tcaagtttca
82980cttctctctg gaagctctac tctaggctgg attcagggcc ttgtccacat acccaccaaa
83040tactctgctt acctctatgg aagtccccac actgatctag aataatcagc ttagttttct
83100gcccccatcc cgccccatga gatgtacatc ttgtgggggc aggaaccacc acgtggtagg
83160tgatttgtgt gcctgctgcc tatcacaggg cctggcgcct aataagcttg cggccaacat
83220ttgttgaata aatgaaaagg gaatggtggg aaaggaagct gaaaaggtag gctaaaatca
83280gtttggaatt acctctggga ggccaaggac tttcagtctt gcagggtagg taacaggaaa
83340ctcctggatt ttgttttctt ttggttttgt ttgtttttaa tgaagggtag cgttatcgtc
83400aggtttttgt gtttaattaa tggagcatat attggaaagg acagagacct taaagcagtt
83460aggagaccac cataatagtt cacattttgc agccataaaa aggaatgagg ccaggcatgg
83520tggctcactc ctgtaatctt atcacttcgg gaggttgagg caggcggatc acctgaggtc
83580aggagtttga gaccagcctc accaacatgg agaaacccca tctctactaa aaatacaaaa
83640ttatccaggc gtggtggtac atgcctgtaa tcccagctac tcaggaggct gaggcaggag
83700aatagcttga atctgggagg cagaggttgc ggtgagccga gatcgtgcca ttgcattgca
83760ggtacatgga tgaagctgga agccatcatc ctcagcaaac taacacagga acagaaaacc
83820aaacaccgca tgttctcact cataagtagg agctgaacat tgaaaacaca tggacacaga
83880ggggaacatc acacactagg gcccgttggg gagtgggggt tggggggtaa ggggagggaa
83940cttagaggac gggacaatag gtgcagcaaa ccaccatgac acacgtatac atatgtgaca
84000aacctgcaca ttctgcacat ggatcctgtt ttgttttaag aagaaataaa gaaaaaacca
84060agaagaaaca aacaaacaaa aataattccc atttaaaaca ataaaaaata ggccaggcat
84120ggtgactcag gtctataatc ccaacacttt gggaggccaa cgcgggcaga tctcttgagc
84180ccaggagttc aaggccagcc tgggcaacat ggcaaaaccc tgtctctaca aaaaatataa
84240aacaaacaaa caaaatagcc aggagtggtg gtgcatgcct gtcatcccag ctactcaggt
84300ggctgaggtg ggagaatcac ttaagcctgg gaggcggagg tagcagtgag ctgagatcgt
84360gccactgcac tccacctgga gcaacagagc aagattttgt ctctaaataa ataaataaaa
84420taataaaaaa cagagaagag gaaagacacc tgagatatat ttccatatct gaatcaatag
84480gatttatcaa cgttctcctc tacccccaaa actaattcct tcctaaactc tgttctcctg
84540acactactca taggttaagt ataacagcat tatcacattg gctgtcatgt gggctcctgg
84600ctagaggctg cttcacagct taatggacaa gagcactgag acagggtggg tctaaatcct
84660ggctctgcag ctgattattt gtgtgatttt gtccaaatca ctccatctca tgagcctcac
84720tcttctagtc tgttaagtgc tgaaaataaa agtatccaat tcaattcatt atttaatgaa
84780ttatttagcc taacaaatag ctattataaa tatttaggct gggcacagtg gctcacgcct
84840gtaatcccag cactttggga ggccaaggtg ggcagatcac ctgagtcagg agtttgagac
84900cagcctgacc aacatggtga aaccccgtct ctactaaaaa tacaaaaatt agctgggtgt
84960ggtggcatgt gcctgtaatc ccagctactc aggaggctga ggcaggagaa cgcttgaacc
85020caggagacag aggctgcagt gagccaagat cgtgccactg cactctagcc tgagcaacag
85080agcaagactc tgtctcaaaa aaaaaaaaaa aatctctgca tgaagaatgt acataaaatg
85140gtgcagccat ttcggaaaac agtttggcag gtcctcaaat agttaaacat agagttacca
85200ctatagccca gcaattccac tcctaaatat actacaccca agagaattga gaatatttgt
85260taacacaaaa atgtgtatac aagtatttat agctgtatta ttcattacag ctaaaaagtg
85320caaacatccc agcagtccat cagctgatga acggagaaac aaaatgtggt atacccatac
85380aatgtcatat tatttggcca taaaaaggaa gtactgatac atgctacaac atggatgaac
85440cttgataatg ttattctaag tgaaagaaac cagacacaaa agaccacata ttgtatgact
85500gcatttatat gaagtgccca gaataggcaa atccacagag acagaaagta gattagtggt
85560tgccagagac tggagggagg agataatggg aaatgtggaa tgactgctaa tggtatgggg
85620tttcttcttg gggtaatgaa aatgttgtac aattagataa tggtgatcat tgtaaaactt
85680tgtgaatata caacatgctg aattttatac tttattatat tttatttttt ttgagacaag
85740gtctcgctct gtcacccagg ctggagtgca gtggcacgat ctcagctcac tgcaatctct
85800ctgcctccca ggctcaagca atcctcctgc ctcagcctcc tgagtacctg acactacagc
85860atgtgctacc atgcctggac aatttttgca tttttagtag agacagggtt tcgctatgtt
85920gcccaggctg attttgaact cctggactca agtgctccgc ccacctcagc ctcccaaagt
85980gctaggatta caggtgtaag ccaccactcc cggcctaaat tgtattcttt aaaagactga
86040attgtatggt gtgcgaatta tatctcaatt taaaaaaaac aaaacaaaac aaaaaaaaaa
86100cctttgcgtg tgtcaggcac tagggattcg atgctgaata agacacagac cctaccctca
86160gagaacacag agcccagcag gagagagtca cagatgaatc aagtgttaca tcatctatag
86220gaagcgccat ggaagaaaga catggtgcca tgagaacata cgcttagaga agggaatttc
86280atctagactg gggctcaggg aggaatcttt cagggtgatg cttgtgctca gagttttcca
86340tgtcagaatc agtagaattt atcaatcctc cagaggagga aacagcaaat gaaaaatctt
86400acaacaggag gatgcggaga cattccgaga gctgatcaag ggctggtgtg aacaaagcac
86460ataggatgca gagcctgtgg tgtgaggttg cagctggaaa ggtaaaacac taattacatt
86520ggatcttctg agacaataaa gagtatgcaa taatctcaaa cgaccgaaac tgaccttcct
86580cctccctaac ttgcttgctt ccactgttgc ccgtatcata aaagcaccac cctcttctac
86640ccagtggctt aagacacgaa actcaagtca tcccaggctt tctccccacc tcactctcca
86700catccagcct atcagcgagc ttgtgggtct taccacgtaa agacttctca tctccagcta
86760ctaccatccc ccaagcccag atcaccatca gctcaggcct ggactcctgc aacctttcta
86820accgggtctt cccaatccta cccccgcaac atgaccccaa tagcccatca gaatggacta
86880atcgagatgt agatttgatc aggccacatc ccttgaaagg cttcctgtga ccctcgggga
86940aatgcacaaa ctcccaatga tggcccctga gtcctgtgcc atctgggtct gccctctgcc
87000ctctgtgtct ttgccatggt aacctccttc acacccatta atactccatg ctctctccta
87060cctcaagttc ttcctgggct ggaacattct ctgcactagc ctagccaact aaccctttag
87120atcttttgtt tgtttgtttg tttgtttgtt tgtttttgag acagtcttgc tctgttgcca
87180ggctggagtg caatggtgca atctatctcg gctcactgca acctctgcct gccgggttca
87240agcaattctt ctgccttagc gtcctgagta gctgagacta taggcaccta ccatcacgcc
87300cggctaattt ttgtattttc agtggaggtg ggttttcacc atgttggcca ggctggtctc
87360gaactcctgg cctcaaatga ccaccctcct cggcctccta aagtgctggg attacaagca
87420tgagccactg tgcccaggca acacttcaga tcttaatgat catttccttt aagtgcctga
87480cctcttgtag taactagcct gactccagca atgaatcctt ttgcaatgta acctatataa
87540catctgagtt tccctttgat aaaactcatc atatatttgt tcctctgaca gttcagaggg
87600caagggcctt tgcccacctt cctcaccact atcctctcac cacttaacac agaactcacc
87660acccaccatg cctcctgcct gacaaattcc taaccatcct tcaaatctca ctcacctatt
87720accttctggg aggcagtctt ccctgagcac caagacaatg ggacacattc ctttatacac
87780cctgctgaac atctcttttt tgaggggcgg gtagagatga gtgtctcact atgctgccca
87840ggctgacctc aaactcctgg cctcaagcga tcctcctgcc ttggcctccc aaaatgctgg
87900gattacaggc atgagccact gtacctgacc gcaactgggt tagnnnnnnn nnnnnnnnnn
87960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
88020nnnnnnnnnn nnnnnnnnnn nnncctgggc aacaaagacc cttcctctac aaaaaaaaaa
88080aaaaaaaaaa aaaaaaaaaa aaattatttt aaattagcca gacatggtag tgcatgcctg
88140tagtccaagc tacttgggag gctgaaggga gaggatcact tgagcccagg aggttgaggc
88200tgcagtgagc cgtgatcgta ccactgtact ccagcttggg caacagagtg agacctcatc
88260cctaaaaata aagaagaaaa tatggcaatt tgactgtaca tctctaatgg gatatatcct
88320aaggatgaga aaggaataag gaaggacaga aaaaaggaaa caaagaagta gcaacagtat
88380ttagcaattg tattgttatc aagtaacatc aatattggta aaaccagtaa ttatatttaa
88440aatactatat atgtgtatgt acatttacat atgcatatgt taggaaccaa gtttatcaga
88500ggaagagaaa gggctacaaa tgtaaaatca aggaaataaa aatttgaata aaaatatcag
88560tattaagtat ttatgatatt tttcttataa aaaaattata tatatgttaa ctctatccaa
88620aacccaaaag cagtgacaac ccaggagcaa taaaaaacct cagcatccag actgtagtct
88680ctaccatttc caattaaaga aacccagggc tagttgggaa aaatgacaat ttcatgtcta
88740gggcaagaaa cacacctagt gaaatggacc tgaacattta attgtgttag aaagtaagga
88800aactctctag aaataatgtg atttcatcta aaagacacag attctgggct ggtaaagttt
88860tcaatggcca aaggtgagac aatttgagca tcaagaagaa tcatgacaga acagattaaa
88920acatgtcaaa tatattttaa aatgaaatat tataaaagaa acaattagta gccatccctg
88980aaggtcacta gggcaccaac tcatatttca aactggtaaa taaatgtgta agccaagcat
89040ttatttctgg gtaacaaaat agtaaggaat gtttttcttt ctagaagaat tctagtgatt
89100aaaagtagaa gatagaaata gaaaatcatc cttttggcca ggggcagtgg ctgtaatccc
89160agaactttgg gaggccaagg caggtggatc actggagatc aggagtttga gaccagcctg
89220gccaacatgg tgaaacccca tctctactaa aaatacaaaa attagccagg tagtgggtgc
89280ctgtaatgcc agctactcgg gatgctgagg caggagaatc gcttgaacct gggaggcgga
89340ggtcgcagta ggctgagatt acgccactgc actccagcct aggcaacaca gtgagactct
89400gtctcaaaaa aaaaaaaaga aagaaaagaa aatcatcctt ttgcgatcct aatgaaataa
89460tgggcctagg cattgatcat taatggctcc taaaatcact aagtatatgg ttgatgggaa
89520actttatagt ggatggatca gactcgcaat gtctaaacca gttgatcaat cttaacatcg
89580taacaagaca acagacacca ggggctgctg acaggagaac agaggaaacc catagctcta
89640ccactgagtt attcacggca aaaaaaaaaa aaaaaaaatt aaactgcgtt tcctccaagc
89700ttctaatcct gttgtttaca ggaaataccc aaggaaagga atacttttaa atgacacatt
89760aaaacaacgc caaatccaaa atatggggaa atgacccagt ttcttcaaca aataaacaag
89820aaaaggtagg ggggaggact gttctagatt ttaaaagcta tagaagacac agcaaccaaa
89880tacactgcat ggaccaggca tggatcctaa ttggaacaaa ccaactgtaa aaggatgtat
89940ttgaaatgat tggaggaatt tgaacagtga ctgcacagta gatgatatga agaaattatt
90000gttatttttt aggtgtgatc atgattttat ggtgatgttt aagtaaaaga ggccttattt
90060gttagagata catgtacggg tatagagaaa tatttacgga tgaaatgata cgatgtctga
90120gatttgcttt gaaaactcta gcaggtgtgg gagaagcagg tgcatgggtg ggggaaggga
90180tagatgaaat aagtatgcaa aatgttagtc tacttttgtc cctcctgacc cagcaggtta
90240aaatacctca gcatacctct actcctccaa ccaggtccaa ggatcaggcc aaaactccct
90300gatgtggtaa acagcctgac cccttcttac ctctctctct ccagccactt ccctaagatt
90360ccccagtgct ctgtgcccta gccagcccga ctcatctgcc cagattcctc aatgtttcac
90420tctctcattc accattttga ccccactgtg ctcctgggcc actctccaag gccccgcctc
90480ttcatctcct ccctccttac tcatccttca ggtcttggct taggtgccat tccctccagg
90540aagccttccc tgacaccaat cccatcctca cctagaacag attatgtgcg cttctttgtg
90600ccccccatgg ccccctgtgg gtttgcttca cggattataa ctgcctgact acctgccttt
90660ctccaccctc tagactgaga acaccttgag aaaaagaaca catctatctt gtctgtcatt
90720gaatccctgg tgtctggcac catggctgac acataactat cactcagtga ctagtgtttt
90780aatgaatgaa tgagtgcaac tagacagggt taagaacaaa agagaagacc aggcatggtg
90840cttcacgcct gttatcccag cattttggga ggctgaggcg ggcagatcac ccgaggtcag
90900gagttcaaga ccagcctgac atggtgaaac cccgtctcta ctaaaaagac aaaaattagt
90960gggggatggt ggcacacgcc tgtaatccca gctacttggg aggctgaggc aggagaatct
91020cttgaaccca ggaggtgcag gctgcaatga tctgagatca caccactgca ctccagcctg
91080ggcaacagag taagactcta tcacaaaaaa aaaaaaaaaa aaaaaaaaaa gagcgagaga
91140agatgtcatg gggtaaatga agacctccct tcctggttcc ctgaccagcc cctgccctcc
91200cccgcatctc acctgtcttt cttgcttcct tctggtactt ctgtttaagc cggtccatga
91260gcaggccatt ccagggggca cacagcactc cgaactgagt gaaggcaaag gcatttgtgt
91320aggtgctgac tgcagaagga agagagaggt tggttgatga gaagtttcca aaactccctt
91380ccaggcaggg actctcccac cttacccttg tctgcatgtc cctcctcccc acaccatcag
91440accctcctct ggtgtgtaca gccctgctgg gaggctctgt gttcccagct gggacatgca
91500gatgggctac ctcccagccc taccacatac ctcgtgccat gtccccaccg gccatgttgg
91560tcagcaagga gttgagagtg ccaatgaaga ggtagtgcca caactgtatc acagacagcc
91620acaccaggtg ccaggcaaag cgccgagaga aagcgtagct ccagaaggag cggagttcct
91680gcttctgccc tgcccctggg gtctctaatg gggagaggag gatctgggcg tgaattacga
91740ggaaagtgga caggtaggat ggggagtgtg gaggcttcaa tggaacattt cagatccggg
91800cccaccttct acccttggct cccagaactc acccggcgtg ctctgcacct gcctcggact
91860cacacatccc cacctccctg ctttgtcatg ctggccctac caccttggat gaccctctgt
91920gttcttctct attgaaatcc gatctgtctc tcacagcctg gtcaatgaca cttcttgcag
91980taataccttc ctgatctttc tcagcgagaa aggtgaaagg aacgacaagg agaggagaaa
92040gtcagaaggg agaggagaat gagtgtggat actctgttct aacctgctcc tcagcacctc
92100cctttctttt gataccagta tcctgagttt ctttgggaaa tcttcctcta ccctaatctt
92160catggtccag atgggaccat gaattcagtg ttctgttcct ctctcaaggt taaccaatga
92220gatggttcct cctaacaagg cagaccggcc atgagtttag attaggatgg acttaatcta
92280aaataggtcc tacaccctgg caagttcaat gtcctccctt gattttggaa gcttcccaga
92340accctattct tctttcttta aaataaataa ataaatacat gttttggatc caattgtcag
92400atggtaaaaa taaaaacaaa aaaatcaatt ttattctgta tatttaagat atacaatatg
92460aggtcatagg atacatatag ctactaagat ggttactaca gttaagcaaa ttaacatatc
92520catcatctca catttctacc tgttttgtga caagagcagt taaaatctac ttgtttagga
92580aagtcccaaa cacaatgcag tttgatgacc tacagtcttc gtgctgtgca ttagatctct
92640aggcctgttc atcctgctca tctgctcctt tttgtccttc gacctgcatc tcccatctcc
92700tctcccaccc cgttcttatt tctactgtag ctagctgcgg tttgtgatgt gtgtaaccaa
92760agacgcagaa cagagaggaa ggaaaggaag cagtgataga gttgggacaa taagagaggg
92820cggacccagg agacctggag aaatgggggc actgtaccag acttagtgca atggcatcac
92880agaagagggc agaaccgagg agtgggggga agggaaggca acccatggca ggcgggcttc
92940aaggggtggg gaagtgatag gatgcgaaat agagaaaaga gggacagaaa agagacgaaa
93000gccctggacc ctccattaag tgagagggtt gggaagatgc ctaaggccct ttttctgtcc
93060tgcctttcct gattctgggt ccctggggga gctctggagg tgaggggcca ggaaaggcac
93120aaggagaggc ttgggtctgg aggagagatg ggttagccag cagggctcac cttccttcgc
93180tgaaaggaac tcctttgact gtagctccct gttttcatgc tcagctgttt ccttctcttc
93240ctttgtggtg ccattcccag ggcacaggct atggaaacaa aagccccacc agcaaggcca
93300aggactgtga gccgaacctg agactcagac tggagggaat agcatggtga atcccacatt
93360ccaccgcact ttggaatcac cttttagcca ctctgatgcc caggttgcag accagaccag
93420ttaaatcaga atgtctggag gtgagagcca ggcttccttt tctaagatct ctatgtgaat
93480ctagtgattc taataagcag caaagtttag gaagcatgaa aagagtaggg caggcccagg
93540ttcaaatccc agctctgcct cttcctagca acagaaagat ggctcagact taacccttct
93600gagcctcatt ttttgcattt agaaaatgga gataaggata tctcagagga ttattgtgag
93660gatgaaatca gagagcacat ggggtctgac aattagtaag tgagcagcaa aggaatgccc
93720ttcctctact ccttgtggca aatgactgca aaaatgatca catttcttca cctcctctgt
93780atttccccca atttgaatga gactgcagct ctatttcccc atgccctgaa tctgggccag
93840ccttgtgaac tgcttcagcc aaaagaatgc agcagaagtg gctgtgccaa ttccaagctt
93900aaatctcaag aacgcttgtg catttctgca ctctttcaga accctgaaat cacggtgtga
93960atgagcccac gctggcttgc tggaggatga cagccacgtg acccaggcat ccctgtcact
94020ccaaacctat gtgagtgagg ccatcctagc atagccagcc cccatgtaat cctccaaatg
94080atcagatgta tgaatgagcc ctgtcaaaat catctacatc tggccctgat cagcggaact
94140agccagctac ccacagactt gtgaaaaata ataaatgctt aacattttag gctgctgagt
94200tttgagatag tttgttatgc agcaatagct aacagatgca ctgctccagt cctcctcctc
94260tcctgtgata ggtttgcttt accctgtcca tcccacccta gggccaatga ggggctctgg
94320cccacaatca ccagatagtc cttacccata gctgtagttg gggggcagtg ggtatgggat
94380gtgcccccgg ggcatcagga ggaaagtgcg tgctacatgc caggtactgc agacagagat
94440gaagatgaag gaggccctga ggctgatgcc tttttcataa agaagctgca gaaggagaag
94500gaaaaagtca gtgtcacacc cacgttcata gcagcactat tcacaatagc caaaggatgg
94560aagcaaacta agggtccatc agcagatgaa cagctaaaca taatgtgatc tatacacaca
94620atggaatatt attcagcctt aaaaaaggaa agaggcaacc atgctggctc acacctacaa
94680tcccagcact ttgggacgca cgaggatcac ctgagcccag ttcaagacca gccttgacaa
94740catagtgaga ccctcacccc ttctctagaa aatttttatt taattagctg ggtgtggtgg
94800catacacctg tagtgccagc tactcaggag gctgagtggg aggatttctt gagcccagaa
94860gtttgaggct gcagtgagtc atgactgggc cactgcaccc cagcctggac aatgaaacat
94920gaccttgcct ccaaataaaa aaaaaaaagg aaaggaaaga aattctgaca catgctgcaa
94980catggatgaa ctttaagagc actatgcagg gccaagctca gtggttcctg cctgtaattc
95040tagtgcttta gaagaccaag acaggaggat tgcttgagtc caggagcttg agaccagcct
95100gggaaacagc aagacctcat ctctactaaa aataaataaa taaatcagct gggcgtgatg
95160gtgcacgcct gtaattccag ctacttggga ggctgaggtg agaggatatg attacatgat
95220tacatgcctg taatcccagt actttgggag gctgaggcaa gcagatcacc tgaggccagg
95280agttccagac cagcctggcc aacatggtga aaccccgtct ctactgaaaa tacaaaaatt
95340agtggggcat ggtggcacgc acctgtaatc ccagctactc gggtggttaa ggcaggagaa
95400tcgcttgaac ccgggaggcg gaggttgcag tgagccaaga tcctgccacc gcactccagc
95460ctgggcaaca gagcgagact ctgtctcaaa aaaaaaaaaa ggttaagata gtaaatttta
95520tgttatgtat attttattgc atacaaaaac atcagcagaa gaggcagggg ctggaaccct
95580gttttctaag gagtcctagt acaagccatc acctactatc ctgtaagctg attagggaca
95640cctggtacac acatgccccc acccacccca agacacaccc ggcagtagag gagtcctcat
95700acgacccatc cccacagccg gtggagcctc ctcgtgtggc tccccagaga tcttctagcc
95760cagtgccttt tttcccccaa cgacagcaaa ggccttttgt tcaaagaaaa ttttacacaa
95820aaattcatct tacaaaacac accaatgggg agcttgccag tcatctccct ctttattctc
95880cttggtgact ggtatgacat caaagagaat ccctaagttc ctcaacagct cagtttgaaa
95940accaccgacc tagcccaacc tcctcccatt ttacagagag tgacgttgag gtccagagag
96000gtgcagtgaa ttgctcaata aattgacaga gtaagcagca gcaaagtcag attaaactaa
96060gaattcctgt tcctgctccc tttccccttc caactctaga gagacaggag agaggctggg
96120catggtggct catgcctgta atttcagcac tttgggaggt caaggaaggc ggattacttg
96180aggtcaagag ttcaagacca gcctggccaa catggcgaaa ccccatctct actaaaaata
96240caaaaataag ctgactgtgg tggcacgcct atagtcccag ctactcagga ggctgaggca
96300ggagaattgc ttaaacccac taggcagaga ttgcagtgag ccaagatccc accaatgcac
96360tccagcctgg gagacaaagt gagactccaa ctcaataata aaaaaaaaaa aaaaagagag
96420aggaaagaaa gatgaggcag ccatctgggt tctccagggg aaggagggag aacccagaaa
96480gtgactctta tgccaggagt agaaaggctt gagtgcctca ggggctcagt ctctgcataa
96540ccctccaaac ctccaaagct tatgggacta agctagactc atgtctgggt ggtgactgcc
96600agagatcctc ttctctgccc ccataacctg caggcagtgc caactgcctg tgacctaaca
96660ctaagcccag agagaagtcc caggttggat ggcttgagat ccacactctt cccttccttt
96720cactcagcca tctgtggtgt gctggcttta gtcctccagc ttgctgcctc ataattgaag
96780catggttgcc acaactccag ctatcacatc ctcacaccac aacattcaat gaggaagact
96840ttgtttttac tctgctttca ccttgcgtca gggaagaaaa gtccccttga atcttccact
96900atatacactc cctttatctc attaaaaagg actggatcat atgctgacct ccacctatca
96960ctagcaacgg gtaaatggat tgccatggtt ggctttaatc aatcaggatt catcccctgg
97020gctaagcggg tcactgccca gataaaactg ttcgcaatga ataagacaga atggttgttg
97080attgacctct aatagccttg gcaacagttc atcccctgat accccaacat cagccactgg
97140gacagctgga caagcctctg tgtctgcccc tgctgtaccc actagccact tgccaccttc
97200ttgtccaaac tagaagctca cagcagcaaa cgccccactc taaaggtccc ccagcctcta
97260cccaacactg gcccaagcac attatgacca ctgccacaaa agcttgggca agtctgaaga
97320aggggcttag cggttacaag ctcaggctct agaaccgaca agcctgggtt caagttccag
97380tatcatggct actagctgca gaaccttcaa caagcttttt aacctcagag actcaaatgc
97440ttcatctgta aaatgggggt aacacagtac ctacctcacc gagttgatgg agacaaataa
97500tgcaggttca caagacaagt gtctggcata tacaagtgcc cagtgaatgt aggctgttgc
97560tatatttacc ttaataataa ggaagactgc cgaggaagag tcaaatgctc cattgtacag
97620agtgatgatg gtcgaacggt gttggccaaa taggttccca atctggggat gataggacta
97680gcctggatca cttatttatt catgaaacag atacttcctg agcacccagc atgtggcaga
97740ccctccttat acccaaactc accctccacc gctagagctc ccacctcagc ttgggccaac
97800cccatctgag gcagccaatt atagaaaagg gtctctcctt ccctccacct tcccgccacc
97860ctgccgagtg cctgggatta gggaaggctc ccacctgcag gttggtgatg agaaacagga
97920ttcccccaat ggtgagcatt ggcatggcca ggaagagcag cacggctgag cctggaccat
97980caaagtcaga ggtagggtgg tgtcatagtg caacccaaac atggagcccc aaactctgct
98040cccacctgct ccaaattccc aacaatcctg gtatccaggc cccaattcta gccagcgttc
98100cagcgtcctt caagggtttt taggataccg gccaaggctt ccccagatat ctctgtggaa
98160gtcttctgag ccaccttctt cccaaccaaa gttggtcctc agtctgtggc aggccaggaa
98220gtctacagac agaggcagag ctctaagtga agccacctct ctcttccctc agtaaaccac
98280aagctgcctc tccctttcat ccttgacact cctggaaaag aagaccctgg actcaggtcc
98340ctggctcaac cctctagccc attccctaat tcatggtatt ggccttgagc ttcaatcatc
98400tgtttaatgg gaacaacagt tcctgctctt cctgtctcag gtgctatgag aactgagtga
98460gaaaaggacc atggtctttt ctttgttcac taaactctga gcacttcttt ggtgccaggc
98520attgtgcttg gcactggaaa tgcaagatga atcagatagt ccttgccctt aaatagactg
98580acatgcaaac aaatggttat aacaggtctg gtaagtgtga gaccacagca aaaaagctca
98640agagctgggc taggggaacc cttgacaaat tcttcctccc caaaccagac ttctgcccac
98700cattattctg gccacaacct atgcctgtcc tattatttgc taaaatgttt taagttgact
98760cacttttatc caaaaagtat ctatttttaa aggacacttt atatcactac tgtagatgaa
98820aacactggca ttacttgtca tgaatagaaa gtaactgtca aaataaatac aatgaaagga
98880aaacaatgtt attcaattgt agctggatgc atttgacctt agaatgttca aagcctaaga
98940cctgctcttc ccatcagtgt taaaatcaca ctggccccac atgaagacat tctttcatga
99000aatcagaagg actgaaagag aaataaaaag ggaatagctg ttctaccagg tgatttgatg
99060tttgttagtg tagttcacgt agtatgcgtg tgcccctaac atcctcttaa ctaccgtgct
99120ataccttaag aagcactgcc aagagctaat tttagagtat tcacacagtt taccattcaa
99180tttctgtctt tataaaatgt acatctctcc tactactaaa ggttggagac tcctttcaca
99240atagagtcct tatgggctca atgctttttt caaaactgaa aagccctata ttatggagga
99300agaggaggat tgttgctcag acgatttgca ggcacgagtc aaacattacc cagccaccac
99360ctccacattc agttgcttaa aaatcattta caggctttta gagtagatga tgctggtttg
99420ataaggagag tggtttgaaa taattggttt gaggtgctgg gccatctcat gagatctgtg
99480tgaacaaaga cactcagcct ctgtgtttgc ccagcatgag tgcagacaat ctcatgatgc
99540tgtcagcttt agcatagctt acacacacaa gagtaatgta ctttctttcc taaaccaaaa
99600attgagccac gggtctaaca ctaggaagga atattgggag gcatctcgtg gccaccataa
99660ccaaggcaat gacagaaaga agagtgaggg atcaggaggc ctgcacatca ggcccacctc
99720ccacttgctt tctctgtggc catggacatg tctttgcaag gggtcctgct gtggcttcag
99780tttctcctct gtgtaatggg tggaagggtg gtggaaaata aaccagattg gagttccaga
99840cttaacagac tggtgaaatt ttaaaacaaa gattttgagt acaatagggt tgtcaacttt
99900taccctgcta agtaaggata tttgcaaaat ggtcattcat ataatcattt cattaaaaag
99960agaaagagaa cattttaaca cataggagaa ggatgtaaag gttttttgtt gttgttttgt
100020tttttagggt tttttgtttg ttttttggta gagtctcact ttgtcaccca ggctggagtg
100080caatggtgtg atcttggctc actgcaacct ctgcctcctg ggttcaggca attctcctgc
100140ctcagcctcc tgagtggctg ggattacagg cgcgcaccac catccagcta atttttgtat
100200tttttttagt agagaaggga tttcaccatg ttggccaggc tggtcttgaa ctcctgacct
100260cagatgatcc acccgcctca gcctcccaaa gtgctgggat tacaggagtg agccactgca
100320cctggccaga tgtaaagttt tgaataaatt ctactctctg aagtaatccc tctccatcat
100380ccttgctttt cacattttct caataaactg ttttcacaga ccagcaatag ctcaagatcc
100440ttccaggatt ctttcaagct gcagatctct gaataaccat gtggtctgta tatcttgcct
100500atagccctct gctcacacct gccccagcca ccaggtgcct ctgagcttgc atccctccca
100560cccacctgac agcactcacc tgcagaggtg aaggctatga tgagtgtggc ggtggtgtag
100620aaaaatctga aacacataac aggaaaagca gaatattgtc aaggagggag aaacctggga
100680gaaaaaacat gattctgctc agccagccca caagtgtagg acttgaccgc accctcagcc
100740tgggatgcaa cgggcactga tgcctctgag ccccaggctc aaaaccaggc gcaagaagcc
100800gcgatgagat tgagatgtgg tcctgacctc atggacagtg cattttgctc attctgaggc
100860ccaaggctag catggaaagt cttggacaat gagctcagct gacgatgtga ttggcttggg
100920acttagccag gacagaatgg gcaaagcgaa ggtcctccca cctggaagcc ccaacagccc
100980aaccccttgg agaaaggggt tagtgcctgg tctgcaaatc aaggccttga gttctaactc
101040ctcctcactc tgtgaccttg ggcaaggcgc tgtccttctc tgggcctcag gagccttttc
101100tataaaaaga aatgatcgga ctgatctagc tcagagtgct atgatttcag gactacagtc
101160ccaaggttat caggctccct tagcatttgg gggtcttgta aggcatggag taaaaaaaaa
101220aaagcaatat cctaaggctg gagaagaggg aggggacaaa ggaaggggag gaaaggggag
101280gtagcaggga gccaaggacc aagaaggact gaggtacagt cattctgcat ccaaaggctt
101340aaattgtaag ggactggctt tactctggct gtttccggaa aggcaggccc agccagccct
101400cccgtctctc tctctgacag ccaatctcac atgtgcctcc ctgggagcac ctgctctgag
101460ctgtatcagc ccccagcagg ccgctgatta ccactgagcc tggccacaga gcacgagatt
101520aggatgcagc aacacactgt gtgtgagatc acgtcccgaa ccttctgact catctgcaca
101580ggaaaccccc ccagtctccc ctccagtcag aagggacctg aaattccacc agtggcaata
101640ccaaagaaac ttcctattag ctaagcccct agggagtgat tggctgttgg ggcggggagg
101700gggggcggtg aggaggatga ggatgaagcc tgggcaacct ggatgtgagg ctgtgcaggg
101760gatgaggaca aggatccttg gggtgaagga agagaagagc aattttaggt tttgctaatt
101820ttgtaaccct ggctccaagc cagcccttac aggaagtcac cctggcctcc ggctcaattc
101880agcacgtgat agggaagcca catttatgca gagcagggaa cgaggtaagg aaatggaagt
101940ggggctgtgg tgaagtgggc aagtctagag agagtcccgc tgcctggggc tgttcctaac
102000agctgctggg agcgagctgc aggtgtggtg cctggcaggg tggccgggct gtctgactct
102060ggatttcact ccaagctagg ctgctgcctg aaggattcct cttacccacc tttgcctggg
102120ctggcctttg ggacttacat ggctatgagg cgtgccacgg tggtcttgaa ccggtcaaag
102180atgtagccag tggggaatgt catgaagttg ttcatgaagg accccagggt gaagatgagt
102240gagaacctct catcctgggc tttgcagtct ggagtagaaa aaaggtctcc catgcatccc
102300agccttcctg ccaaatgagc acacaggctg ggctcccctc cacctcagac agcttgtcgg
102360tcgcaaactt gtcccttaag ctgagttgaa atgtggctgc ccctaattac ccctcaggag
102420ctggtgcctc cctcccaggc acttcccaga tcaagtgggg tgagagctgc tgacccttcc
102480tctcatcata gaaagagggg tgggcagggg gcagagtcct tcctgctcct tgccaccacg
102540tgggagccag acttaacttc cttagaaaag tcatccctgc ccttaccagc ctgccctgtg
102600gcattgccaa tcggcccagc atctggtcca cacagatcct taaagtaatc ttcattcttg
102660aagacaaaca ctagtgaagg ccagccaaag aggacgccag caaagcccag gcattccagc
102720agcccagtca gcagtgtggc cacgtgcagg ggcaggccct ggcccgccat gagcagaagt
102780ggagtggatc ttcaaatccc actttgtcct cctggacgga tcacaggcgc cgtaagcctg
102840gcgtttgagc acttggaaaa ttcctctggc aagccaagcc cttcctttcc cgtagctctc
102900tggttgtttc aggcctgggc aaaaaccatc agcgggtgat tctctggatc ctgtagaata
102960aagatagagg ctgctggaag aggaggcctg cgggaaaggg aaaggtagac tagagttatt
103020tgtgaggtgc attaagaggc aggatgatca tggccgctgg cagcaaatgt ggggaataaa
103080tactccaata catcatctta ggcactgcat ttgagtaacc acgtggcaag tagagaggca
103140ggtcttgatg gccacctgga gtcacaggtg agataaacga cttacccaat agctccccgg
103200gagcaggtgg agaagcggag ctcctgcgct cgaattctga ataccgtccc ctaaaataat
103260gacagcaact caaccaggtg cagaggcagg gagatttcat acagaagaca caaactcccg
103320ctgccaagtt ggtgttatct tcagtttact gacaatgaaa caaaagctcc catggatttc
103380aggaacttgc ccaaggtcac agggctagtt tagtcacgac gcaggccatt ctactgccag
103440aaataccccc aactcccatg accctcgcct aggactcgca aacctggtcc ccgccgccct
103500tcctcgcatc aacttctacc aggaaagcct ccgggggccg ctccccgcca gcctccgcac
103560cccgctccag cctgcggcct gccctccccg cagaggagcc cgaggggcca ggccgcgctc
103620ggcgccccat ggcgcccgaa aggggaccct tcgccctacc cgcctgctcc gcgccggggc
103680tctccgcgcc ctttccgcac gggccaggtt cgcattcgcg cctctcgcag cccctcccag
103740tcccctgctc gcctccgccc cctcctgccc gcccggaagg ggctggggca gacctcccac
103800tctccatcac ttccttcttc ttttcccttg ctcacagcct cccgcgccct ttttacctct
103860ccctcttgaa acttctccct ctagaacccc ctagaacccc agcggtgtct ttccctccct
103920cctcgctgcc tttcagcctc ccagccccct tgcctctgcc tcccctaacc aagttagttg
103980aatgctgtta ctcgctcagg cccacctagg gaaaatgtca cacccagcac ccagaggaca
104040cacagacagc acatgagggc atagggacac acacactcta tttgtgcatt ttgccttgac
104100cgctgggttg gcagggaaca tatttttcct atttgctcac cagcttaacc gtctctccca
104160gtttcacact cccagagctg ccaaaaaaat cccaaccaca gaatcaggaa gccaagaacc
104220aggactgagg gcttttcaga aaccatcccc tggaggactg ccccatattt tcactcccaa
104280aaacccctta gatgactccc tgcctcaccc ccgcccccca ggttctgaaa gagccttccc
104340gccagactgc attgattaac cattcattgc cccatttttt attaatcaaa gacatatata
104400attgctcatc ggagcttgtg atcagcgtga ggccttacta agcagctgcc ttactatcct
104460tccagcccag agcacgtgag ctgacgtctt ctttggcctg tgtggccgtt tccttgccaa
104520aagctcagtt tggggagagc ttcttgcgta ttagatgcag tctgcagact cccaacccca
104580gctacctgga tcccctgagg gcccaggaac tccagctatt ccaagcccac tcctcttttt
104640tttaagagga agaaatagag gttacgatag gggacagcca gaactgagga ttttccagct
104700caccaccaaa gcacaaaaga taaaagtctg caaccaccct agtgacttga ctgaatggag
104760gaagggtggc tggggtcctg taccccaagc tactcactag ttatacaacc tgaggcaagc
104820tctttggctg ccccacctgt aagacgagga caatagtacc ttaattatag gaattgtcat
104880aaaagaagta taagatgggt gtatgaggtc cctgcatggc gcaggtgcta taggcagatt
104940gtagggtagt agattttcta gtctgcagtt atgtagacag agccagagaa gcagctctgg
105000ggaggaattt caaaggaact tgcccacggt cattctacaa agctgcagta ccttcccaac
105060tctgaaacgt atgctctcat caccccgtct taacaaacat ttggacatta gagaaaacaa
105120gtcttttctt aaaataacat tatttatggg agaaaatcca caaaaatata gcatcccagg
105180acaaacaggg cttaagatgc aagattttct attttactgc aagacacaaa gactctgaaa
105240ttaatgcatg ccctatcttc tgctctggca tacattttag tctcctgggg ggatcagtaa
105300gtgtggaagt agcaagggag aaacagaaaa aagtcaaagt aaagagacag attttagaat
105360gttaatctgc aggagcctgc cagaaagatc tagctcatgg gctatctgta catccaggac
105420tgaagcacgg gacacggggc aggtcgtcca gggttctgtc caccttatct tgttacctct
105480cttgactctt agagcctcca ctccacatct cccatcaatg tctgcagaag acgtggcctc
105540cactaacaca agtcttactg aactgatggg acaggaaatt agaatatcct ctgaaccatt
105600cccatgttct ttggttcgaa ttccagcagc tagaaaaggc agatgctatt ctgatcactc
105660tcctgcgtgg ctccaatgag gattaatgag taacatcaga gagagaagtg attataataa
105720ggtctgacgg tgcacccgat gtcttcatcc ttttctcttc gcctccttcc tcatcatctc
105780acaccttttt ttttttaatt gactgattgg ttcaacaaat acatgtggta cctcaggctc
105840tgtgccaagt gccgggattc gtagagaaga gattcagtgc ctgctctcaa ggggctcatt
105900ctcttgtggg agagacagac aaagaaaccc aagatttctg gagtgtggga atggtcttcc
105960aggcagatgc tagcacagca cattgaaagg cacggaacct caacaaaaca ataacattta
106020ggaaccagct agagcacagg gtggtgaaga aagtggaaag atttgaggcc agcgtcgcca
106080tctaagtgag ggcattaaga attcagccca catcaatcaa tcatgtccta ttgatttcac
106140cccttaatat ctctcctatc tatccgtggc cactgctcta tgcagacact catcatctct
106200cacagaggca tcatctgctt ccaagccatc gccattctcc tgcaagagtt tatttccatg
106260gttcccactg gatggcttca cttaactgct caaaaccctt ctgaggtcca gtcaactggc
106320tggtaaggac cagtccaggg tctggggatg ccagccatga gacattgctt tgaggggaag
106380agggagcata gaactggatc tcctgcatcc tactgcccaa gtaccaatgc tggaggtggt
106440tttccttccc atcatcagca agtctggata tccaggatcc accctatgga tgtttttatg
106500gacagagtgg gaagatggat atgtttaggt tagggaaaga gggtttgcca aagagggcag
106560tataagtgag ctgcactcca tcattcccct ggcacaaaca atggctagta tcctctagtc
106620ctcaagagca ccaccttcca atgcagtccc tgcctgtcca cagacctctc tcctcaaact
106680tcctctgaac aacctcagcg agggcaattg ccactctctg ggcagagtcc agatattctc
106740tcctaccctc tgacatcact ttctaaattt gtatatgtag ataaactctg agccattcac
106800ataaagggct ttgatttcgg atacgccaaa cacataaaca aacaaacata agctttcctt
106860tcacaatggg ctcatgtaaa ttaaaatgtt tggttttcca cctacggtct tgaaaggggt
106920ttctacagcc tgttttggaa gtcagaaagc aaaaggtaaa tgcaaacatc atttcacctg
106980cagagaaaat tctaatcctc ttgaggcagt gccaaaaata atacaagcac actgctatcg
107040agccaattac tggtatctct gagcttccgt ctcctcatct ataaaattgg aatcgagctg
107100tatggattaa agataatgta tgaaaactgc ccaattagta cactattaat aaatagcagc
107160tactgttgtt aacaaatatt attgacttac tggaaaacaa agaggaaata aagtcacatt
107220tagggagaat ttcaaagtgt tcctaaccta aaaaagaaat aaattagggg gaaaaccact
107280aagtaatggg tgagctcagt ttaccttgct taagaagtcc caccctagag aactgatctc
107340tagatgacac ccaaatgcac tcagtacaac cccccaagac tgtctgggct taaggcaggg
107400gcttggattg tcctgtaagc tgtgggaagt ctgttcatga gccacagtag acaggaaggg
107460gatggagtct tagagctggc tcttcagggt atctcctagt gtgttcaaag cagttctcag
107520gagggtgggg aactactaca tagccaagta aatatgaggc ctccttgctc tggggagacc
107580tttctcttta acagaggtga atctgaaagg atacccaaag aggcactgga gggtgggggc
107640cactctggcc cctcagagca gccagctcag cttcagtgga tgctagaggc agcagaggat
107700cagcctggat cagcctccct ttcaccatgc agaaaacaga gctcccccac cagaccagat
107760actggaagca ctgggccagg cctaagagaa agcagagccc caagccccac cacaccaggg
107820cttatgaggc tactgctacc caccctccca agccccagct ccacttctat gttcatcaag
107880caactgttta ctggtaactg cacttcccga tagactttgc tagaaaggaa tgcctcagtg
107940cactgacaat atctaaacct gcaactctaa ggactaggct ggggaacact gtgtcaacat
108000ggaggcacgt cctacccctg agaagaaaaa taaggaatat caataatacc tgctgggcac
108060tgagcactga ctatgtatct gattcgaagc gctttttgct taatctgtca ctgaatctca
108120cgataggtgt tgttattagc atctattatc tgggccaact gaggcctaga gggacgaagc
108180aactccccca acatcaccag gtagcagtgt caggactggg atagaaacct gatgctctga
108240ctgaaactaa tgcttttttt tttttttttt tttttttttt tgagacagca tctcactctg
108300tcaccaaggc tggagtgaaa tggtgtgatc tcagttcact gcagcctcca cttcccaggt
108360tcaagtgatt ctcctgcctc agcctcccga gtagctggga ttacaggcgt gcaccaccat
108420gcccagctaa tttttttgca ttttttgtag agatggggtt tcgccatgtt ggccagactg
108480gtctcaaact cctgggctca agtgttctgc ctgccttggc cccacaaagt gctaggatta
108540caggcgtgag ccaccatgcc cagccagctg atgctcttaa tctgtgccct acccagcctt
108600cctgggaggc ttcccaagag ctacacagag catgagttct ggaatcgggt tgatgggggt
108660accagttatt actaatagga atgaagatgg gtaattcttt cagacagcac ccttgattaa
108720aacaagagag tagtgctgcc tctctgtgat tctgtgtctc cctgccctgc tcacacagac
108780accacaccca cccacacgca tgatcatgaa aagaggaaat ggatccagga gaaggagacg
108840actcctgagt gaaaacaacg gggtttttca cattgagagc tttgcccaac accccaaaga
108900tgaaaagagc aggaaactgc tggggccgat tgaacactgg acttttgttg tggaaaaagg
108960caaagggaag ccggaagaga ctggaacagt ttccatggtg ctggaggatg gggaagtggg
109020tagggattag ctggagggag aaggagaagc tggggtggga ggggaacctc cacttgccag
109080gagagcacat gtaggatggg aaccccagat gatactcaag gcatggcatt agaccagaag
109140caagtctgtg gtgaaattag ggaaggctcc actgcggact gtagacagag cactggacaa
109200ggaagtggga gacccagggt ccagtcctgg ctctggagcc ccctgggtgg gctgccccgg
109260gcacctttct ctcttggggc ctccattcct acctctgtga agcgagtgct gaacctctct
109320tagccctgac ttgctgaaat gctgggactc tgtacagagg ctgacattaa gcagggatct
109380gtcgtggggt gctgcaatgt tcctccagat gctgcacggg agagggcaga aaaggcctat
109440atggtgagtc cgccctggga gcctctgctt ggaagctgaa gtggcctgag agtgactcag
109500aaaccacgga agttcccggg gctgatgggt tcttatagat tgtacatgca gctctcctcg
109560tgggctgcaa aaccgcaaga tgggctgtga ccactctcaa ggaaagagcc ctatctgcaa
109620aaagcattct gccctccagg tcttaaagca aacacagact caatccttat tccttttaag
109680acaaaattgc ctcaggggca tcagggaggc agcaggcctc aaatgtgtgc ctttctagaa
109740ttctcaatga aagcaccctt ttgggtatta ataatgacaa cagtaatgac agtcatttac
109800tgagtgctgc ttttgggaca ggcattgggc taagagctat atgtaatata tattattatt
109860tgatgcccac agccacccca taaggaggcg gaggtactat cattatgcca actttaaaga
109920tgaagaaact gaggcctcaa gagatgaagt aacttggcca agtcactcag ccagtaaatg
109980ggaagagata gacttcccag tatccagagc ccatgttttc accattatgc tgaagtacct
110040cttttcctgt gccaatgtga tctgcctcca ggaatcctgt cttgatgttc ccttccccat
110100acagaagtcc tctctgtgtc ctcttcagcc tgatagtata tcttttcata ccattctttg
110160gacatctctg ttatactact ccaatggtgt tccctcccct acccctccct gggagcttag
110220ttgttgtgat taagtatagg ggaaatgacc cacactaaac aaactcataa gagactgatt
110280gataaacctg aaatgcaatt tattaattaa cactgagaaa tgaaaccacc cagcagatgg
110340gaatcctaag gctgactggt cagcacaatc tctttcagga aggacaggct tttgggaaag
110400gaaatcaata ccagaaggtt ctttgttgag tacaaagtca gagggaaggg agttgatgga
110460ttgacacata ggtgaagctt gacatacctc tataaagcct ccatcctgcc aaggatcaga
110520atatccaagg cagggagcca tctgggtgtc ctctcctttg gacagtgctg gatttttctg
110580gatcctatga agatcttacc tttctggctg catttatcat gattgtggaa ggctttttgt
110640ttccttgttt gcttagatta atttctgcgt atttaataga actgaaaggc aatttcccat
110700tgagacccac tgaagaggaa taatcaatac atactagttg tgttgccctt tgcagagaat
110760tcacttctgt gttgtcactg tatcctcatg cttccttata atggagggac agagatggta
110820aaaacatgga cttggaagcc agaccgtctg ggtttgaatc ctggctctgt tacttataag
110880ctctgcaacc tcgggcagat tacctaagtc agtttcccct tctctgaatt ggggatataa
110940tagcacccac ctcaacatct gtcaagagga ttcaatgagg gaatacacat aaagtgctca
111000gaacagtgtc tgccatctgg taagcagtcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
111060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
111120nnnnnnnnnc taatttttgt atttttggta gagacagggt tttgccatgt tggccaagct
111180ggtctcaaac tcccgacctc aggtgatcca cccgcctcgg cctcccaaag tgctgggatt
111240acaggcatga gccaccacgc caggccccac acacctttta aacaaccaga tttcattcat
111300cgggaagtgc ctgtggggct ggtgtggaca tgtgggtgaa ggtggcactg ggagaagtta
111360ggattctcca tgacctctgt tactcatatt cccacactcc tcaaattagc ctgagtctcg
111420aggacagtct gatggctggg caaaccctgc ggcaaaccat tccccagccc tgccctctca
111480accagagtcc ttccgataca tgattctggg cagctgttgt tacccgtgtc ctccatgttc
111540ttccagagat atccatgcat gcatcggcat atgtgtataa ttattatatc tatatttcat
111600cccacaagct tttgacatca atagtagcat attattaaat tgttctgtac attattttat
111660taacttggta tctctggtac tgctcaatat aagaacatat agacctggcc aggcacagtg
111720gctcacatct gtaatcccag cattttggga ggctgagatg ggtggatcac ttgaggtcag
111780gagttcgaaa ccagcctggc catcatggtg aaacccccat ttctactaaa aatacaaaaa
111840ttagccaggt gtggtggcag gcgcctgtaa tcccagctac ttgggaggct gaggcaggag
111900aattgcttga acctgggagg cagatgttgc agtaagccga gatcacgcca ctgcactcca
111960gggtaggcaa caaagcgaga ctctgtctca ataaaaaaaa aaaaggaatg tatagacctt
112020ctttattctt tttgatggct gtaggtggat gttctaaaat ttgtgtaacc aatctcctat
112080tgataatatt taagttatgt cttcagcatc atatgaaact tacaaacaag gttgcattga
112140ctatccatct gtaaatgtct ttttgaacat ttctagaata attgcaggat aaactcctaa
112200aatgagaatt tctgggtcaa agaggatatg cattttacat ttaatagata tttgtcaaat
112260tgtcttccaa agtggtcgta ccaattaaca ccccgacctg taatgaatga gagtgccttt
112320tttccccaca ccctggagag atgaaaaatt tatgggccca ctttggagtg catggtggag
112380gaagctgttg gccgttatat aaccctcgtc attaataagc ctgggggtgg ggggggagaa
112440agagaggtta gttagtgggt gcaaacatac aattagatag aagtaataag ttctaatgtt
112500cgatagcaga ggagggtgac tatagttaac aacaatgtat tgtatatttc aaaatagcta
112560gaatggagga cttaaaatat tccaacacat agaaataata aatgcttgtc tgcggccatg
112620ccaccctgaa tgtgccagat cttgtttgtt cttggaagct aagcagggtt gaacctggtt
112680agtatttgga tgggagaaat gataaatgct tgaggtgata gatatcctaa ataccctgtc
112740gaacattata cattctatat atgtaacaaa atatcacacg tatcccataa atatgtacaa
112800atataatgta tcagtaaaga gagggctggg cacggtggct cacatctgta atcccagcaa
112860tttggcaggc cagggtggga ggatagcttg aggccaggag ttcaagatca gcctgggcaa
112920catagcgaga ctctgtctcc acaaaaaata aaaataaaaa cgaattagcc aggcgtggtg
112980atgcatgctt atagtcccag ctacttggga ggctgatgca ggaggattgc ttgagcccag
113040gagtttgagg ctgcagtgag cctacgactg caccactgca ctctccagcc taggcaacag
113100aggaagacca tgtttctaaa agaaataaat taaataaaat aaataaaaat aaaaagactg
113160aaaagcagag tggtaagaga aaggactttg gggctcaaca gtactagcct tgaaccctgg
113220ctgttactta cccatcgtgt gataagcaaa tgccttaacc cctgtgtgcc tcactttctt
113280aacatataaa atagaagtaa aaatcatacc cacttcaagg gtcattataa aaagccaata
113340gagataatgt atataaagct tctggaataa tgcctggcac acagtaggag tttaataact
113400ggaaattcat tgttgtagtg ggcagccttc tgaatctgtg tcctctttgt ccactaatgg
113460ctttgatctg gatttggctc aggcaagacc tggggaaggg cagagactga gggcaactgg
113520aggtataggg tggtctgagc ttccccagca gagtgaggct gggaaaggtc tgggagacag
113580accaggcagg tgctgataag accggaatgg gaggctggag cataaggcag ttcagttttt
113640cccaaagggg ggtgtacaaa acgatctcgt atgactcctt tatactgtta atgttttcat
113700tttatcgcgc actgaaaaac aaaaccaaca tatttaatga atgattccaa ggggattctt
113760gcttttacaa aaaatgctaa agtaggcatt cacatgttta aaaattgagt tgatttaaat
113820tttaaaatta ctaagtcata gtacataatg tgtgagccac agctatcccc aaaatcatga
113880tagcgataca ttaatgactg aagttcttta aacatcaaca tacaatgcca attccagaat
113940tcagctcaaa ttctgcaatt acacaggctg gggttgaaac ccagcttttt tgctaactgt
114000gtaaaattag gcaggagggc taacctcgct gaatctcaga tgtctagtct gtaaattgaa
114060gataatgttt gtttttatct cacagagttg ttgtgaagat tcaataaaat cacaacatgt
114120gaggatgatc tggctgtgac acctgtcacc ccactgatct ccagagttga ttcggctgat
114180caggctggct gggcaggtgt cccctttctc cctcaccact ccgcatgcat tcctcccgaa
114240actgcacact tggtcaaaga ggaagacctt tcctgataga ggaggaccat tcttcagtca
114300agggtatatg agcacctgtt ctgtcctgcc agaatctccg aaggagctct cagtaaaatc
114360acaagatttt attgtgcatg gtagcatgag cctgtaatcc cagctactca ggaggccgag
114420ctaggaggac tgcttgagcc caggagtttg agaccagcct gcgcaacata gtgagaccct
114480gtctcaaaaa aaagaaagaa agaaagaaaa gaataataat agtaataaat cacctgtgca
114540acgtgctcac ttctctcttt ggaatgtagt aagtgtacct aataaatgtg atcattgtaa
114600tcatcacagt gagcacaggc taaagcatct tgactttatt ctataagcaa taaaagagga
114660tttgttttta cagaactcat tatgttgtga aaataatttt ccaacattaa caaagaacat
114720tcttcaagta aaaggaaaac cacccatcat tctcccaacc ttcaataatt ttcaattttg
114780catattctcc agactttgtc aacatgaata cttactttac atggtcgcaa tcagtgttca
114840tgcaaattct tttatcctga cttttataaa caaatatgat gttataaacc ggtctccatg
114900tttctgcata ttctttataa ttatcatttt gtggctgcat aatattgcat tgactatgtt
114960aactgcagtt ttcttaacca tttcactgtc tggggaaatg gaggataatg ccagggtcat
115020gcctggagct tttttttgtc tattgcatta tattcttaag atcaaatccc agcagtgaga
115080ttagtcagtc aaaaagtaat aatattttca aggctcttgt tatattttac tagattgttt
115140tccagagttt tgcacactgc tcccagagat gtaggaacac agacgtcatc caaccttgcc
115200agtgctgggt gatggtgttt ataaacttct gctaatttaa taagtatgaa atgctatcct
115260cacacggctt tcatttctat ctctttgatc attaacaggt tgaactattt tccaagtatt
115320tgtttactct ctgcataccc tcttgggtga agtagtcatc cacttccttc acctgtttat
115380ctgttgaagt cttgaggctt gttttataaa tgtgagcgag cacttcagag tcaatagaca
115440ttaattgctt ccagccagat ttggccactg aggctcctga gcaggggaat gcatgatcaa
115500aactacaccc tggacagatt aaattaattg gagaaaatgg gctgagaggc agagatatgt
115560gtcactggcc tactgtgttt gatcctatag tgggggcctg aactggggca acggcctgag
115620tcccccacta ccagtagcag gaggctccat gtgtccccca tattagagct tgcggcactt
115680ccatttgccc cacctcctac aataccccac atacatgtac tcactctccc ttgcaaatct
115740agtggcttca acccacagaa tttaagggga aaggaattgt tctgtcctgt tcacttactg
115800cagaaatgag aaaagcgttg ttcacatggg atcacctaat gaagggatgc catccccaac
115860ggtgcctata aggaaatggg ggagggttgg agagttgtgc aaaatgcaac agggaatcat
115920cagagtctct tgccccatga tagagggttc tcaaattaag agagtctaca gcaactaatc
115980tcacggccac tctaggcagg gcttcccaat gcttccccaa ccccacctcc atcctagact
116040ttacccactc tgctgaacac agatgttacc catagcacct tgcaccatga ttgtttgatt
116100agcacctccc acagtagact gtgtttctga taggtcagca acatttgctg agcacctact
116160ctgcagggct gtgccaggtg cacaaaataa acaaagccaa agacaacatg gaccctgaac
116220tcagcaagtt cagagtcaag tgggataggg aggctctctt cactggaagg taactccaag
116280aaacaatggg actcaacttt ctaaccaaga gaactccagg gagctaaaat tctgacttct
116340ggttaagact ggtgtggagc ttcattaaag aagaaaagat tcacccagac ttgagttcat
116400agcctggctt tgcagctttt aagtcatgta acctttgatg aagttatgtg acctctccac
116460ccagctgccc ctaacacctt gcaggggcag ggctggagtg caaagggagg cactggtacc
116520acagcctggg aggcaccacc ccactagtgc aagccgggca acctctgccc ccaaggcatc
116580cctagcctcc caactgcaag catcaatctt gcacttggaa aggaacctca cctttgaaat
116640ctaggttcaa atttagaatg atccagctcc ttgaagttct atacagaaat acagccagca
116700gccaggcccg gtggctcacg cctgtaatcc cagcactttg ggaggctgag gtgggtggat
116760cacttgagga cagaagttcg agaccagcct gaccaacatg gtgaaacccc gtttctacta
116820aaaatacaaa attagccagg catggtggta cacgcctgta atcccagcta cttgggaggc
116880tgaggcagga gaattgcttg aacccaggag gcagaggttg aagtgagcca agatcgtgcc
116940attgcactcc agcctgggca acaagagcga aactccatct caaaaaaaaa caaaacacac
117000gcagcttccc tccacttccc aaccacagct ccatctcaga caacaagggg cctcatgtcc
117060atgcacatga atatccaacc aacatgtcta aggcccaacc acaccctctc caaacatctg
117120ccccttggcc accctttggc catgggttca tgcactggca gaaaggtagt tcagagaaga
117180agcccacaaa gggccgggaa gtccacttgg gctttttgag attccagggt ccaggataac
117240ctaagtgtgg tctagaagag agatgcagct tctgggaggc acattccttg gtcttaggga
117300cttcttgccc ccatggaggg aaactggcta gatgagggcc aaagcagagc cctctaaagc
117360acagggctca gggaaggact ctttttgacc agatctaaga gcagcactac ctctctgagc
117420ctgtttctcc atctgtaaga aggggacatt aatagactct ccccgctaga gttactctac
117480atcagccagc acacgtaagt tcatgacatg aagcaagggc ttaatatata cccgttgtac
117540tataaataat aggccaggcg tggtggctca cacttgtaac cccagcactt tgggaggccg
117600aggaggatgg atcacaaggt caggagtttg agatcagcct ggtgaaacct catctctacc
117660aaaaatacaa aaattggccg ggtgtggtgg cgtgcacctg tagtcccagc tacttgggag
117720gctgaggcag gagaattgct tgaacccggg aggcagagtt tgcagtgagc caagatctca
117780ccattgcatt ccagcctggg tgacagagca agactgcatc tcaaaataaa taaatacaca
117840catacataca tacatacata catacataca tacatacata catacaatac atggacaggg
117900accctaaaaa tgagacaggg aaagagaaaa acatgttctg acaaccttgc cctttatact
117960aatttaggtt ttcttgcctg ttttagaaag ggcctggaca ggagccctgt tcccctcagg
118020ccaggcagaa caaggtgtgg aactcactgt ggaagggttc tgggtgacaa gtgcagcccc
118080gtccctccac ctcccagcac agtaggcagc acgtgtctcc attgactggc tcaggagcag
118140gcctggtgac cagtgggaga gctgaggagc ccagggtggg gtctgaagga atccctagaa
118200aatctgattt tcccccaggg cccacatcac gtgcccagag ctgggaaagt ggaggcagca
118260tgggatctag ctgagaggct ccatttttgg tagcttctag tttgggagtc acagagacac
118320ctggatgata cgaagatgta gctttgcagg actctctaga acatggagtc caagatattc
118380ccttcaatga tgggacactg aagcccacag aggagaggtc tgtcccagtt actcagccat
118440tcggaggcag agaccaggct agaactcagg acttttaatt tggaccagga ttccttttac
118500cacagtgggc agccctagca agtgccaggt agggtggaac tgtgaaggtc atccgagggg
118560tagtacacgt gggtaggaag tcatatctaa gaactgaccc ccagacctgg ctctgccact
118620cactccttat gagaccacag gtgctgggtg cagtggttca cacctgcaat cccagcactt
118680tgggaggcca aggcaggcag attgcttgat tccaggagtt cgagaccagc ctgggaaaca
118740tagtgagacc cccacctcta ccaaaattag ccaggcgtgg tggtgtctgc ctgtagtccc
118800agctacttgg gaggctgagg tgggaggact gcttgagcct gggaggcgga ggttgcggtg
118860agccaggatc atgccactgc acaccagcct ggatgacaga gtgagacaga atgacacact
118920gtctcaaaat aaataaataa atgacagcag atcatcattt ttctttctgc ctctagactg
118980caatgcctat ttctccaggt agtcactagg ataaaagtaa aaataatatt atcagcattt
119040accaaataca gggtcagcta ctctgttatg ttctttcatg ctttgtttct tttaagcctc
119100aaacaactct atgagctggg aacaagtatc gtccttcttc ctcccatctt atttatttat
119160ttatctatgt atttatctat ctattcattt atttatttat tttgagacaa ggtccttcta
119220agtcaccagg gatggcctca aacttgagct aggaactagt gtcaccaccc cccaatttct
119280tttattgatt gattgattga ttgattgact ggttaatttt gaggcagggg tctcgctaag
119340tcgacagggc tggtcttgaa ctcctagtct taagcaatcc gcccgcctca gcctcccaaa
119400ttgctgggat taccaacacg agccaccatg cccagcccct cccatcttct gaataggaaa
119460actggggttt gaaaaggtaa gcgacttgcc caaggtcccc tccctagcta gagagcttca
119520gagccagggc acaaacccat caaagcctgt gctctcgccc attgagccac cggacctcgt
119580acactaaccg ccaagtgttc tacacagtga aggtgacaaa gaggtgaagg gaagagccag
119640ggaggttctg tggactcact cggtgggtat gcccagaggg aagggggatc ttgggtggca
119700cattgagagt agctgcgctg ttagtaagtg agaactcgga agtccagact catccagtct
119760gtgccaataa acccctcctt ctacctggtc tcctttccaa agccagctgt tctccagaca
119820atggggtggg cgggggcggg tgtcctcctc cttctcaggg aaaatccgac gctgagccca
119880tctccagaga tcttggcttc ccgtggggct gcagatccac ctagagccac cagagggcgg
119940gccagcactg cggccaaggc ttgaagaccc agcacaccaa agcccggcca agcctccagc
120000ccagtgtcca agagtccagc cagaggccga gtcctcgatc tcaaaatgtc taactgcaga
120060agcccaactc atgttcaggc atgatgtgtc tgattctact gggacaatca ttgccaccaa
120120agaattactg ccaaaatagt aacgacatta gctacctacc acccctccac acaccaacac
120180acctcatttt accaagcact ttctcatgcc tggggtgcct ggagacttaa tgcagcctcg
120240cgatgaccgg gtagcctcac cgtacagatg aggaaactga ggcacaagga aggggagtac
120300attgtctagg gtcacctgga gaactctgat ctccagactc aaatttccaa ttcgtccccc
120360ctccccccaa ccctaaccgg agctaggtgg ggtggggaca gcaaatgtgg atggggggag
120420gtaagagggg tcagagtgct ctacagagaa gaccaaatgc attgtggcac ctactgtaaa
120480atgagaccag ccagccaccc acccaccagc cagccaccta aaagtcttca gtgggcacct
120540gctggaaaca cgacaatgga tgacacgagt tccctgccct caaaaagctg gtagtctagt
120600tgggggtgga gggggtgagt cagcagataa ttatgggaaa ccgtgacacc tgtataaggg
120660gcggggatga gcagaggggc tgcgactgcc tggagccagg gattcccgga cggggcttcc
120720ctttcctcgc agctcgtccc aggaggagga gctccccccc agcttcgggg ttccgcctgc
120780cttgggggcc cggggtcccc tcccacccct ccccgaagag cgcgggcccc gggaaccgat
120840gacagcacac ctgagtcagc ccgccgccca cccgcccctc agcgtctgtc tccgcatctt
120900gtgatatttc gctccccggg agccagcccc actgcgctcc ggaggcagct cggcaaacaa
120960acccagcgac agattgtgcc gcggctcatt ccggggaagg acgccaaacc ccaccctgct
121020acccccaaca ctccctcccc gccgccgcct ccaggccctc cccccaggcg caggccctag
121080tcggggtggg tcctggggaa acgcagggtc ctgtcctgcc tcctggaaat agggggagcc
121140ctgggtgagg gaagacggga gccccagaga cttttctttc tgtttctacc tgatccgaaa
121200acgagagggg cgggaaagga aatttagggg cacagagagg agctgggggg ccgagaaggt
121260ccgaaaatgg aaccagcagg gggcacccga gagccgaggt gcccacgggc cgggagcctg
121320ggaatgaaac tggggaagag ggggagagaa agggaggcag agacaccgag acacacagag
121380acgagagaca gagacgcagg gagccccgcg gggaggagga gagagacgaa gacacagaga
121440gacagtgaga aagacagaag accgggcagg gaaacagacg agtagagaca gaaaaggtcc
121500gagagagagt gagggaggga gggaacagag agacagagac cacgaaatat gagtaagagt
121560cgggggagaa aaccagagaa atcgaatgag aacgcgagaa gaacgagaga ccgtggaggg
121620agcagagaat gaatgggaag aataagacca acatttatca agagccgact gtatgccagg
121680cactgcattg gaccctggca cggataggaa aggaggagcc gcggcgcggg cagcggggcg
121740aggggcttct gtgctcgcgg gagcggcagc ccagggggct cagcagcccc ggcaccgccg
121800cacctgcggc tccagcagcc ccaaccccgc cagcgctgcc tggccaccgt acccgaagcg
121860gctcccccga gggccccgag cctatcctac gccggggcgg ctccgcggac gcgccgggcc
121920gagtcaagac ctgggtcaac cgccctgcag cctttgtagg gaagtgccta ggtgatgggt
121980ctgctgatac cgcctgtgac caggccatga agggccagag gggctccagt gagaccataa
122040tccgcccctc tttaaaaggg ggtagaggaa gttcacgcga agccaacagt cttctcccca
122100gctttgggtc ctctcctgca cccccgcggg agataaggtc tcccctcccg gacacatcat
122160acatacacaa aaaaacgcac acactcgcac gcgcgcccat ctcgcacccg cttgtaaatg
122220cactaagggg catacacaca ccgggcacat atttctttcc acccatcccc aagatcgcaa
122280gcgcaaaacc tcgcacagcc tcacgtttcc caccagctca gacatgcacg ctggcggact
122340ttcagcggct cacccgtgtg cacactcacg tgcccccccc cccgcttccc caagcccgta
122400caaagggtaa cgggcaagca tcctgagtca cacctgcaca agcatccttg cgcgcacgtg
122460cacgctcata tgcactcgat cttgcacgca caaactcttg catatactat tcttatagtc
122520gcacactggg cttgaggtct gggagtggaa ggaaaagtgg aatcttggag ctgtcccagg
122580ggacagaaat gctggaggct gggacactgg cgcgagggac gcggctgggg gcgggggagg
122640gggtgaccca gaagctcatc ttctcctgga aagttgggag gggggaacag gacaagtcca
122700cggcgttcct ctaaactacc gcattccccc aagaagggat ttctctagaa gagtggcgcc
122760gcgaggacga tcgaacacag tcctccgggt cgcttaagcg ggggggaggg gggcggggtg
122820gagggggtta gaaagccgct cccgcctcct agtggtcgag aaagggttaa gtcggcaagc
122880cagcaaacga gggaggagcc agcgagtgcg ggaaggagtg ggggtggttg ggaagagctt
122940cctcgctgtc cccactctcc ctcggctagc agcctgggca cacggacaga cggactgacg
123000gactctcgag cggacagcgc agctagcggg gcgcgggcgc tgggcgtcga cggccagccc
123060cagccttccc cgccccgtcg cgccccgccc cgtcccgtcg gggccgatgg ctcctcccga
123120ggcccgcagc ccgggcggcg cagggtagag cgccgcggcc cggccacgca gcccggggac
123180tcccgggccc tcccggagcc ccgcggggtc cccgccgtgc atccggcggg ctcagggagc
123240gagtgggagc gccctccccc cgctgccccc tcccccgagc atcgagacaa gatgctgccc
123300gggctcaggc gcctgctgca aggtaagaac gccagcggcg ggagagcgga gggcatcctg
123360gggagagaag cagggcgtcc cctctttcag ggattgaggg tggggcagtt ggggaggtgg
123420ggtaacctgg ggaaggggaa aagctcagcg ctggggccgc gcccccgccg ccagggctgt
123480tctcagcagg agggcacttg gctgggagcc cgcgggcgcg tgcgaggagc tcgtgaccga
123540ggtgggacgc agggggcagg tggacccggc ccggagcggg gagggaggct caggttccgc
123600tgtccccgct ccacctgctc cgggggacgc tgaggactcg ggccggctgg ggaagcgccg
123660actcagcaac tcctcctgcc cggtgcctca gcactttctg gccacctggg aagacaggag
123720atgtgggtag ggggctgtct ggggaggtag gaggcgcaga gggaaatcca agtggccctc
123780tctggtagga gagatggagg gcgctagaaa gaggatagtt ctactgattg agtgacagat
123840aagggtgtgg gccagagact gggggtgggg tggggagggg tcagggggag agggatagga
123900aggagaactc aaagatggag aaagtggtga gggaagctca aaggaggagg gagatggagc
123960gggggagggg gagaaggaat aaaggttaga tgggaaaagc gtggagggaa gtgggaccca
124020ggtgaagacc aaggaagagg gaaggagagg aaagaccaga tcaggggagg gatgggaaga
124080agactatgga cagggaccca gaatcctggg atggaggtag cgggaaagag aatcaggact
124140gggaccctgg ggactggaat ggaaaaggag aatggaaaga tcagaaacca gagaaggatg
124200gggatggtga ctagagaagg ggtatcagga accggcgaag agggttggag acagggaacc
124260atggatggga gaggggctgg agaggaggga agaggaggag gaagagaaag gctgagagag
124320agggactggg gattgggggt gctgcccagg gatgagacaa agaggcttct ggtaaccact
124380tccacgtggg aagccctcca ttcccaaagc gcctgcctgc cacatttctt ctctcaggga
124440gtggctggtg ggccagatgg ggggtgcttt gagctcaggg ccctgggggt ggctgtgagg
124500gacagagggt gaggactttg gaaggggagt gacagcctcc gagggtgggc aaacaggctg
124560gctcctgtgc tgccatttat ttatccggcc cggacgttgg attctgcagc cgctgccgcc
124620accacggtgg ctgcttattt tggggtgtta cattctggca gagtgagaag ctgtttgcag
124680cagctctaaa cctccgtcac ccgcgtcagt gcctccccag gcccctgcgt cactggcatc
124740accaccacct ccatcccact cctcagctcc cacctcctca gcccctgccc cctcagcatc
124800tgcccgcagg ccccagccct tccctgaagc agcccgttgg gtgtggagcc cttgcttctc
124860gtctgggacc ctgtgcccct ccttccagag cgagaggcct ctgctgcctt tccagggagc
124920atcctttcct gggaccactc tgcaccagcg actctgccct gtgggtgggt agcctggatc
124980ctgcccccta ctttgggtcc agttttcttc tcctcaagtt ccttcttcta caggggcctc
125040cggcccaaag agtggcctgt gggctgagaa ctttgtttct gagccttggt actccaaggt
125100ttgatagcca gagtcctgga cagtggtccc tcagtgaaca gatacttttg gctctggaca
125160cttcagcctt ccgggatcaa taccatgttc tggcctctct tggctccctc ccctggtcag
125220ttctggccat atattctgga caggggtcat ctcttcttga ctcccacatg taatcactac
125280tctagaacaa ccgcaactgg aagcctagga ggtgaaagtt gcagagagag ctggagtccc
125340ttccttgcct tgaccctgaa tagccaaaca gactcagcat tgtggctggc ccagccctag
125400gcacctgggt gcaatttctc tcctgtcttt acctcaaggg cagtgtctca cacattcagg
125460cgtggtttct gcggaggatg tggccacctc ttaaagaaag atcagagtgt ctctctgaca
125520tgggcttgat gtccctcttt tccaatctgg gttccacctt gtactagctg catgacctga
125580ggccactgtg tcatgtttct ggggctccct tccttcatct gcaaattggg ggccacaata
125640ttgacctcca ggggattatg tgtgttgtgt tcaatgtata aagaagttaa cctgtacaaa
125700tgcagtgcct aggacaaaat aggtgcttct tggtttcctc ctaccctgct gtactctccc
125760ctgcagctct agccatcccc tgctgacttt agaggagggg gtgagcagag agggtggggg
125820aggctgctac aaagggcttt cctctgtcca tgaagtagtg gagggatgaa atgaaggctt
125880ctgagaaaga caatgaaggc gagctgtaga gacctggtca ggaggcctgg ggtgctcaga
125940aactcacact tcccctcccc agccctcaat ggtgttacct atgatgtgag gggtcggctc
126000taggtggcca ccgaggtatc cccctttcca gctctgatac tctgtgcatc ttgccccagt
126060ctccaccggg aattcacaaa atgaaggcca ggagtggagc cgtggtcctc gggagagaca
126120ggaggcctgg gcctggaggg aaggagtggt ggtgctgagg aggagtgaga acagggggtg
126180gggaagggac gtggcaagaa agaaaagggc acacactggg cagggcaggg actgagggcg
126240ggggagagag ggaaaggcac agctctctag tcccccaacc ccccagtccc accacctctg
126300ccctggagtg ctcgctccag ccccagcagg cctggggcag tgaagcccag agccccctcc
126360cctcccctcc tccttgcctc cagtgagagc cgctgcgtga attatggatg agctccttgg
126420gttacagctg ctttgcacgg cagtggcaag ggccagaaat ggcaacagag tcactgttat
126480gcagcagctg ttatggagga gcccccagca ccgggtcgct cttcagagag cctgcaggga
126540ccactatcat gggctggggg aggtgagccc tggttggggg agacatggga acaagatgga
126600aggagagtgg ggaaagagaa gagaagtagt ctaatgtggg caggtgggga gcaggagagt
126660ctagggagag aaagaggagt aggcaccctt gccagctcct gcagagttta ccctcaaggc
126720cggaaggaac cctgatgcca ggggaatggg ccttgcctct gagattgcac atccttccct
126780ctgtctctcc tggggcagcg gtcagtccgg aggctggggg aaagctctgt aatcctccag
126840gggctagcgg ccatcagggc tcacactctg gtgagcttgt ggataagggg taggattaag
126900ggatcagaga aggatttggc ttcttttggt gtcaagtcct tagggaagtg gagatcagag
126960ggtgactctg acaggaaggg aagtgccctg gctgggcatc aagagacttt tctggccctt
127020tccctgccaa cactttgctg tgtgaccttg ggtaagtcgc ttgctctctc tgagctccag
127080tcatcacctc agtagaactg atgcttgaac cagaggaatc gaggggacct ttgcggcttt
127140gaaatctcca gttctaagcc ccaaacctca accctcatga aacccactca gggtccccac
127200tgtgcttcca cactccacct ctgcctggtt cagatgaggg gtaagagaca ttgctcctcc
127260accccacgtg ggtctaagaa actcgggagg agaaagtaat cgtgaaacgc cgcacggggg
127320aggggtgaga agggccgaga aacgcggagg tggtgtgaac gaatggaaca gcagccgctg
127380tgtcactgag tattacatca cacccagcct acacacgcac ggggcccggc gctcacacac
127440acgcggagga cagccagcac gcaccgacgc agcaccgacg cagcgccagg aggggccggg
127500gacactcacg gtggggccca aaagcgagga gcagcacact gggagtgtgg atcttccacc
127560ccgcacctgt gtgctccccc ctctggagga ggaacaccag ggcagctggg atgccagcgc
127620cacactcggg gcctgtcagt cccatgcgtg cacacctggc tgagcagcac tgcatttggt
127680gagcacctgg ctcacgccac tacccaaaat cacagataca tacacacatt cacgcacacg
127740gcaacctcag gagcgtgaca caacacacac aaaaccacca ctaagcaagt gcaatttgca
127800gccttggaga ccccacactc aaaatcacca acccctcagt ctctcccagg gtctctgaac
127860cccaaggagc cccaggatgt cagagtgcag aaacaagtct tcctcccctc tgccttcaaa
127920agcctaggac gttgcttgaa gcagaaggtg ttcagtcact gtgtgcccag ggaatgactg
127980cctggctttg ggggtgcagg ctcccttttt ccccaggcaa aactgccaga agaaaatccc
128040aggagtcacc tggaaatcat aagaaagtgt agaggtcaag ctagttccgg cctagaactt
128100tatcagctat agtgacggca aaggccaggg atgatgggag gccctgcacc cctattaaaa
128160tatgagtaca gacacctgca ctccactctc tagcccccag gctctctggg cctgcttttc
128220catcagtatc ataataagga tggatcatat ccaaccttca aaagttactt tgggggaaaa
128280aaaaaaaaaa gctttggctg gatgcggtag cttatgcctg aaatcccaac actttgggag
128340gccaaggtgg gaggattgtt tgaggccagg agtttgagac cagactgagc aacatagcaa
128400gaccccatgc ctacaatttt tttttttttt tttttttttt tttgatacag agtctcgctg
128460tgtcacccag gctggagtgc agtggtgcga tctcggctca ctgcaagctc cgcctcccgg
128520gttcacacca ttatcgtgtc tcagcctccc aagtagctgg gactacaggc gcccgccacc
128580atgcccggct aaattttttt tttgtatttt tagtagagac ggggtttcac cgtgttagcc
128640aggatggtct cgatctcctg acctcgtgat ccacccgcct cagactccca aagtgctggg
128700attacaggcg tgagccaccg cgcccggcca aaaattttta aaaaattagc tgggtgcagt
128760ggcacgggcc tgtggtccca gctcctcagg aagctgaggc aggaggattg cttgagccca
128820agtgatccaa gctgcaataa gctgtgatcg taccactgca ctccagcctg ggcgatggag
128880caagaccctg tctccaaaag aaaaaaagaa agaagttttt aagtaactgc gaatgaggag
128940agcctggggt gtaaaatgca gattcccagg ctgtcccccc aggaattctg catagttcct
129000aggactggct ggtggcctca cttagagacc cgacccttaa ggcccctccc ggcacaaaga
129060ggctctgact ctgcaagggc gaaaagtaca ggaaagtaag ggcactgggc accagtgggc
129120tggcaagacc agaccccaga gtgagtccat ttcacacggg cctcagatct ccaaagggtc
129180ccaagttact tccagtcatt ctccaatggg gtgactttgc cccccagggg acatttggca
129240atgtctggag acattttggt tgtcacaact ggaggcaggg tgctgctggc atctagtggg
129300tagaagacag agatgctgct aaatgcctta tatagggctg cccccacaac gaggaactat
129360ccggcccaac tgtcaatact gaggcagaga aaccctgacg ttagtctttt gacattaatc
129420tctagacaag gtcaaacatg caatagtgaa aacaggaatg aagagatgat cattcttcaa
129480ccaatttgca gtgctttcta caatggcctt ttggcattat tttttaatat atgagaagcc
129540tcagaaagtg gaagtggcca ggccacttga ggctataacg ttgtcccctg agcccccaga
129600catgggagca ccagggctct aggcctttat ttttattttc tattttttcc cctgaaacag
129660ggtcttgttg tgttgcccag gctggagtgc aagggtgtga tcgttgctca ctacagcctc
129720aaactcctgg gttcaagcga tcctcctgcc tcagcctccc aagtagttgg gactacaggc
129780acatgccacc atgcctggct aatttttttt tttttcttgt aaagacaggg atctccctta
129840tgttgcccag gatagtctca aactcctggc ctcaagcaat cctcctgcct tggcctccca
129900aagtgctggg attacaggtg tgagccacca tattcagccg ggtctaggcc ttttaccaag
129960ttggggggct ggcccccagc tggcactcct gccctggaag cccacctagt aagttctgct
130020tcccctcccc acagctcccg cctcggcctg cctcctgctg atgctcctgg ccctgcccct
130080ggcggccccc agctgcccca tgctctgcac ctgctactca tccccgccca ccgtgagctg
130140ccaggccaac aacttctcct ctgtgccgct gtccctgcca cccagcactc agcgactctt
130200cctgcagaac aacctcatcc gcacgctgcg gccaggcacc tttgggtcca acctgctcac
130260cctgtggctc ttctccaaca acctctccac catctacccg ggcactttcc gccacttgca
130320agccctggag gagctggacc tcggtgacaa ccggcacctg cgctcgctgg agcccgacac
130380cttccagggc ctggagcggc tgcagtcgct gcatttgtac cgctgccagc tcagcagcct
130440gcccggcaac atcttccgag gcctggtcag cctgcagtac ctctacctcc aggagaacag
130500cctgctccac ctacaggtga gcctgccctg cccccaccct cagccccttt ctggtttcct
130560ctctctgtgg gcccctctgc tccccgaccc tggcgtgcgt ccctcctctc tccccaggcc
130620acccttcctg cctcagcatc tccatttctc tctgtctatg tctcttttct ctcttacatt
130680ctccaggggc tttacttttt cccttctgcc tctctacctg tttaggtccc ttgctgttcc
130740tctctctctc tctccctcta actccacaac cttcacctct ctgcctctgc ctgtctgtct
130800gtctatccct ttccatccat cactgcctct ctcactaact tgcctccccc atctgtcttc
130860tgcctcttct gtctgtctcc cttcacacac ccactccgca tacaccccca tgtctgtctg
130920cgtgtgtgta tctgtctctt tctgtgatct cacgtgtttg ccttcagggc actctgcctt
130980cccccagggt cccctgccca aaggcctttg cagctgtttt tctcacccac cctcaagtct
131040gcccacatca cggtgaagta gagagagaag gcagagccac agccactggc atcccacaga
131100aagttgcgct tctctccaat tcactgggca atgggacggg agaagcccac accccttcta
131160gattcccatt ttccaaacct gtcatctcaa tgcaggggaa gaaagaaaag ggtaaatctc
131220tgttatgcag ctggagaatg gatgctctga aaatggaagg aataccagta attgttattc
131280attgttatta ttattgatct aattattgtt tattgttgtt atgctgactg tttgacacgc
131340aaatcatccc actccatttc cccaggaagc aataacacac cctccaaacc accctgagag
131400aaaatcttcc cttggctaca gagcctccgg ctggaagggg gtgaaaatat ccaaattctg
131460ccctctccct acttgaacct ggaacgtgct tcctctgcct catccagggc tagtgcctaa
131520ctagttatca atctgctagt tggaaaatca ggtcagtgct gatgatgcta atgataataa
131580caatagccat aacaacctaa caaacatact gagcacccac tacgagctag atgctaagaa
131640tacagtagtg aacagaacag accaaacccc ctgccttcac agagatacca ttcccatgag
131700gagggaaaga agtaaaatgc acggtatatt ggaaaaatat gtcttatatt attcttattg
131760ttgcctaaat agtgacagta atagcagtag ccgccaccac ttagtgggta cacagggtca
131820gccacagtgc caagcacttt ataggtatcc actctgccat ttacaagcgt gtgacatttt
131880ttttttttac ctcctcagac ctcagttttc tcatctgtac aatggggtag caagagcacc
131940catctcctag ggattttgaa agcattaaat gcatgaataa tttgtaaagc acttagaata
132000gtgcttggca tacggtaagt gctatataaa tgcttgttaa aatactattt taaaaaaaga
132060aacgagcctt atttaacatt ggtttcagtg aagtggccca acttggactc catcctgaag
132120atgtgggtca acttcaagga ttatactaag gtcatgagtg agtcccagaa attgcacctc
132180acagtttatg aagtgcactc agccacctca tctcatttct acagcccagt tgggagatta
132240ttttcacctc cttgttaaca atggagaagc tgaggctggg ggccctgaag accctataga
132300gatatagtca cctccaatca taaatctttt caaccattgt cggtgtgacc ggaggcttat
132360gtcttctcac catcatgttg agcctcacaa caacctggtg atagggacag ttaggggcac
132420tagggacatg gaatgaatgt tcctgaggcc acacacccag gaagagctgg cgcttgaacc
132480tcatggtctg gctacaaggg gacagtactc tggagtacaa ttgagcaggc tcatttttga
132540aagcacacag tttggactca gcaagaccta ggttcaaatc ctggctccta tatatatgac
132600tttggacaaa ttacttaacc tctctcagtc tccatttcct catctctaaa atggcaatca
132660ggatagtact taataataat cttttttttt tgagacgacg tcccactcta tcgcccaggc
132720tggagtgcag tagtgcgatc tcggctcact gcaacctctg cctcccaggc tcaagtgatt
132780ttcctgcctc agcctcctga gtaactaaga ttacaggcat gtgtcactac acccagctat
132840tttttgtatt tttagtagag aagggtttca ccatgttggc caggctggtc ttgaactcct
132900gacctcaggt gatccactca cctcggcctc ccaaagtgct gggattacag gtgtgagcca
132960ccatgcccag ccaataataa tccttattta agaagttttg taaggattaa aatgtaaggc
133020atttagcaca aggattaaaa tgtaaggcat ttagcacata tgggcactat aataataatt
133080actactacta ctactactaa tactgagatc aaatactact acaaattgat catgcattta
133140atgctttcaa aatctcctta tcaatatata ttagttattt aggaggaatt tggagtcaga
133200gggcctgagc ttgaatcccc gatctactat tttctgactt atttaacttt aagcaggttg
133260ctaaccctct ctgaacctca cttactttat ctgcaaactg ggaataatga aaataatacc
133320ttccaccaag aatggctgta aataggaaac gagttagtgt atagaaagcc catagttcag
133380gctggtgtgg tggcccatgt ctgcaatccc agcacttcgg gaggccaagg tgggtggatc
133440acttgaagtc aggagttcga gaccagcctg gccaatatgg tgaaaccctg tctctactaa
133500aaatacaaaa attaggcagg cggggtggca ggtgtctgta atcccagcca ctagggaggc
133560taaggcagga gaatcacttg aacctgggag gtggaggttg cagtgagctg agatcgtgct
133620actatactcc agcctgggtg acagagcaag actctgtctc aaaaaaggaa aaaaaaaaaa
133680aaaagcccat agttcagtgc tgaagaaatc atgttattat gaccccatcc tccattgact
133740ctcaggccaa caacagcaat caggacctga ggtcagcaaa ggcttgggca gaggggacct
133800caggtggaca ttggggtctt ctgaaatggg aagtgtttgt tctctacgcc cctggcatga
133860atggtaccag gcatcatggg aaggaagcaa cttcacacct ggccttttat agaggagatg
133920gaaaacacag cctctgcctg tgaactgcct ggtagggctg ggctgggaga tgccacaggc
133980aggtgaggaa acatgggctg gggtgagatc cgcagggtgc aggtgtgacc caagatggag
134040ccaggcctgc cccaaagggg agctttggag gaaactccac cagaggacca cagcttttca
134100gaatggggaa gggccaggca ctgtgccagg tgagttcatt catcaacaga tatttactga
134160gtatctacca catgccaggc aatgttccag gtgccaggga ttcaggagag aacagaaaca
134220gtggccctgt tctcccagag catattccct actcaagtgt agccagatga taaagacact
134280tgttttcttt cttttttttt tttgagacga agtctcgctc tcttgctcag gctggagtgc
134340agtggcacga tctcggctca ctgcaacctc tgcctcccag gttcaagcga ttctcctgcc
134400tcagcctccc aagtagctgg gattacaggc atgtgctacc atgcctggct aatttttgta
134460tttttagtag agacggggtt tcaccatgtc ggccaggctg gtcttgaact cctgaccaca
134520ggtgatctgc ccaccttggc ctcccaaagt gttggattac aggtgtgagc caccgcaccc
134580gccgacactt gttttctctt tcagtcatta cagtggcctg catggttttt gtttgttttg
134640ttttgttttg tttttgtttt tgagacagtc tcactatgtc acccagctgg agtgcagtgg
134700cgcaatcttg gctcactgca gcctcacctc ctggggtcaa acaattcccc catcttagcc
134760tccccagtag ctggaactac agacatgtgc caccatgtcc agctaatttt tctattttat
134820agagacgggg tttcaccatg ttgcccaggc tggtctcaaa ctcctgaact taagcaatcc
134880acccgcctcg gcctcccaaa gtgctgggat tacaggcatg agccaccgta cacagctggc
134940ctgaatggtt taaaaatagt ctttatgctc aagcagatca gatctcagtt tgaattccag
135000ccacacctct aatttgctct atggctttgt gcaagttatt taaccactct gagcctcgat
135060ggacccatct gtgaaatggg gataacctgt accttggcga gcaggggttg tgaggattaa
135120aggagatact actgagctca cagcccaatg tctggtacaa agtgagtatc caatgaatgg
135180tagctatcca ttaacaccag ggaggacacc aactgaagct cagcaaaata aaagcacagt
135240ccaaggtcac ccagctagta aggaacatga cctagaattg gcccaggtct gtctgactcc
135300agagtgcagt tgttcagagg tctctggagt tggaagccac gttccactgc atattagctg
135360ttggacccta ggcgagtcac ttcacttctc tgaggctcca tctcgtaatc tctgaaatgg
135420agataataat agtatccacc tcatagggtt gtgacaatta agttactata taggatctgt
135480gtagcacaga gcttggcaca tggtaagagc tcaatcagtt acctgcttga caatgctgac
135540gccgatgatg acgatgatac ccatcctaga ctgatgagct ctgtaagcgg gggtgcctgg
135600cacagagtag acactcggta cagctctgtg gaatgaatga ggcacatccc agaactcacc
135660aattcataaa aatcagatgc agatgggatc ttaaagatca cctatcctaa gtcccttgtt
135720tcacagatga aaagacccag gcccagagag gtgcttggag ctgcgcaagg tcacacagcc
135780aagcagctca tttgattagt gtcagagcca agagctggga gtttggaggg aggcaaggtt
135840aagaacagga tgctgtcagg gaagcaggca gggatgctgt gttaagattc caaatggatg
135900cagagagctg tgaaccggcc agtggggagg caagggaaat gtggtttttg aaatggaaga
135960ggatgacttt agcagaggct ctcagcccag agggagggga gatagggagg ggagataggg
136020aggggcgggg ggagggctag ggctgtgaaa gtcaagagct tattaatgca tagagaacgg
136080ttttaacagt ggagagagga aggaccggat ttgaaagcta cattcaagga agtggcaacg
136140ggatttggca acagcttgga tggggggagg aggcaatgga ccccaaggca gaggctcaga
136200gaagggaggg gcaggacttt ttgcagagaa acaaaaggag aggagaggag gttagaatca
136260agaaattctg tgggccaaaa cctggggctg tgggtcaaag gcacctgaat tccctaggat
136320ctctggaact ttggtctact cttctgacct cccgaggtcc cccaaaatgt ggattacccc
136380tgctcactct cccccaaccc ccggcccctt atcgatcctc tgaccataca tctctgggtg
136440tgtcctactc ttgctgacac ttcataaaaa gaggaacccc atttaggtgt tttgagtggc
136500agggattcca agcctacccc ctggatgggc ctggaagaga acaagagcac caggccatgg
136560tgagtcaggc tgaggccagg gaggtgcaag gagccagctg gaggcctgag ccaggatttg
136620gggtggtggc agcagggggc ggagaatggt ggtgtcagag gcagccgaga aggttgaggg
136680ggacggatct caatgtggcc aagaggaggg ctcttggcac gctcagttcc tgtagcgaag
136740agggcggaag ccagatggga gggggcgaga acaggcagga gcacaggaag gtggaggctg
136800tgggtgtagg ctgggagtca atgccctccc ccaacctgag gcctccgacc aggctcctgg
136860gtggcaggca tggggaggaa agcgtctccc caggcagtga gggagggaga gccacagtca
136920gggaacaggc cccctgggtg aactggcctg agcagagtgg atgctcctgt tctgagaccc
136980agacctcctg gaacctgctg accacagtga tgccctgcac aagaggggag gacctcaagg
137040cagtgaggtc agggagctga agtcctgctt ccctctctgg caagccctta tctctttgag
137100ccccagtgct ctcctctaaa aaagtgagct gggctgatgg gtgccaaggc attagctccc
137160aagtcagctg atcatcagaa tcccctggtg agctggttat aatgcagagt ccaggaatcc
137220ccactggccg tgggccacac acacccgccg ccccccgctg ttaattctga accatagttc
137280caaggtcctt tctgcactaa tgtggcctga ttaggtgact ccctagcacc aggcaggtgg
137340gacagcgcct ctaaggggag tagtaatgca atgtggcttc cttcctctcc tcccctgccg
137400cctctggggg tggagctgat gcccctcacc ccaataccca gcctagtagc agtactttgg
137460ttcccccagg gagctcctct tttaaagaaa agggacagga cccaattgtt actgagcccc
137520tattgtcata gtagccacca tttattgatg gttgactatg cacctgccag atactgtacc
137580cttaacagca tttatcatcc aaccctcctt tagcctgctg agggggttat acataataag
137640gaatattgta catactgagg aacctgagac tccatgaggt taaaacttgc ctaaaataac
137700acagctaggg aaaaggcaag ctggattttg aactagggct ctaagtgctg agcctgtggg
137760cttcataatt ggaccaaatc cctgtgtgct gggcacgtgt ccagcacttc cctcatatga
137820tctttatgtg aaccatcctc tggaatcctc agaacaaacc caggaagtag gtatactcat
137880ccccatttta cagatgagga aacaggcaca gagagatgac tggcttggcc aagttaagaa
137940taatggctaa caaacaaaaa caaaaacaaa aattaaaaaa aaaaaaagaa taatggctaa
138000ctcatggaac tcatagaact ccacaaggaa aggtgttcta agcaccttca tacatgctgc
138060ttcatttaat ctctacatta tacagatgag gaaactgagt cacagatatc ctgagtgact
138120tgcccacggt ggcatcagtt aatgacagat ccaagatttg aaatcagaaa ggctggctcc
138180ccagtctcca tacttcacca aaccagaagt tctgaaactc aaactgtggt cctgccaatg
138240gccacactgg cttccctggg gaacctgtag acatggggat tcccaggctc caccccaaac
138300ctcctgaatt agaaactctg ccccccgccc caccccgctc agagatccgc aggggatcct
138360aatacacccg aaagtttagg aaccactgac ctcaccaata ccactttttc cacagcaaat
138420aggttagagg aggcagaatc caaatccagg atgctatgaa tcaaaaggtc aaccctttct
138480cttctgccac ggtgcacccc cttccctccc ccggccaagg ccccagcggg gtctgcaccc
138540tgcctcaggc ccattctctt cttctgtgcc ccactccacc ccacccagga tgacttgttc
138600gcggacctgg ccaacctgag ccacctcttc ctccacggga accgcctgcg gctgctcaca
138660gagcacgtgt ttcgcggcct gggcagcctg gaccggctgc tgctgcacgg gaaccggctg
138720cagggcgtgc accgcgcggc cttccgcggc ctcagccgcc tcaccatcct ctacctgttc
138780aacaacagcc tggcctcgct gcccggcgag gcgctcgccg acctgccctc gctcgagttc
138840ctgcggctca acgctaaccc ctgggcgtgc gactgccgcg cgcggccgct ctgggcctgg
138900ttccagcgcg cgcgcgtgtc cagctccgac gtgacctgcg ccaccccccc ggagcgccag
138960ggccgagacc tgcgcgcgct ccgcgaggcc gacttccagg cgtgtccgcc cgcggcaccc
139020acgcggccgg gcagccgcgc ccgcggcaac agctcctcca accacctgta cggggtggcc
139080gaggccgggg cgcccccagc cgatccctcc accctctacc gagatctgcc tgccgaagac
139140tcgcgggggc gccagggcgg ggacgcgcct actgaggacg actactgggg gggctacggg
139200ggtgaggacc agcgagggga gcagatgtgc cccggcgctg cctgccaggc gcccccggac
139260tcccgaggcc ctgcgctctc ggccgggctc cccagccctc tgctttgcct cctgctcctg
139320gtgccccacc acctctgact gcggtgctga gatcgaagag gccagtgtcc gatccccgct
139380tcccgtccac ccggggctgc ggctccggcc ccagtcgccc caccttccct ggccttgctg
139440cctccctttc ccctcccagc tcctctcctc cccggggagc aggccgcctc tccttgcctg
139500ccccctgggc tgtcctgact tgtggcagcc ccaagagggc gtgtgtggtg gctcagccct
139560gccctcccca gttctggcca ttaactcttc cccatcccaa ggctggggtg gggcccccca
139620ggcagccgct gacccgcact cctaagggcc cacagcggac accagagggg cttttgtctg
139680cagagcgtct tccaccagca gagcctttgg aagctccccc agggagcccc acccaggacc
139740ctttggggga tgcctcagtc agggccaggc tgaccctgac ccctgcttac cctagtcccc
139800tcaacctcct gacactggag gaatactttt ctcctaagtc taccctggac actttttagg
139860gcacctggag agaactttcc tctccactgt ggcccctgcg tggtgaagat caaaagaagt
139920tgtttgggaa aaaaaattta ttaaaaaatt ctattatttt atctactgta agatttgttg
139980acttgggacc ccgaaagcgg gatgaggtct cagaatgtaa ggattgcagg gccaggaggg
140040ttggagaagg ggagccgtcc cccgccatca aagagcttcc tggtggctgg aggtggtgtg
140100cgctcccccg ccatgaggag gagctgaagc cctgcattct aggtgaggcg cagtgtggca
140160gccaagagtg ggtgctggtg gcacctcttc tcttcatttg tccaggggaa gagctgcagc
140220caaccctgag tggtctggcg cctgaggaac taagcctggg gaagacctgc tgtctggtta
140280acagccctct tccagaccct gttccttcag gaaacaagag cagttctcct gcaaggagga
140340gtcacataca cactcctggt cacagacagc cccaacatgg ctttgggtaa atgtgaacaa
140400ggcactgctc cctcagggaa acacagcccc atgccagagc aaacacctta gcaaacagag
140460accaaggctg ggtttccgcg tacacttgcc tccttggcta agtgcccttg tgcagtgcac
140520agcgtacaca cctgcacaca gcaaccctgt gggtatgtgg tctctctctc agctcctgtg
140580aggtagaagc catcagggat gaaccaggtc agagaagcag gtttccaaac aggctagaag
140640agggaccgag gaactcgggt gatcagaggg acaggaatcc caaattggga tgcattactg
140700gcttgaggta caatcagaac cttcatcttt ctggtgtgtg gaagagaggc tggggactgg
140760gaagagctca ggctaagaag gacttgggtt gggatttagg ggtgagtctc atcagactga
140820gcacttggag agaagtttgg tagtttgaat ttggagctaa gaatctagct tgggcagggt
140880gtggtcgctt gcacctgtaa tcccagctaa ttgggaggct gacgtgggag gatcacttga
140940ggccaagaat ttgagactag cctggacaac atatcgagac tgagtctctt aaaaatgttt
141000ttttaagaat ctagtttgga gtggggtgtg atgtctcaac gtctgtaatc ccagcactct
141060gggaggctga ggtggacaga tcacttgagg tcaggagttc aagaccagcc tggccaacat
141120ggcagaaacc ccgtctctac taaaaattca aaaaaattag ccaggcgtga cggcgggtgc
141180ctatagtccc aggtactcag gaggctgagg cacaagaatc actccagcct gggtgacaga
141240gactctgtct aaaaaaaaaa aaaatctagc ttgggaggtg ggaatagaaa gatagagggg
141300gcctagatgc tagggcttga ggaagcaggc tgaggttctg tgattctggc tagggaggtc
141360aaatgatctt gagaagaaga gaagaaagga gaagaaatca gcatctaagc ctgaggcagg
141420tagactccgg ttaagggtgt ggggtgggct gggggagagt gagagcagct ggtcagaaac
141480ccagggagct cggagtctgg ggtcttgcag gggcttgtgt caggctggct gtgaggaggt
141540taatgggttg gattggaggg acagccagac aagagctctg gtggaggagg ggctgctggg
141600gcctgggcag ggggagggga gctgctggta aattagaggc aggctgtcca ggtcatagaa
141660ttatcattgt gaaatattca tgggccatcg gtccagatgc tatttcagaa cagtgaaagc
141720aagaggagtg tgtgagcctc aggaagaagc ctgaagcaaa gccactctcc accaaccccc
141780acccctccca ccaccagccc agacagaccc acggacgccc atcacgtgca cacccacact
141840cccgagctct cacacacact cgcaccaagc agagccatgt agcacgtgca agcacaccaa
141900ccacccacgg gtcccacaaa caggcaggtg tcccctaaat tctgacatgc acactgacat
141960gcacacccac tcaatcagga cccagcagag atcacctcca gcgatctcac atgcgcagac
142020ccccaaactc tccaaacaac ccagattcac caccttgacc cacacaccct gagataggag
142080ggatgttcaa ggccatccag cccaaccccc accaatgctc tgatggggaa actgaggcca
142140tagaaaggaa gggatttgtc tgagattcct ctatcccctg aaaaaagcaa aattcattca
142200cctcccacat tctgagtgta cccccattct gcattttcgt ctgccagaca cccagcctag
142260ttgtaattaa ctcctccctt tctctaattt cctgcatcta ttcagttacc cagtccccca
142320cccagccaca gtctatccct tccttcccat tctccccacc acctccctgc tccagctact
142380cattacctca tgcctggaat ataaaagaaa actgcgataa cctcctcgct ggtttcctac
142440atggaatctc tccctccctc ccacccagcc ataccgtggt gaccagattc atctgatcaa
142500aatttgcata tgttatgatg tcactcagga gcctgtaatg gcttcctaat gcctataggg
142560taaaggtaaa acaccttagc agagcatcaa agatccctca gagtctggta ccaactgctt
142620ttctagcctt ttctctcaca atctcatccc aaaccttcac tccagctaga acgtttgtat
142680catactggcc accagttatc atgtatgtga aacccaccaa ccgactttga gtgcccccct
142740aaaatttctc agtctctcct gaagtaggaa acctcttccc cctcctcaga tctcagactc
142800cagagccctt tcccaaggcc aagactgcac ctctctgacc atatacaggg gttcttcaaa
142860gcagcagaca gaggctcagg ctctggctcc ctccaagcag acggctgccc ccgactggcc
142920accttgggaa gcacagccag gtttcagtcg tctagaacag agaatgagca tctaaccgcc
142980tggggagagg actaggacac cagatgataa ggtttataag cccttaagcc tctaaggttc
143040ttacacccag agtagggggg ggacggttct cagccctgtt tccctagctg cgggctccca
143100attttcgatc cctaatccga gaggaactcc tctccaatga aatacagact tgggactctc
143160aggacactgt ggaagggaaa tttcccaaca gactctgaga gtccaggagg ccagggatag
143220accaggtggc aggcccaagg tccagctggg gtcaggtttc tatatgaatt tttaatgctt
143280ccagatagac ttgtcagatg ttctgaaaac tgagcatctc ctttcacctc tgtacatgat
143340gcccttctcc aaccccattg cccctgcagg agggcaggcc tgggacagat attcagtggc
143400ctctggagaa acggttttgg gacagtagaa gggtaaatga cctagttatg ttcccactag
143460taagctgtgt gaccttgggc aagttactta acctctctga acattagagt tctgtgggtt
143520tgtttttgtt ttgtaagctg gggacaatag tgccagccta aatcaatttg ttgtggggac
143580tcagtgcaat agcccatggc aaagtgacct acatgcttgc tgttattatt ctctttcctc
143640aagttctgcc tccctcttcc agcttttctt ccaaccccaa agatgtctct ggctattgct
143700tcgaaggtag gaactttggt tggttctccc ctttctcttc aggcccaaac tccccacctc
143760aagatccttt ggcctttgta gaaacttcag gtgaggaggt ggcagagaaa taagaaagtg
143820tgcaaggctg gtggagtgag agaggaggat agatggcgaa gccctagcag aggggaggga
143880agtgggcagt ggagagagg
14389916215980DNAMus sp.misc_feature(1001)..(1100)n is a, c, g, or t
16ttgggggtat aaacccagaa gtgggattac tgcaccatac aataatcctc taacttcaag
60caatttttcc acaatggttg tatcatttta cattcccact ggctacgaga agggttccca
120cttctacaca tcttcaccac catttctgtt tttgtttttg agtaacagct gcctaatgac
180tgtgaagtgg tatcttatct cagtgttgat ttgcatttct ctgatcatta atgtgggaag
240gcatcgtttc atatgtttat tggctgtttg tgtatcatct tctttggcga tgttgattca
300agttatttgc ttgttttttt aattggagtt ttaaaaaatt gttgttgagt tgtgggagtt
360cttcattagc tctgcatatt aataccctga tgaaaatgat taacaagtat ttgcttccat
420tttgggggct tccattctgg gctgttttta ttcttttgat actcttttga ttctcaacag
480tttaatctga ctaaaattca gtttatttct tcttttaatg gccatgctat tgacacatcc
540cgtaatcact gccaaatcca gtcatgaaga gtttctttca agagatttat agttttagct
600ctttaagttt gtcatgtctg tttcacttaa ttttgtatag tgtacaaaag tctaacttca
660ttcttttcta tatggcttgc tactagtata cgaagagcta aatttctctt tccttgagtc
720tcaacctctg atgtgtagca atttcttcag aggaaaacat ggtgggaagt tccttaaaca
780taggatgctc catggaggtg aaatagttca tcctacaggg aagcttgtta aacacaggaa
840gtacatactc agcagctcta gtaagtgagt gaaactgact ggaggcacta ggtccctcct
900tccctacgca tatagaagct gtaaggattg ggaagagata ctgtcaggtc agctcagctg
960ctgcccggaa gaagctcaga cccactggcc tggctccaag nnnnnnnnnn nnnnnnnnnn
1020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1080nnnnnnnnnn nnnnnnnnnn atcactcttt actcaggcca cctacacgct gtttatagcc
1140tgcctttgtc tctttggcta tacttcctgt ttatgtctat gcctcccctc tttctttttc
1200tttctcttct cttctcatct catctcatct ttcttcaggg gggagcctgg tctagaactc
1260acaaagattt gactgtctct gtctccttgc actaattaaa aaatctttta caagcatctt
1320ttagcaattc ttacagggaa attttggaat gttaaactct gattgttagc gggctgaaga
1380taacaatagc tctgatgata aattgcttgc caggcaagtg tgaaaatctg agtttgatcc
1440aaaaagccgg gtacagaggc caaagagtcc ataatcctag taggggcagg aatcagggat
1500gggtgggtcc ctggggtttc ctggtttgtc agcgtagccc aattgggaat agccaggttt
1560cagtgaacga tgctttctgc aagctgagag aggtccttgt tcaatctctg tgacccaact
1620ggagggagaa gagagccagc tctccagaag tggtcctctc aactttgtgc atgcatgtcc
1680atgttcacac agggaatgga taatgcttaa aaggaagacc ggcagggggt tggtaatgca
1740cctcctttgg tgacatgctt tcctcttgtt catgctgctc caggtgtggt cggcagcacc
1800aaaaaccagg tgtatgtttg taatcccagt attctctggt cgtcagtagg aaatgaaaag
1860cgaggtcatc ttcgtataga gttagcaaac tctaagccag cctcggctac atgagacttt
1920gtctcaaaac aaaggaaaaa tcaaggagga cggctcccga gcactgtcac ctgaagctga
1980cctctggcct ccacatgcat gtgcgcaaac acatgtcctg cacaaacaca cagacacccg
2040catctgctcc ccgacaaaag aacctgaaac cagtatactt tgagaatttc ccattcatag
2100ttaccattgt gtgttccttg tgnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2220nnggtggtgc ctttctctta cccagtctag aagggctgga ggcagggtgg atggggcact
2280ttgaactccc acctaggcaa aaaacccagt gatctctggg ccagtgtgtt gtttgcaagg
2340gaataaggta gagagccgcg gaggaagaca ttgggggttc tatgagtatg tgaaggggtg
2400cacacaccac acacacacat tttttttgtt ttaaatttac aaacattaaa ataggctgta
2460atgtggctca gtgggtagaa aaacctgctg tctaagcctg gtacgagttc aatccctgac
2520aagctggaag gagacaacca accacaactg ctagcagcca gaagcactgc ttgctaacac
2580tcaagagagc ctggagtgga agacactgga tccccagcag gcaagcctgc aagaagatgt
2640gccttgccta gacaacggca gaacaaacat caaggctggc agagctgtcc aggactgttc
2700atattaatca tgtatagata agagggaatg gcacagacag aacaattcaa cacacggggt
2760atgaaaggaa aggaacaagg cacacaaagg acaaagaacc tagcatacaa gaaagcctaa
2820gcagagagtg gcacttccca gaagggagtc ataaaataga ctgaattcat taaaacaaga
2880gccaaagata aacggctcaa aaaactcacg gaaaacaggt caaaataacg tcacccatct
2940gacagttgat actgtcaact taaccgtatc tagaactcca gcaggcacat ctccaggcat
3000gcccctgaag gggtctttgg actaggttaa ctgacgtggg agtgacacca tctatggacc
3060gaagcctcag acagaataaa aaggagccag tgagctgagc gtcagtgctc attgcttctg
3120gcttcctgtc tgtggctgca gcgagacacg gtgcttcctg ctttagctgc catgacagac
3180cacaccctca aaccgtgaac caaaataacc tcctctctac attgctttta ccaggcattt
3240ggtcacacca atgagaaagg ttaactaata cagcactcaa tacttaaaaa cataaacacc
3300aaccttgttt gcatgtgtga gactttgaag ctcacgggcc agttatgccc aatgccaggt
3360ctgctggcta agggtgagag tgcacaccta taatcccagc tgctgtggaa tcagcaaaag
3420cgctacagat ggaaggcagc cagggcagct gagactgact caaactgata gaggtgggag
3480gcatagagaa aaccagatta atagagtgtt ccccactatg caagaagccc tgggtttcag
3540gacgagagaa ctaagaatac agaagtctac tgtgtagaag cactgctagg tcacacagaa
3600acatcactca agtgtctctg gatgctacac ggagggcgtg tgaagtattg cttcctgatg
3660atctgtatct actacagcac tgctgtttta gtatgcgctc ctccactaca gctcctcacc
3720acaccaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
3780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaat taatcaaaga
3840aaacacacac caccagttag agaaagttaa tcaggccgaa tggcggcttt cccctgtatc
3900caggctaccg tcaggacggc tcactgccac tggcaactct gcctgaacaa agcccgcagc
3960caacgtgggc ttcaggggct ctaaacactg caatcaaagg ttgtgtgtgg gggtgggggt
4020gctgctgcta ttcaaggatt cccaaagctt agatgtattc atcatactca caggaaagcg
4080tgttcaaccc atcactcatg agcagtcggt accggggtga cctattccct gtagaaatgg
4140gacggatgtt ctggaaaagt tgacagaaaa gttgattcat taggcaggct ctttgcccaa
4200gccctgaggg taagcaaagc taactggcag gagactaggt ttgccattaa tctgagacaa
4260gatgaaccac ttgcccatcc tcctgacacc taaatactaa tgaaagaaca atggattgag
4320ctggcattat taaaaacgat agaaacagaa gtatcaatag tcatgtgttc tttctcccat
4380atgtcaaaac aatgtgtaag atggcatcga acacatgcag aaactgttta gggaacatgc
4440tgaaaatatg aagtaaaatt aaaattggaa agaaagacaa tttgcctaaa gcagctcaga
4500gctggagaag ggaccgaggc agagataaca gcaacgtgtg gacatacgga tctggggcag
4560agcagtcacg gactcagccg gaaagggtgg ggcagcctct gaaggaagtt aaggtaaata
4620gagccacaag gtgattggcc caggagtggt gccaccttca cctcctgcct caaagtctga
4680aggaatgatc ctggagtctc ccatctattg atatatgaaa ttcacagtat gttttagaac
4740ccactgaatg atgggtagat taactaaaag aaatttaagc ggggtggtgc aggtctttta
4800atcccagcac ttgggaggca gaggcaggtg gatctctgtg agttcgaggc cagcctggtt
4860ccaggacagc cagagataca tagagaaacc ctgactcgaa aaaacaaaat taaaagctca
4920tcaaaacaac aacaacaaca aaaaaaacaa aaaaacaaaa caaaacaaca aaacacccta
4980tagtacctgt tggtgagttt gagtgagtga gtgagtgtgt gttagagaga ggggcgggga
5040aagtgtgttc tggaaatggg agaaagagaa tgtgcatgtg tgtttctggg atgtagacaa
5100aactacatgt cttccatcaa atgcaatgtt taattatcta tgagttgaac catcttcatt
5160ctgctaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaac aaaaataaac
5280caaaccagta aacaaaatcc tgtaagataa agcctaagac aagacacttc ctggggctgg
5340ggagttgctt agaccataag gagttcataa tccaggcgtg agagcccgag ttcaggtccc
5400tgggcttcca agtcaggagg agaccaagga atcaacaagt ctcgactttg gtctctagtc
5460ccatgcacac acacgcgtgt aaatacgtag atgttcactc acacacagaa gactgcacct
5520ggctctctca catctcagcc aacatataaa gcctgcatta tcagaacatt ctaggttcta
5580gtttcagtca actcttacac agaatggcca tcatactccg tctacaactt ctcctgatct
5640acccacgtgt cattgcttca gtattaacaa aacccagaat aaccagctgc gtagatcctc
5700cctgatgccc cagtcattgt cttactgaga ctactaagtc acaaggtagc actctggatc
5760caaaaagcaa tatccaattg agagttacaa cctataagga ggagtttacc ttcattatag
5820ggcactggat tcccaatctt taatccaacg tcttcagcag atttcataac ttccaagtcc
5880atcaaaacaa ctactttcct acaaagacag acacaagtta gaattaagaa ctctgcagcc
5940tttcagatga gttactaaga agcttacttt agtagttgtc tggctaaaac tgtatccttt
6000accaaccttt tctcattctg gactaacttg agaagtatta attcctaagt aaatacttca
6060cttattcttt ccccacatct ccaatgtttt tgtctttaat ttattatagg gcaattcatt
6120tcctatctag ttccctgatt aaaacagtag accttgctgc atgccattat cctcatggag
6180gcactgatac aatttagatt attaaataca aaaccctaaa acacaaaaag atgatttttt
6240tttaaaacaa gattttaaaa aaagcatgtg ctacgcttcc ttctgccact aagcctacac
6300atggtcctct gactgaattt ttcccctcat tctgcttcat ctaatatgtg cttttcaaac
6360ctggaattga accagggact tattcatgct aggcaaatgc tctaccatag agctataccc
6420ctccaactcc catctcaaat atcatttcca aagacatttt cttggtctct tatttagatc
6480aggtttcttt gtcctcctgc agctatgact tcattccttc agaacactcg tcttagcttt
6540aagttctgta ttaattagtg attgttttca ttctctctgc tagaatgcac tttcaataaa
6600ggcaggtagc cagccacagt gcttaattaa gcaacagccc aacgatgtca ttcactacat
6660actgggacaa gatgcctaac atcatctgca gataaagacg aactactggt gtcaggagac
6720agctaagggg tccagggctt gggcacgctg agtgtgagca ctggagtccg ggtgcccaga
6780aacgcacata aatgcaatat ggatgtggca atctacctct aattccttct ttaagacagt
6840ggctctccag agcaagctgg ctagcaagac aagccatatc agtgagctct gggcttgacc
6900aagaccctgc ctccaggtgt aactcccaag caaaaggatg atggctcaca aatctcaggc
6960tatcatgttc atgtacaaaa tgtcaaccgg catacacaca tgcacacaca tgaaaactgg
7020gagaaaataa gaagaattgc aaccaaaaaa tgtaatttga ggacacataa ttgcaggcgg
7080ggagtggggg gatgacagaa ggtgaactga gtggaccgag ggaaagctgt gctagcggca
7140atgagaagaa gggtggggca gtctgagcaa gggttcagca atcaccacgc tttactgtct
7200gcacagcctg gctgtagaat gctgggcttt atcacacaga attattcagt atgtgctatc
7260tttacagtaa agttattcta tcaggctatg ctacttcaat agaacaagcc tgaaaaagtg
7320gtctgctgct gagaacctga caaagatgac ctgttagaac tgtctgccaa gtgtggaatt
7380ccagcactgg ggaccaggag ctcgagggtc accccagatg cagggagtta gaggccagtc
7440ttggcaacat aacatcatgc ttcagaaatt aaaaacaaaa nnnnnnnnnn nnnnnnnnnn
7500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
7560nnnnnnnnnn nnnnnnnnnn catgagatag ttaataaact gaagaaagcc atacaaggag
7620taaagtagat agttgcaagc atgaagaaag acaaaccact tgagcttttc ttttgtcgta
7680aggaggaaac cagacaggtc cagagagatg gctcagagat taagagcact gactgctctt
7740ccgaaggtcc tgagttcaaa tcccagtaac cacatggtgg ctcacaacca tctgtacagc
7800tacggtgtac tcatatacat taaataaata aataaataaa taaataaata aatcttaaaa
7860gaaaaaaaaa aaaaacctaa ccaatcagcc aggcgatggt gacacatgtc tttaatccca
7920gcacttggga ggcagagaca ggtggatttc tgagttcgag gccagcctgt tcttcagagt
7980gagttccagg acagccaggg tgatacagag aaaccctgtc tcaaaaaaca aacaaacaaa
8040caaacaaaca aacaaaaaag gaggaagcca gacaggatgc actttatacg tgaatggaat
8100tgacaaaaga caagttctat aagtgttagg gaaaggggga ggacaacggg ggttcatgtc
8160tgtggtggaa cacgtattag aaggctctgg gtatcctgtt tccgacaaac aggcactccc
8220aatcacacag gccactggat gtctcaggca gagaaagatg tgatagattg actttttaac
8280aatcacagac tgtgtggaaa atatttgtaa ggttgtcatt gtcacccagg atagagctga
8340tggttattca aacgaggatg ggacaacaga aatgggagag agggatgtga gaaccatttt
8400gaaccagggt gatttactgc gcacgtgtat agggtctaca gggagtggga tatgtagagg
8460aggcctatgt tcctaacttt ggtaatgagc ttattacagt tactatgcac agcctggaag
8520atactggaaa aggtgcaggc taggctagaa aggtactaac tgagggtttg acagcccctt
8580ggatgtcagg atgcagcaag cctacctctg tatgtagtca atcccttctc aggctatggg
8640tcctgcagat catccgtctc tgtatccatt attcccagtc catcctctga gtggctccct
8700cttatccagt ttaacaaaat gctgactgca agctcccaag cccagggctc tggctccttt
8760actccttgtt attgtacttt accctgtttg cttgggatag agtgtgccct ttataaacat
8820ttgtgaaagg gggaatgaag aagaataann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
8880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
8940nnnnnnnnag agagctcaat ggttaggagc actggatgct cttccaaagg tctccagttc
9000aattcccagc atcaccatgg cagctcacaa ctgtctgcaa ttccagttcc aggggattca
9060acactcagaa acataagtgt aggcaatcta cgtaacataa aaataaataa atgagctgga
9120aaagaaaaca tgtttcaaaa tatacaagta atggggctgg aggagatgtc tcaatgggta
9180agatcattgg ctgctctttt ggaggttctg ggttcaattc ccaccaccca catgacagct
9240cacaactgtc tgtaactttg gtcctgtggg agctgatgcc ctcttctggt gtgcagacat
9300acatgtagac aaaacacctg catacataaa ataagttttt aaaaaagtta cacatacacc
9360cgtgtgtaat ataacacaca ctggcttaac ttcctcagca ctgactgttc accatacgga
9420ttcccatgag gttttggttg cattctatca ccgaaaaaaa aaaaaaaaaa ttagaagaaa
9480gtatatacat ataaacctct ccctaaaata aagttttctt ttctaaaagt acatccttat
9540ttttttattt tttttttttt ttaagaaatg ggaacaacag ttctgctcac actgtatttc
9600tagcatgtaa catcttgcaa gtacttaacc gtattctata tcagctcaac acacttacta
9660ccgaagactc aagatcacaa aaaaaaaaaa aggacccaga ctggataatt aaacgtttct
9720tttgttgtag taagcgacct cttccttaga agatactaca gtaatgctga agaaatgaca
9780catctactgt aatctgttct ctgggattcc aacttgtttc ctctgctact cctcccttgg
9840cggcaatgtt cgtctgcatc cggctgagct cctcgctgcc ttgttaaacc tccttcctga
9900acttccgacc tgtagttccc gctctacagt gcaagcgagt ggataaggaa gcgcatacct
9960gccgtctttc agggtgttga cgatgaactt gtggacctgg cagacacagt tgctggccag
10020ctgccctccc tcgaccaggg tgttcagctg cgtggccagc atgaacgctg caaaagcaga
10080gagagagggg ctcagtctcc aagcctttcc ttaacccgaa agctcatcac aaggagaacc
10140attaaataca gctgtttaaa actcctccgc cctgcagaga ggaaagcagc atcaatccgc
10200cccatgtaaa agtctgaggc tcttcctaaa tggtatctgt ttctcacagt ctccaaatca
10260tttttactgt aattctagtt tctggggaaa gacctttctc ggtctttagc cccgtgacta
10320gagacaacag gcaaatattc cagaaaggcc cccattttct ttttaaagct tctannnnnn
10380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
10440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngcacat cttgtgaagt gtccacatct
10500ttcggtccct cgaatttggg tttcttctgg gacgtggtag catgtgactg tcactccagt
10560gcttggagca gcagaggggt caggaactcc aggctggcat tagctgcaga gctggagcag
10620gtcctggaga acagaaactt tggttgcagc attaatgaac tagaagaatt tttttgtctt
10680ctgttaaata taaatacctc cattatcttc tcataaacag tgttgccttt ttatttaagt
10740ttttaaggat caggcacaga gactccatgc cagactacca ctcaaccact gagctacacc
10800cccaacttgc ctttctgcta ttttttaaat tgtatcagtg gccaccaaac atggggagag
10860gtcagggggc tacgtggagg aattgtttct ctcctaccaa gtgggcccca ggtttcaaat
10920tcaggtgacc tggcttggca gcaagcacct ttacccctaa gccatctcat tggcttcatc
10980ttttaatggc cccttcccct gctctgaggc aggctctccc tatatagccc tggctggcct
11040caggctcgca ggtccaccag tgagcaccag gtttctgctt gtccttacct ccccagcact
11100gtggttataa gcatgtgcca ctgtgtcaaa ctcagtcact aagctttgcc aagccatagc
11160ccagcccttg agtttactgt ttgtctgtgt ggtgatttgt caaaccactt ttgttccact
11220gaggtatttt gtcaagtttg acaaaattag ttgagtatgt aggtcttttt ttctggaatc
11280ttctgttata gcttagtctg gtcttgaact catgatcttg cctcaacctc acgattattg
11340aggattattg agatggacag gctgtgtgac catgctcggc tgtgtgtttt agcatgcatt
11400agtcatttga aaaacgttgg ctcatgacac tttacaggtc ttccatgttt gatatgtttt
11460atttaatcca aagtaattcc agcaccagag gctgagacag gaggatctca aggtcaacct
11520agagatgcat agcaggcggg gccccactcg gttaggttaa tatcatcact gacttcagga
11580gaaaagtctt aagtattggg gactaaaagc aggaggatct gaagttcaag gtcatcttta
11640ggaacttagc agacttgagg ccagcttggg cgctgtggga ccctgttttt aaaccagaaa
11700acaaattgaa aggaaaaaaa aaaaaagctg gaggaagtga atgtgagtgt tcacatagtc
11760ctgtttccac aagaaaacag ggttactttt ggcaacaaat aggtgctttc tttgaaggct
11820ggcatttttg tgacttgtca ttggagaaat gatttaatta agacttttct actgagtgcc
11880tctgaagagg ctcttttaaa tttagtttaa ttttatctca ttgttagtgt ggtgtgcttg
11940tgcacacaga aggcagcttt ctagagtctt ttcactctct cctccacagc tcctggagtc
12000aaactcaggc cctggctagg caagctctta ggacagtgtt agctgtagct tattaagttt
12060ttaagaattt ttataagact ctgtttttct ttctcaggtc atgatacagc aggaaaatac
12120atccataaag cccatcctgc aggtcattgt aagtaccggc atgtgtgttt agcataatga
12180agatggttca cttatagtta attaaacatt ggattggatg gaagacatgt agttttggtt
12240acttcccaga aacacaaatg cacattcttn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
12300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
12360nnnnnnnnng aattcagagc tgatatgtag tactaactcc tactcaatga atcctttgtt
12420cttctattcc ttcattacat tactgttaat agtggtaact atgtaccaaa gagtcaaata
12480actcttggac catccaaggc agaaggaagg ctggcaaaaa tgtatgatga tctgggatgg
12540gaatgtactt cagtttgtac aggaggccct tggttcattc catttctggc aatgcataga
12600cctgtaggat ctcagcactg gtggggggtg gggggtgagg gtgaaggggc gggaggttaa
12660aggcagaata gtcataaatt caaagtctgg gtcctggaaa gaggactaaa cgattaagag
12720ctttagctgt tcttctagag aacctggtgt gatccccagc acatggtgcc tcacgactgt
12780ccgaaactct gattctaggg ggatctgaaa accctcttct gccctctgta gatacagaac
12840acacatggtg cacatacata catgcaaccc aaacaaccca tatacataaa atattttttt
12900ttcaaaaaga cattcaaatt cttcctcggc tatatagtgt ttaccaaacc tcaaaaacaa
12960aacaaaacaa aacaaaacaa agaatcatta atgttttgcc ttcatgtatg tctgcccacc
13020acggacatgc ctggtaccca gggagattaa aagaagacat tagctcccct ggaatggaga
13080taggtatgat ctaccacttg ggtgctggga acctgggtcc cctgcaaaag cagtaaatct
13140ttttaacccc taagctgtct ctcccaacgc ctaaagattc ttgtaacaca gcatgatgag
13200cactggcaag catagcatgg taatctgact tcagggcgcc agattttgag cttaatgctt
13260gattattaga agtaacgtac tagatttaat gcctggagct tcaagcaaca aaattaactg
13320aagaataaaa ataaaaaccc tgccagccat gatggtaatc ccagaacttg agaggcagag
13380gcaggtgatc tctgtgtttt gcaaggccag ccacaatcta catagcacgt tgcagtannn
13440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
13500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnncga ataaatctac acatgtaaaa
13560agaaattcaa agaaacaaat gccaaataaa tacacatatt gtaataaaga gataattgtc
13620taaaaaactc aaggctttaa atggtaagat atcatattct tggatgaaaa gatctaatgt
13680caaaatatat caatttaatg caattatgta tattcaggag atctctggtt ggcttttgaa
13740cttgatagca ctcttataat tcacatagaa gaaaaaaaac catgaaaact gccaaacatt
13800attagaatac tccacagatg gtattttggc agcacataca tcgaagggct gtgaaagatg
13860tgtagatcat ccacgccttg ctagggagag ggcgggtgtg tgtggggggt atagctgttt
13920gggaaaataa cctggtaatt cctcattagt taaatcatag tcagaacctg gactagcaac
13980ttctctctaa aatacattca ccctcagcat ctgcattgcc aggaaaccac tcctagcagg
14040atctgtacgt ggatcaaggt agtagcatct gcatttaatt gacattctcc taaatgcttt
14100aaattatctc tagattactt atagtagcca agatgatgca aattatgtta cactgtatta
14160tctggggcgt aacaagaaaa tgtctctact caggttcatt caggtgcagt acttcccctg
14220aatacttctg aatacacgga tcaagaagcc acagaaagag ggctaaccat atacaagcat
14280atagtacact aataaccatg tacaaccata tagtacacta atattcagtg cattactcaa
14340aatgcaaaca gatggaaaca atccaacagc ctgtaagctg aaaaacaaga taagcaaaat
14400gtgctgggcc tagaggccca ggtctataat tccaactaag gtcgaggcag gaggatctca
14460agttcaaggc cagcctagac aacttagcaa gaccttgtct caaaacaaaa agtaaagagg
14520ctgaggatat agctcagtat agagcatctg cttagcatgt gcactgacag ccgtatcaca
14580gaggaaaaaa aaaaataagc aaaatgtgat ctgtctgcac aacaggatat cacagccccc
14640taccacaggg gaacgacaca gtaacacaac aaaaacttag ccctgaaaat actatggtaa
14700ataaagaagt gtcactgagg atcaggaaat gcatgactcc atttacatta tatagaaatg
14760agaagatcag tgagcctcta ggactcaaga gatttgggat tggcagctaa agggtactgg
14820gtttctttat gggggtaaga aaacattcta aacttaactg tgagaatgac tactcaacaa
14880tgtcaagtgt tcaaaaatca tacttttttt tttttttggt ttttcaagac agggtttctc
14940tgtgcagtcc tggaactcac tctgtagacc aggctggcct cgaattcaga gattcacctg
15000cctctgcctc ccaagtgctg ggattacagg catgcgccac cattgtccgg ctcaaaatca
15060tacttttaaa aattgcccag tgactcatga atacaatcag aggcgggaga ggacagtggc
15120aaactcagga taccagtgtc ttttatgtct gctgcccaac tatcaatttc ccatagttac
15180cagagaactt tttggtttgt ttcatcttat ttgttgcttt tggtagaatc tcaatatagt
15240aagatacaag gctggcctca tactatatag ctgaggacga ctttgaactt ctaatcctcc
15300tgcttccatc tcccaagtgg tgggattaca ggggtgtacc gctatgccca gcaagcacaa
15360agccatttga accacacccc agccttttca gagaaacctg tacaagcctt agtgccttag
15420catattaagg caacaaaaga cataatgcgt ggctaccata gagtgtttgc ctaccatgtg
15480tgaggctcta ggctaaatgt ccagcactta taaaaaagag ttaaaaacac tcatgactca
15540aggatgacta tgcagtcttg tgtacaaagc cccgcattca atccccagca ccgtgcacat
15600caggcaggct ctgtagagga cccagcttaa ggtcatcctt aggtaagtta gaggccttag
15660atggctacat tagatgagac cctttctcat aaacagaata aataatttaa agctcctgat
15720caaacactat gccttcccat cacactcaga ataaagcact ctactggccc tttaaggact
15780gcccatctgg aagagaaacc taagttacat tccttgcttg tgtcatatgt gataacaaac
15840tcactggaaa tacgaaaata cagtcttaag cttggtcaga aagcttcccc agcaacatga
15900tntcagagga cataatgcag aaagtggaca aatgcaaann nnnnnnnnnn nnnnnnnnnn
15960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16020nnnnnnnnnn nnnnnnnnaa tcagaggaca tctttcagga gttagttctt tcctccctct
16080atagctttca gggatcaaac tcaagtgtgt actgagcgct tatgcccagt gcgccatcgc
16140accaggcctg cttctttgtt ttttatgggt ctgaatcaat tagcaccatt acaacaatgt
16200tgacaatcag caagtacctt tctctacctg gctagtaaga gaagtaagtg cctttggtgt
16260gtgaacgcag tttctcttgt gaagtgcatg gacttgatct ttgctcacaa cgttttttag
16320gtccttaagt tgcttgggtt ttatgggaaa ggctcttggg ttttttgaaa agattttact
16380acaacttgat ataatcatta tttttaatcc tttaaatagt atgacttatt ttaacagatt
16440aatattgaac tgttctttca ttcttacata ataaatcctg ccttaaaaaa taatcctctt
16500agcttccttt ctctattttc aaatttgttt tatatttttg catgattttg aacatttata
16560aaagtaggca gacaacacag tagaaccaag tccccatata gctgtgcaca tagcttcaga
16620ttattgcctg ataataggtc ctgttttgtc tctgttttct cacagggatg tgttattgtg
16680tgtgtgtaca catacataca tatatgtatg tatgtatgta tgtatgtatg taatgaactc
16740cctttaacaa aacaagtact gggctgggaa gacagcacag ttagttatgt gtttaaccgc
16800acaagcatga caaccagagt tgagatcccc accaaccgca taaaaagctg ggcatagtgg
16860cattgacctg tagccctggt gctggatgaa agctggggag gcaggtagat cggcagagct
16920tactggcaac aaatctgccc agtaggtaag ctctgggctc agacatccta tataggaaaa
16980agatgaaggg cgaggcgcag cggcacacac ctttcgtggt agtgcttgag aggcaggggc
17040aggccagtct ctgtgaccag cagcctggcc tacatgtcaa gttgcaggac agccagagcc
17100accacctact gagactgtct cagaaataag ttttttaaaa aattgagatg aaggagctgg
17160taagatggct tagaaggtaa aggcacttat cactaagcct gaagccccga gtttgaccct
17220ggaccccaca ctgtagaacc aactcctcca agttcttctc agacctccag cagagcacaa
17280gtgtatgcag acacacacac taagtaagtg aatgtaaaaa acatgacgta gtggcactgg
17340cctttaaacc cagcattggg aggaagaagc gggtggatct cttgagtttg agaccagctt
17400agcctacata aggaatttca aggcagccag ggctacctag aaagtagctg tttatgaatg
17460aatgaataga aggaaggaag aaagagagac agacttaaaa aatatatgct ggagagtaac
17520agaagaggac accggcttgc tggtgtcttg acctctggct tgtacacata cacatgtgta
17580gtgcatacac ccacatacaa ttgtactcag acacacacaa acatgtactc attcatatac
17640tgcacacctc aacactcaga aaatgaaaaa acaggtacca tttacacctc cgtgttcggt
17700ttccaaccac tcatatgtat gggttgtaaa tgcttatatc tgtatgtgtc tgtatatttg
17760tgtatacatt caaagttgag tcaggatcca acgtaaactt ggatagtagt gggttgatgg
17820tctggaagcc tgctcgcagc tgtctttttc tcctcgtacc ttttcccctg tttgtttcta
17880cgacagcagg tcatttgtct ctaagtgtta gtttcccatc ctctctcttt tgctgatggt
17940agccttgtag tagtcacctg tgttctctgt aaaatggctt tgccgtgtta tttcaatatg
18000ctatcatcct catcttgcta tatttcattc aatatatgta tatattacaa gatagattaa
18060aattatttta attttatgct tatgaatgtt ttgcctaagt atattgcacc ttgtgtgtct
18120agtgtccaca gaactcagaa gaaagtgtca catattctgg aactggaatt gcaggtggtt
18180gtaagccacc atgtgggacc tggaaaccaa atccaggcgc cannnnnnnn nnnnnnnnnn
18240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18300nnnnnnnnnn nnnnnnnnnn nncctttggt caaagatcct cagtttcgac tttgattacc
18360cagacttcct gtttctctca tggaacagtt tccccctgag atttactagt ggaagaaagg
18420cactcaaaaa gcagggagcc ctcgtacaaa tgagacttcc tagctatata attaggccga
18480gatgcacaca tccacagtca ttacccttct tcagagcctt tgtcatgtca agtgtatttc
18540gcccatgtga actttagaac tggcttgttg tgtttcataa aaagtgtagt tgggctcttg
18600attgggattg tgttaaattt atagattcat ttagggagag ctgacaactt tacagtatta
18660aatgttttca tccgcggaaa aggttgtctt gccacttact tgggctttct tttatgccct
18720taagtaaagg tttatagttt tctttatatc agtcttgcac atttcctgtt agatttattt
18780ttgcttgtaa ggtttccttg gttggtattg tgtataaaca ttttctccca attacatacc
18840ataattgatg gttaggagta taaataaaag gtagaatttt aaaattgctg tatgaatgac
18900tgctttgcta taattcaaca tcttttcatt tttatgccaa tctacttgac atgtttaggt
18960taaatgatga tatctgtaca gagtaatcct ctgatccagt atttgcacat cttactttct
19020aacgtccata gcatagatac acatcttata ctgttgagta catatatatt taaggtattt
19080accatagtct tataatatgc agcgtgcttt ggttcaagac agttgccctg tgttcctcaa
19140cattaacatt tttttcatca caaatacaca ttgaccttta tcaaattttt aaaactatct
19200tgagagaaat gaccattttt cttaatctgt taatgtaaaa tttttataaa aatagttata
19260aataatatta gcctacatat ttcttctgtt ctctttttca actcttagaa tcagagtatg
19320gtagtctcag actaaaccag gagcttccta tctgtttctc tgttcttaag tcacttatat
19380aatgtaagga tgctgtgtat atctgccagc taggccttat atacaaaagg cacccatcac
19440aaccttctaa aacagtctta ccacttagag accatgttca aacatatggg cctttgaggt
19500aattgccaca ttcaagctat aatattgtta tctaagggaa tatcttcact tctagcagat
19560gcctaaaaat atctaaaggt aaacactggt aattgctgtg tttgttgatg ctgctcttcc
19620tcctcctcct cctcctcttc ctcctcttct tcctcctcct cttcttcctc ctcctgcttc
19680tccttttctt catcctcctt tcttttctta tttttgaggc atgatttcac catgtagccc
19740taggtaaccc gtaacttact atgtatgtag accaggctag cctctgtctc ctgagtgctc
19800atattaaagg tgtgtatcac catatccagc aacacttgct ttgagatggt tagaggaaaa
19860aaaaatatac gtaaataaag atggatgcca attactaaat tgttacttcc agtcaaactt
19920tgtacctagt ctaaggccaa aatagggatt ttttttctac tttgcaagtt ggctccatta
19980agaggctttt cttctcttgg tctcactaga taggaaggag agagaggagg gaaggagaga
20040aagcggttga ggagtgggag gtagtgtgac cgagaatacc cagtaggctc atatatttaa
20100atatttggtc cctagttgat agaactgttt agaaagatta ggaagcatgt cttaggggct
20160ttgaggtttc aaaatttaat gctagaccca gtctttcaag ggagggggcg gtctgtctct
20220ctctgcctgc tgcatgcaga gctctcagct actactctag tgtcaagcct gtgtgcttcc
20280tgcctcaatg atcataaatt aactgtaagc aagcctccaa ttaaatgctt tcttttatag
20340ttaccgtgat catggtgtct cttcacagaa atagtaacct gtggtgattt taatatgcct
20400ggaccaggga gtggcacttt taggaggaat ggccttgtta agaggaagtg tgtctctgtg
20460ggggtgggca atgagaccct cgtcctaacc atgtgagaac cactcttctc ctattggcct
20520tcagatgaag atgtagaact ctcagatcca cctgcaccat gtctgcctgg aagctgcctt
20580tgttcccacc ttgctgcccc aattaaatgt tgtacttata agaattgttt ttggggggct
20640ggagagatgg ctcagcagtt aagagtactg actgctcttc cagaggtcct gagttcaatt
20700cccagcaacc acatggtggc tcacaaccat ctgtaatggg atctgatgcc accttctggt
20760gtgtcagaag acaggacagt atacccacat acattaaata aataaataaa taaataaata
20820aattcttttt aaaaaagaat tgctttggtc atggtgtctg ttcacagcag taaaacccta
20880acataaccct gactaagaca acaagtgagg aaaggtgttg tgtgacactc tggatctctg
20940gaagctcacc tcagcatgaa gcttgtcgaa gcgnnnnnnn nnnnnnnnnn nnnnnnnnnn
21000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
21060nnnnnnnnnn nnnatctaag tacactgtac tgtcttcaga cacaccagaa gagggtgtca
21120gatctcatga cagaggttgt gaactcagac ctttggaaga gcaatcagtg ctcttaactg
21180ctgagcatct ctccagccca aaataattct tactagtaac atggaacaat caagttttat
21240tatatgatac atattaatca acttataagt acatgattat gcacatttat catatcgtgc
21300aaccatcact gctgtcgttt tgttttgttt tgttcttttg aggcccggtt tctgtgttgt
21360tctggaactc actctgtaga ccaggctggt cttgaactca atgatctgcc tgcctctgcc
21420tcccaagtgc tgaaaacaaa tgtgtgcacc accacctctg gctatcactg ctgtcttttt
21480ttttttttta acagttattt atttcgtgca tgcatgtgtg tataagcatg taacgtatgc
21540catggtatgc atgtggaggt cagaggacaa ctttcaggag ttagttcttt cctcccactg
21600tgggttctag gaaccaagct caggttgtta gacttgcatg gcaagtgcct ttaccacaga
21660gccatcctgc tggccctact ataggtcctt atataaaaag atcatatgcc gggcaaaaac
21720caaacaaaaa ataaacctca aaaaacaaaa ggaccatata atattgtggg ggagtggatg
21780aagtcctgaa cgaatgtgtt ctgttgacat gtctgtactt cagacccatg ggaattggca
21840aagccttcct ctggtcctgt gaggatgctg atagtctgtc taaaaactag agatcacagc
21900tttctcctct ggatgactgt aaccccagat tgttcctctt cagagactgt ccaccaagct
21960accctgccta cttaagctgt acacaatgaa tgagctgagt ttccaggtta cagcacagta
22020gacactgtcc atcagtgaga gcacagccta gcctaacagt acacatgtct gctttcttca
22080cgtttccaga accaagcctt gctggataga gcatatttgt ctgtttggct tatttcactt
22140gataaaaagt tttcaaggag ggccaggtgt ggtggcacac gcctttagtc ccagcactcg
22200ggaggcagag gcaggcaaat ttctgagttc gatgccagcc tggtctacaa agtgagttcc
22260aggacagcca gggctataca gagaaaccct gtctcaaaaa accaaaaaaa accaaaacaa
22320aacaaacaaa caaacaaaca aaaagccaaa aatccaaccc cccccaaaaa aaaaaccaaa
22380ccaaaaacca aaaaacaaca acaacaaaaa gtttttgagg tttaatttat tgcatgtcac
22440agaatttcac tgtttaaaaa aatggctgaa taatatttca ctatccattc acgtatttgt
22500aggcattcat gtgtgtagtg gtttaaataa aaatagcccc cataggcttc tacagttgaa
22560tgcttagtca ttgagtagca gtactagaga gggaattgaa ggtgtggcct tattggagta
22620ggagtggcct tgttgcagga attgtgtcac tttgaggtcc cagcaacaag gttgctctga
22680tcacatccaa agacattcta ggtctatgtg atctggctgg aattcagaca tgcccttaat
22740acacaccttt aatcccaaac aatgaaggta aagttagttt ataaaaagaa gcacccatgt
22800ttgaaagtga cgtttaatta agagtgatga attagagaaa gatctgctgt cacagagcag
22860agaggaaaga gaggcagcat aagagggagc atggcagagg gagagggagg aggggttttc
22920accagggcat ttgtacagag acaggttgca gagctagaac aggtgaagac agaacaagcc
22980agagaatgag aaggagccag gagattagga cagattgcca atgttaatag gctaagcaga
23040gcattttagt cagaaactga gagaagtcaa attgaatcag ttagcttgga aaggagtttg
23100agcagcaaca gctgagttaa actagccaac agaatccaga aagaactaga aaagatgagc
23160ttactcagca gcaaatctca gaggctaaaa acatcttaga cctagattag actgcatgga
23220ggctagacgc ttccagggct aggcctaggt tagcagacgg agagagtaat aagccttgga
23280gacaacagtt aatacagaag actatgtaca gacatggata tgaacctctc agccacttct
23340ccagcgtcat gcctgtctgc attgttagga gtcatctagg aaaggctaag ggcaggcaag
23400caacttttcc agagatggtc cactgttttt tgcatggctt ttgagaggcg agctctgaga
23460gggaaggttc caagagactt catcccagga ttgctgctta attacgacat gccttttctt
23520gtcactgtta tttagtataa tgactcctga gctttagccc atcctattgg gcatatttcc
23580tgcagatcaa cataaagatg aactttcaca aattaatgct gtttagatga ataaatgatt
23640ttataaaatt cctgatttga tttaaataat tttaggaaga aagctttagg agatagttta
23700gttggtttgc cagaaagatg taataacgtc agaatcaaga atagaatgtg gctgggcagt
23760ggtggcagat gcctttaatc ctagcacttc ggaggcagag ataggcggat ttctgagttc
23820gaggacagcc tggtctacag agtgagttcc aggacagcca gggctacaca gagaaaccct
23880gtcttgaaaa acaaaacaaa aagaaaagta agtaaaggct gcataataaa gaatacaatg
23940agctttcaca actacaccaa aaagagacat gcttgggaca aatttgtgat caaggaaaaa
24000tattcattct agatcaggtc caaggatgaa gccacaagtg tgtgatatga tgaacaagac
24060catggataaa ctgttgtttt gagcttaaag aataaaacac tgctttgaaa ttaactatca
24120acattctact gtaactttcc tttttataaa ttttatctat gagataattt tctaaagaac
24180ttgtgtctat aaaggtatag aaggacagag agaaagaaat aaggtgtggc atctgggctc
24240tgctccatcc acccaaataa atatgtgtgt gtgtgtatgt atgtatgtat gtttatctat
24300atgtatgtat atacatacat gtgtaggtag gtatatgtgt atgtatataa gtatgcatga
24360acacttggga agttgatgag acaagtgaga ggttgggccc ccnnnnnnnn nnnnnnnnnn
24420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
24480nnnnnnnnnn nnnnnnnnnn nngaattcac tctgtaaacc atgctggcct tgaactcaga
24540gaaccgtgtg cctctgcctc taaagtgctg ggattaaagc atgtaccacc acaacccagc
24600tagtttaaat gtttcttatt tttttgttta tgggtctttt acctgtatgt atgtgtgtgc
24660accatgtgga tgcatggtgc ccttagagtc cagaagaggg tatcagatcc cctggaactg
24720gagtgacaga gggttgtgag ctgggacttg aacctaggac ttctaaaaga gcagcaggtg
24780ctcttaatag ctgagcctta tctccaggcc gtcccatgga tttggggggc tttgtttcat
24840tttattttgt tttgagacag ggtgtgtagc tcatgcttga atttactatg aagccctgac
24900tcccctcaaa gtaaagatcc tcctgcctct gtctacagct gctaggattc gaggtcttgt
24960accacatgct cagcacagcc atgattcata acaataaaaa aagaaagaga gacctaaatg
25020gccttagaga taaataaatt attttttttt taaagattta tttatttatt tattacatgt
25080aatgcacact gtagctgtct tcagaccccc cagaagaggg agtcagatct cattacagat
25140ggttgtgagc caccatgtgg ttgctgggat ttgaacttcg gaccttcgga agagcagtcg
25200ggtgctctta cccactgagc catctcacca gccccgagat aaataaatta taatgtatgc
25260gtaaggtggg atcatctcag tctccgggaa tcttgcctgt tactccttcg ctctcccttc
25320tattcatgct tgggtaactg gccctggctg attgatgaga gctgatttcc ccactgccct
25380gtggcaggga ccactgcgcc cacagggctc cctcaggatc ctcagtacag agctgcacag
25440ctgggtggaa gtagagggct gcatatataa cacgatctca actttatttc tttaaataaa
25500aattttattt aaattttata cagctctata taaacgaagg aactattgaa ggttcagcaa
25560ggacctgcca acggttgtca agggtaatgg cgatgtagtg attttttttc ccccttccat
25620tttacttcca tactttctac attaccccac aactggcaag tattatttta aaatgaaagt
25680aaatagtgac agatgacttt gaaggaaaat tgaatcggta aaaagaaagc tgagagacca
25740cccgggaagc ccaggctaaa tgtaatctgg gtcaggcctc ccaggcctgg ggtctcaaga
25800tggtcagctg agggaccctg gtgaccctct tgggccagca gggacgggga ggagccggaa
25860gctgagtacc caaagtgctc ctctgggctc caagggcctg cacagagact gtgtgggaat
25920caaaggatac aggcatgagg actgaggcct gacgaaccca gctatcattc gtcctagaac
25980aggaggcaga gctccaagag tccaaccaag aggcaggaag ctttgggacc cgagatgggc
26040gatgggatta gaaaggcatg tttgcaaata ctttcaaatt tacgatgcac actcactgga
26100aaccccaccc ctgggtgtcc cttccctgcc tcttgccaca cccaatagct gacatcactg
26160gagaaagtcc caagaccagg ctggctggag ctcctgatag gttccaccct cctgcagagg
26220gccctcgaag actagcttgc tcgcccacac cgccagatgt ctgtgtcttt ctctcttttg
26280cctcccaccc tcgtctcttc ctccaacctc agtggagggt cccctgcttc ctggggaaag
26340tagaacttgc cagtgctcac tgtaatgtcg tccctgtagg tgtcatggtc ccccattact
26400gggagcaggt atgcctcaga tctccctcta ttcgctgccc tttcaggctg tctcagtttc
26460tctctgacag ttcctctcct cctgaatcct gcttgttggc atgcgaacag gctcaatatc
26520ttccatctca aaaaacaaac actgggaagg tgttgagaga cagagagcat gggtaatggg
26580tgccccagct tggctgggaa ggggtaactt acaatgctct actgcccagt agggtagctg
26640cagttgtcaa ttaattgtaa atttcaaaat agctagtaga gaggatttta gatgttccca
26700atcccaacac aaagaaatga taaacattca aggcgatggg tatgctaatt gctctgatct
26760gatcaccgca cattgtatac atgtttttga aatgtcaggc tgtaccccat aaatatgtac
26820aattaccgtg cagtgattca agataaaaac tataatttta aaaagctaaa aacagaagga
26880aatagctgcc cttgaccccc ccacccccac aaggtccttc ctgtttgtcc agccacttaa
26940tgtcagagct tcctgtggga gggtggtttt ggtgtacaca gacactcctt cctccctcct
27000tccccataag aggagtcacc cctgtcccac gatgccatgc agggccacat gcgtgatatt
27060aaccagtaag atgtgagcag ggatgatacc tgtctcttat aacaaacgga aaaaaaacca
27120caccaaacca aaaacaaaca aacaaacaaa caaacaaaaa cagggttggt ctgtccctgt
27180gtcttttccc acataaagtt aagcacacaa agtagccacc atttatttat ttgtcccctc
27240ccccacccct ccccgagaca atgtttctct gtataacagc cctagctgtc ttggaactca
27300ttttgtagac caggctggcc tggaactcac agagacacag agattcacct gcctctgcct
27360cccaaatgca gggattaaaa gcatgagcca cgaactaacc agtaccccag agctcttgac
27420tctagctgca tacgtatcaa aagatgacct agttggccat cactggaaag agaggcccat
27480tggacacgca aactgtatat gcctcagtac aggggaacgc cagggccaaa aaaatgggaa
27540tgggtgggta gggaagtggg ggggagggta tggnnnnnnn nnnnnnnnnn nnnnnnnnnn
27600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
27660nnnnnnnnnn nnnggcagga tcctgtgttc atgtgcaaca ctcgatgcaa gctgtgtagt
27720gtttggttct gagcacctga aggggaccaa gcaggctgat gcccaggcca cgggttcttt
27780ctggccccac tgcccactcc caccctctgg catccccatg atgaacatgg ccacagatca
27840cactactctg ctcctctccc agatccacgg agccataggg tccccagatt catctctgca
27900gctaacaagc tgggcagtgt cacctccctc aaggttcctt tcctgctctg agcagcagtg
27960tctcccacag tgagacactc atgtccactg gaagatattg tagccattaa attcctgtgc
28020taaaataact agggggactt gtcaatcact acactcttag ccccggactt ctgactcata
28080gagggtggtg acagctcagg gacctgcatt ctaccaaata gccatgtgtc cctgatggag
28140gaactgcccc tggacaacct ctgcagcaac tgaaccctct gtggtctcct agttcttctg
28200gacaggtgtg accccagtac ctagtgccag gtgagagagt gctagggcca cactaagggg
28260tgacaggaca aggttggagc tggtagatgt ttgggccacc aaagagaaca ggtcagtagt
28320aaaagccatc atggcctgag ccagcctgcg agtctcctct gcagttggga cactcttgca
28380gtgtcctggg gacctcttga gggtagcatg gtcaccaaaa tcctacaagg acagatcaga
28440agtcagtgag gtcaagggaa cagctctagg ttctctgtgt ccctcacgga cctttttttt
28500tttttttttt tttttaagat ttatttattt attatatgta agtacactgt agctgtcttc
28560agacagctcc agaagagggc atcagatttc gttacggatg gttgtgagcc accatgtggt
28620tgctgggatt tgaactcagg accttcggaa gagcagtcgg tgctcttaac cactgagcca
28680tctctccagc cccctctcag tcctgatgcg acagggcagc aaaggccttg tcccagatct
28740gaggagagtc atgctgaagt ccttcctacc ccaccccttc cgaacccctg aacatcagcc
28800ccataactac tgactccccc acccccattc ccttgcttcc actgatccgg tcctcctctt
28860ccctctggcc ccacccattc ttccccagcc ccacctgatt gtacctggtt gtccaacttg
28920aagagggcag gcaggggcag cttctgctgg gcctgctcac tcactggctg tagaaatgag
28980aaaggagatg aagaaaaggc ccttcccatg ggtccccatc ttgccaagac ataggtgagt
29040ccctttggct cttcccccta aacctctcac ttttgagtac ctgctggccc gggagatcca
29100cggcgctcac cggagagaac tgttgagaaa agggagaaca gagaactcag cgttcctccc
29160tctccaccct tctggcctct cccagatttg cccccgcccc ccagcatctc cttcagcctg
29220actgaccact tcccactcag acctcagctc tgcctcaccg tgaaacaggg accttgcagg
29280caggacaagc tgagtacgag gagcccccgg agcagtgcca tgttcctgta tccagaacag
29340ggagtgttag ttcctacctc acgctcgaag gccaagcagt agactgctat ccatgggttc
29400cttgaccgca ccaggctgcg gaacctggac tcaaaacata gcagctgtgg acctcactca
29460ctctgagagg tgggatttcc ataagctttt tttttcacct gtacatttag tcttcattct
29520tttcgtctta cactgtggat cagtcctggg ttcaaattta aagccctcat cttgcaagag
29580gaccttgcgc atctcccttc atgcctttgg ctttaccctg tcttggtaat tcatggcaga
29640agttcttcct gctcccatgt agatgttgag gacccaaata agaatctctg taaatactga
29700gcatgatgcc tggcccccac cctagcaaag ccacctgacc tgttgttcat ttcatccagc
29760ctttctcagg ctgccctggt cctacccaaa ggctctgaga gctaatctgg gctggcaggg
29820cagccagaaa cttctttgtt gaccaatgaa tgactggccc agacaccttt ggacttacgg
29880gaactacaag cctcatccca cttctgctcc aagttctgat ccagggtgct tcggggaagc
29940ccagctggcg gaagggggga ggctctcagc ctagagagcc ttcctttcca tcctcagccc
30000cctacccagg ccttatttca ggcaccagct cttctaaaag gtccttctgt tatccctaga
30060cctccacaac tgtgttcaag aaccttcagc cagggcctca tctccaatct ggatatatga
30120tttttctcgc caagagtagg cctccaggtt ttggagttct agaggtttct cctggagctg
30180cctggacctc tgctcctcac caccccagga cgctgtgaag ctgcaggctc cctgaataaa
30240ttcatccaga ccccttgcca aggtgccagc tgtctacttc ctctgctgcc caagcagcag
30300gctgcaccac ccctccatcc tacctcttca ggcttcttag cgcagcacac gcagcacacg
30360gtgttctcct ggaccagctt gctccccacg ctcccccagt gcagccagca gggcctagct
30420ctctcctccc acaggacctt tgctctcagc caacccccgt tcagcttgtg ttcagtgctg
30480gtaaatattg acctgtacat ccggttaaac attgatatgg gggccagaag accctttccc
30540atcaaggcta cccagaaccc tgcctgagcc tggagaaggg gtttacagga gcagataagt
30600gaggaggttg ggcctggcaa gccttctaat gatccctcaa cataggggat tatccacagt
30660cagtgaggct cagagaggct gtgtggcctg tgtaagggcg cagagtgggc tccagagtca
30720cagccaaagt cccaccacca ccaccaccac caccaccacc accaccacca ctaccaccac
30780caccaccacc accaccacca ccaccaccac caccaccact accaccacca ccaccaccac
30840caccaccacc accaccacca ccaccaccac cacctcatct acccatacta anttgaggct
30900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
30960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tctatgaggg ttacatttta
31020gaacatctct ccttttttcc tttttgagac aatcttacta tatttaggct ccccttgaac
31080gtgtgatcct tctgcctctg cctcccaagt gctgaggtta caggcatgca cagtcacatc
31140tgcctacaca atgtcttagc agcccttagc agcaccaggg gtcaggaagc cctcaactgt
31200ccctttagct ggcttctctt gtgaagggct atgtcttctt ccccttccta gcatggagac
31260ggctcttagc cccagagcct tccttccttc aggttaaaca gcaccagttt ttggtgggac
31320ctcccatttc cctctatctc cctaagcaac gaccttttct gctctgactc tcatctggca
31380cttggaccac aagacaaaac tgcagcctgg gctgtgtgtc ctcgcacatc attcctgtgc
31440ccccctggag tcaggtctag gggaggaaga cagggttcac gactcagaaa agaccactgg
31500ctgtcctagt gtgccctcac ccatcctata gcacgcacat gctgatgtgc cccctccgct
31560ccatcaccat cctctcatgt acacgtgccc tccctcgcca gacacatgca tcactaactt
31620ttctgacttc ccagaaaaat atctgatctg agaagttagg agtctgccat catcagctat
31680ggtccttaaa attaagtcag acaatccatg ggacatgaag ggcaacaacg agaagactcc
31740tcgttccttg ttcactctgc ttttggcagc accaccagca ggaaccaacc tggctctccc
31800taatccctca tctatagcag gtctcccggt gggaatttta gggacctctg tgttctcatc
31860caggggcact gccactcagc tgctcaggga gagacccctt agaacaacaa agaaatcaat
31920gcagatttag gcttcttgtt tcccttccca gcccctccca tcacaggcaa cagcctccct
31980tggctgagcc tcaggaggct gatttatcag agaggtgctc agagaggcac ctctggtccc
32040tctgggtagg tagcaactga gacaggagga gatggtcacc ctgggcatcc tctaccagga
32100agtaaatgag atacccttgc agatgggacc cctgaagttc ctcccggggg cgggggtggt
32160ggtggtggga gtctaagtca cagatctttg ttaccacgtg gttagactga ggactgaatc
32220tgaggtggga aatctgatgt gcatggggaa acacagaggt ccaatgctgg ccaagagcta
32280caagcaggga caggtgctag ggggatgtct gaatgttcca ccccaagcca caggaataac
32340ggaaatggag actctaaagg gcagaaagtg agggtgtgca gcaggggctg cacaggacac
32400atgcaaggcc ctggctgcaa taactgggtt ggggaggcag tcattggcta gccaggggca
32460ccaggacagt gatgccatcc tgtccaaagg gcagtgtcca agccagattt ctaggctcca
32520gggggaggag ggtccgggga gaggggtcaa gattctcccc ctctgagtca aggttggcct
32580tcccatgtgc cccaaatcag gaggcacaga aactgggatg ttgtggtctc acatccaagc
32640tgagaagaca agtgggagcc agtacatgtg tttcagatta aacccagtcg gagacaaaca
32700tgttgctcct cctcctccca gagccaagct gccttcaagc cacatggcag tgaatatgcg
32760gacagtgcag gggaggacac ctctctctcc actggctcaa ggacagtttc aaggggttca
32820ggctggctgg ctcatggcta cgccgctcac cccctggaca gtttggggtt tttccctcct
32880gaaatcttgg aatctgaatc agcctgagat accccataat tgtacctccc aacaccccca
32940gaaaggtcag ccctgcagaa cagaactctt tggtccccac ccatccccct cagccctgga
33000ggctgaactg atgggcagct aaggtccaga cagtggctgg ctcttggaaa gcctgtctct
33060ttcctttgac tcagaccact ccctgccgtg gcttacatca ggaggtgcaa gggctgcagg
33120agggcagcca gaccccacaa accagctagg ctaaatggtg cttattgttc gcaagaggcc
33180atgacctcat ttgtctccca gctcttttgg taagagagaa tgagaggaag ctggacagag
33240aacctagcag gcctcaggca gcccactgct ccttgctgta agggaaccag caccgatggt
33300tctgaaaagc agcgatccga atggagtcag gctgagctgc aggaagctca ccttccttgc
33360tcactgctgg tggaagcaac ttcaggaaga gcccagccta tgggactata gctcctccgg
33420ggtactgctg agtccagccc cagagcttag ctccctgctt cccaccaccc accaccacat
33480cctttcccaa caccattcaa aaccccagtc cagcctctcc tactggtcta cagtgagcgg
33540ctaatagagt cctgggcctc tgtcccccca attctctctc ccctctcatc tgttcacctt
33600ggttcctaaa ctgcaggggc tactataacc ctacctccac ttccttgcac ccctcttttc
33660tgctctctgg ggtgcccctg ccactcccag tccctctagc cagggagcct cttccatatc
33720tgtcttcccc aggctagacc aggcgctgcc ttacctgtgg ttgcggcagc ttctctcaca
33780gcctgcactc tgaggggctc caggaagcag tgaggggagt agctgcctct caaccagcgt
33840ccagcaggct tcagattaca gctactcttt tcttaaagtg acctgactcc atttggaatc
33900tgtgattgca tcattgtctg gtgttaactt taacccactg ctgcccttcc gccatgtggc
33960tccaagacca cacgttggcc accctcctct cccaccacat ctcccttgga tctttatctc
34020tcttcattgg gaccttcatt gggacatgat ggctaacttc aggggcactt gggccagcct
34080ggggtaggtc atgagtctga acttgaacat ctgaaaggat tggctgagag gcaggctgca
34140tggagagact gtgagccagc cggtatggag atgctgggtt cttccaggcg cttggctctg
34200gctcactgca ggtgggagca aggtgattct tctcccctcc tcacctggaa aatgaaggaa
34260tgggactgta cctgacagct ctgaaggttc caaaggacag tggggtgggg actagagagt
34320tggcccagtg cttatgagca ctggctgctc tcgcagagga cctgagttct gttcccagct
34380cacatagcaa ggactcgaaa ctgcttggaa ctccagctcc agagaatctg acgctatctg
34440ctnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
34500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnatgtcagg catggtggtg
34560catgccttta atcctagaac tcaggaggca aagcagatgg acatctgaat ttgaggcccg
34620cattgtctac atagcaagtt ctaggctagc caggtacatc ataagaacct ttctcaaaaa
34680ataaataggg ccagttggca aaatttagct tgccctccta acacaagaac ccaaagtcaa
34740ccccagcaac catgtaaaaa gaagcaaggt gtggtggcac ttgcttgtga tccagcattg
34800tcaaggtgga gacagacgga tccatggggc tcactggcca gccagctagc tggtctactt
34860agtatgctcc cagccagtga gagactgaaa aataaataaa taaataaggg gttaggaaga
34920ggtaacatgg tggctcagtg agaaaagata cttgtcatac aagcctagca accctcaatt
34980ttcagtggcc actaaaggtg gaaggagaga accaactcca aagaattgtc tcctgacagt
35040tttatgctgt ggtacacaca cacgcacaca cacacttgtt acatgcatat gcatacaatt
35100aataatttaa aatgttatgt gtatgggtgt tttgcctaca tgcatatctt tctgtgcacc
35160acatgtgtgc aatgcctgtg aaggctagaa gaggacatca gatcccctcg gagttacaca
35220gggttgttag ctaccatgtg gattctggga acaaaaccat gggttttcca aaagaggctc
35280ttaatcactg agccatctct ccatcccctc aatagtatat atttctgggg ctggagagat
35340ggctcagagg ttaagagtat tgactgctct tccagaggtt ctgagttcaa ttcccagcaa
35400ccacatggtg gctcacaacc atctgtaatg gaatctgatg ccctcttctg ctgtgtctga
35460agacagctgc agtgtactca tatacataaa gtaaataaat aaaccttttt tttttttgtt
35520ttgttttttt tgtttttcga gacagggaga cagggtttct ctgtatagcc ctggctgtcc
35580tgaaactcgc tctgtagacc aggctggcct tgaactcaga aatccgcctg cctctgcctc
35640ccaagtgctg ggattcaggg tttgcgccac cgccaccacc tccaaggctg ctgctgcggc
35700caccaccacc ccaccaccac actacctgac tatttaactt ttaaaggcag ccatctcatg
35760gaaaatgaca cctagcattg tcctctggtc cctacatgac cccatgtgca aacacatacc
35820tgcataaaca cacataaata cataagtaaa cttagtctgg ttgttttgga aatgtgctat
35880ggtttggatt gtgtcccccc aagggaaaaa ttgggtccca gagtagtact attggaagag
35940agtagaactg ttagggttta ggcctggtgg gaagtggcca tggctagaga gacatgccaa
36000ccaaggggaa tctctggctt catcttttcc ctttgctttc aggtcctaag acagtcacaa
36060ggctgcttca ccacatgccc agaagcaagg ggagcagtca tggctgggac cgctaatgca
36120gctgttgatt tccccagata tttgtagtag taagagacag gtgaggaacc ccacagcaag
36180tgttagtaat tgtgtgtgga ggtgccctcc ggggacgggg gccctcctgg ggcaggacgt
36240tcctcttcct catccacctg cactccgaga acaggaaatg gtgactttgg cagagcttaa
36300gcagagcccg ttcatgttac aagtatgtaa attcataagg accagtttct ctccatatga
36360aacagcttca aacaggagaa ggaagaagca aacattaagg aaaagctctt ttattgcaga
36420ggctacactg aagctaccgg ccgccttcct ggaatgtata atcagcttcc ctctgggggt
36480tctgtagagc actgagacat taagtactac tggggtccag gattctgcct atgaagagga
36540gggcccccgt gtccgtgtcc ctcagaacaa agaggaaagg ttggttaagg tgatagtcta
36600gcgggaaggt gaggcggacg ggctggaggc ctgggctggg gctgcttcct gccccctctt
36660cattccactc gaaagcagcc ctgtgttcca cttgggtgag cttcacgggt ttgccagtaa
36720tcttgctgaa gtcgggtgat tcaaacaacg actgtagctc tgtggagatt cagagattcc
36780attaacacca cacacacaca cacacacaca cacacacaca cactccctgt ttgtgtaggc
36840tgattttcaa gaaagcaagc tagaagtgga gtacctcaca gtgacttgtg agctatgagg
36900cactctgtga caggctcagt gacctacctg agaacttata gccaagatgg ctgaagccag
36960acctggcctg agagaatgtt ttgggctgtt ataggacaca tagagataca cacacacaca
37020acacacacac acaccaagga ctgagtctaa tgggaggtgg ttcttcattc ccctcccctg
37080taatggtgtc acatgttccc tgagccaccc tacaaagaaa gccacaggac tcagttctgt
37140cagcaaggtg gcaggctcca agactcagcc ccgagcgcaa agtggccttg caaacatact
37200catgtcctgc agagacttgg taagttcgcc ttcgaagctc agcttcagct tggggacagt
37260cagcacagct tggatagtct tcagttctcg gtcgatgtca tgaatgaact cagaggtgag
37320gctctcttct atcatggtca agttctgggt cacggtcagg ggcaggaaga agatgatgct
37380catacttcct gtcaagggca gctgggcaat ctaacccaac agagatgcgc acaggttagt
37440tgtgagccag aaaaaacaaa acaaacaaac aaaaaaacac caacagctgc cttcccctct
37500gctgtaacgg ggccccagcc ttgtgctccc cagcctcagc ctgggctgta ggctactggt
37560tactggcagt ccttccatga gtagggagtt ttcttctcag cctaaaaccc acagaagttt
37620aatgaacaca cgtttgtttg tggttccgct acggtttcta ttgtgataaa acatgactga
37680aagcaacttg gagaggaaag ggtttatttc atctgacaat tcgcagggtg tcttctcatc
37740actaagggga ctcagggcag gaactgaagc ggaagccgtg gaggaacgct gctttctggc
37800ttgctccccg tggcttctta gcctgctttt ttatgctatc cagaaccact tgcccaggag
37860tgacactgcc cattgtgggc tgggcccccc cacatcaatc actaatctag aaaatgaccc
37920acgggtttgc ccagaggcca gtctggtggg ggcattttat caattgagtt tcacccttcc
37980aaatgactct aacttgtgtc aagttgacca cacgaatcag ggcctggttc ttaggagctg
38040aagtggaatg tcccccagag actgcctgcc agcactgctg accatttgct ttgtatagag
38100cattgaacca gaaatgaaca ataaaatgga tcctttgaac agatgtgttg atcctagggc
38160ctgtggacac agcgactggg cttcccagag cccccatgga atcannnnnn nnnnnnnnnn
38220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
38280nnnnnnnnnn nnnnnnnnnn nnnntggcct catcaggtat cagagagaga gagagagaga
38340gagagagaga gagagagaga ggataaaagg ttagcccagt ggtggtggca cataccttta
38400attccaacac ttgagaggca gaggcagggg gagctctgtg ggccagtttg gtttacagag
38460taagtttcag aatagccagg gctacacaga gaaaccctgt cttgaagaga aacacacaca
38520cacacacaca cacacacaca cacacacaaa taagatcttt aagaagaaaa gaaaggatag
38580tggggaaaca tctgagcaga ggaagaaatg gggtgcgcag gacacccacc ctcagaggag
38640gccctcactg gaggtgtctg cacaggagaa cacttgcact cagcttgccc tagggcgtca
38700gaactcagaa ttcagtttca aagcactgac aggagcagtg actggggacc ccaggttgaa
38760tccccccttt atctaaaatg agtaagaacc aaaaaaacaa aagtgtttgg gatttggaat
38820ctgggttatt tgcatctaca aaagaggtct tggggaggaa acccagttct acccctggaa
38880ttcatcagtt tcctataccg ctgactacac aggggctgaa ggtaatctca atgttttcat
38940aactggtgtg gtgctacttg ctcatgatcc caacacttgg aaggtaggtc agaagttcaa
39000gagcagtctt gactactcag tgaatttgag gctagcctgg gctacatgaa aactcataaa
39060acaataaaag aaaagaaaag gtggcgagtg aggtggccca tcaggtaaaa gtgccaacca
39120cctcgcctga aagcctccac acggaagggg aaagccagct tctacatgtg gtcctctgcc
39180ctccgcatgc accatggctc gtgcaccccc acacccaccc acccacccac ccacatgaca
39240taaatacttg taatgattag tttctgaaga acaatatttt cgttgatctt gtttagggaa
39300caaagttcgt gcacattgac ctgtcgaccg tgtagtacgg gatccgctcc aggaagctaa
39360agattttggg atgcttcatg ttgcagctac tctgatgaac agtgttgctt tctgggccag
39420gtaatggtgg catatacctt tgatcccagc acttgggagg cagaagcatg tagatctctg
39480tgagttcgag atcagcctgg tctacagagt gagttccagg atatccaagg ctatacagaa
39540aaacccctgt ctctaaaaat cactaattta aaaaaaaatt cctttctaaa cctatataac
39600aaatgttttg taggctgcct taacaaagcc caatggccat tcagagaagg ctcaaaagag
39660aaagtttagg ggactctaca agcatcctca ggaaggccac agaaagcaga gcctgggcca
39720gtgagacttt gcagtgggca aggttcagct ctttatgtag gaagaagaga gtcaacagtc
39780agagtccagc tttccataaa acctgtgcag ggcctctagg caaagccctg tgttaggggc
39840aaaggcattt gcagtctaag cccggtgaca tgagctcaat ccttggaacc caggtggaag
39900gagtgtgctg actccacaaa tttgtcctct gatctataca tgtatgcacg tgcacgcaca
39960ctcacataca tgtccacatg cacacatgca tatacatgcg catgcgtgca cccacacaca
40020gggtcaaaag cagcaagaga tgccctgtga aaaacgtctc attcagtctc ccatcatcca
40080gtgccacact ctgagcacag gtggtactga tatcgttcct gattgatcga tcagttgatt
40140tgagaccccg cctcactatg tagcccaggc tggcctggaa ctcacagtga tcctcttgct
40200tctgcaagat gagcccatca tgcccaccat gttattgaag caataccatg ctctataaag
40260caaacctagg caggcaggat ggtggactcc tgtaatctca ggacttgaaa agtagaaggg
40320agatgaggag ttcacatcaa tctcccgtat gcgttggagg ctggagtggc tgttccctgg
40380gcgcttctgc cagcacctga ccaatgcaga tgcagatgct cacagccaac catcagactg
40440agctcgggac cccagtgagg gtgctggggg gaggactgga ggagctgaga cgggattgca
40500agcccatagg aagaacaatg tcagctggcc aaaccaccca gagctcccag ggactagacc
40560acgaaccgag gactgcacat gaagggatcc atggctccag atgcatatgc agcagaggac
40620agccttgtct gacagcatgg gaggggaggc cattggtcct gtggaggttt gatgccccag
40680tgttggagga tgctggagcg gtggggcagg agtgggtggg taggtgggga gcaccttcat
40740agaggcaaaa gggatggggg agaaggcaga tgggatgggg gggttgtgga ggggtaagaa
40800agaaaaaaga tgtctctgaa agtaaaaagt acttgtcact aagcatgagg atatgagtca
40860acccccaagc cccacagggt ggaaggagag aaatgagtcc cacaagttat tttctgatct
40920atacgtgcaa tccatggcat acgcagaaac gcaaagacag acaatgagtt gggtgtggtg
40980gtgcacatgt aattccatca ttcaggagac agaagcagca gagttgttgg aaatctaagg
41040ccaacctaaa gacctacacc caaagaagga caaactataa ggaaaaaggt ggtcgaccaa
41100tgtaacatta aagttagaaa tctctcttca cactgtgtag atactgtaca aggaagagaa
41160aaggcagcca catcaaaaca gtgtaaatca acgagaaaac cagaaacaac tcaagagaag
41220gctgcagggg cctgaattct gttctcagaa cctgcatcaa gccaagagaa tcaaaactgt
41280ctgtaactcc agctccctgg gatccaacac ccatttctgg cctccatcag catcactcac
41340aggtgtgcac acatacacat caataaaaat caaaaccagg gatgaagggg tagggaggtg
41400catgtggatc tgggaggagc tgagaggcac tgggtgaata caataaaaaa tttggtgcat
41460ggtggtgcac gcctttaatc ccagcacttg ggaggcagag gcaggcgaat ttctgagttc
41520aagaccagcc tggtctacag agttagttcc aggacagcca ggtctacaca gagaaaccct
41580gtctcaaaaa aacaaaacag ccgggcggtg gtggcacacg cctttaatcc cagcacttgg
41640gaggcagagg caggtggatt tctgagttcg aggccagcct ggtctacaaa gtgagttcca
41700ggacagccag ggctacacag agaaaccctg tcttgaaata aataagcatt tgttgctgtt
41760acagaaaact ccagcccagt ttccagcaca cacagggtga ctcacaacat cataactcca
41820cttccagggg atccaatgcc ttcttctgac ctctgtgggc accaggattg catacagtgc
41880acagacatgc acataggcaa aacactcaca aaataaaata aatctagcaa aaaaaatttt
41940aactaataat ttaaagaaaa aaataaggaa gccgggggtg gtgtcgcacg cctttaatcc
42000tagcacttgg gagacagagg caggcggatt tctgagttcg aggccagcct ggtctacaaa
42060agtgagttcc aggacagcca gggctacaca gagaaaccct gtcttgaaat aaataaataa
42120ataaaaaata aggccaagta attcttggaa gaatcccaag gggacactaa gtgtatataa
42180aggcgttcca tagggctagg aatgaggctt agcgagagca acttcgctgg tgtatgaaag
42240tccctcagct gcatgtggta cctttaatct aggctctccc gaagcagagg cagaaggatt
42300tctgtgagtt caaggccagc ctggtgtaca tagctagttc caggacagaa agggcgatat
42360aatagaaaca tcntacctag agcccngcca aanaaagggg agacctgaga ccagagagat
42420gactcagtgg ctaagagcat tgactgctct tccagaagtc ntgagttcaa ttcccagcta
42480aaaatttatt taaatgttta ttacttgtat tattatttaa atttaaataa ataagtaaat
42540gggagcctag gtttgagtcc ccaaatcacc aagaaaaaat gttatcattg ctaataatca
42600aattaagagc ataagaactt ctttttaaag aattcttatt tattttatgt atgtaagaac
42660actgtagctg tcttcagaca caccagaaga gggcattgga tcccattaca gatggttgtg
42720agccaccatg tagttgctgg gaattgacct caggacctct agaagagcag tctgtgctct
42780taagtactga gccatctcta cagctcttat caggttgata aaatttaatc tcgtggagcg
42840ctgagaccaa gaactaaagc tgggagattg aaaaatgcag accaccaagg ccctgctcat
42900ttctccagtt ctgatcagct cccgtaccag gggtctaacc aggcctgtgt ctgcttccct
42960gagtagacca gaggccccat ctaaacagcc tgcctgcagc agctcctctc tctaggtgga
43020cagatgggaa tttcagacca atgtcatttc ccaggacatc aacacagcag ccaaatttat
43080tggtgctgtg gctgccacag ttggtgtggc aggatcaggg gctggcattg gcacagtgct
43140tgattattgg ctatgccagg aaccagtctc tcaagcagca gctcttctcc tatgccatgc
43200tggggtttgc cctgtctgag gccatgggac tcttctgttt gatggtcgcc ttcctcatcc
43260tcttcgccat gtgaggctcc ctggggtcac ccagccgtcc ctgctgcctt gactccatgc
43320cagtcctggt gctggagtct actgagattt accattaaac agcaacgttt ctctaaaata
43380ctattaatta attaattaat cacgtgacaa ccccagcgtc catatgggtg tggaaaatga
43440ggaactctac ccatcataca tggcgactat gaagaacaat gtgacagaaa atgctaacat
43500catgtgtgac cgcatgcatc agccctgact gctaaaagtg gacaagcccg aagcgaaagc
43560ccaatgttct acttctaaat gcatgcacca aacgcctccc acaggaccag aggtgcagct
43620ctgatagggt ccttgcctgg catgcatgaa gctgtggaca cgaggcatta ttcgcaagaa
43680cattctagct gtctggaggg ccctcaatcc actgtgttcg cgctgttcca gcaccagtgc
43740ctcctggggc tgcacctgaa aaaggggact gcttaagagg gctcctacca agcctactgc
43800cacagatgca tgatgggaaa gccttctgga agcaactggc tgccaaaggc tctggacaag
43860agatcaccct ctactggaaa ggtggtttca gtctaggttc tgtgggattc caggaaatta
43920gacaacactg gcagtccaac agacagacga tctaaacttc caaggcacag ctggtagaac
43980ttgctgcgga accagacaac aaggtacgag ctactcccat acaacataca aaaaagcaga
44040gagagagtca gagacagaga cacacagaga gagacagaga gagagtctaa agagagtcag
44100ggtctcagga ctgagggtat agtctactgt agagcatttc cccagcatat acaagaccct
44160ggattcaatc tgaacacagg aaaaaagggg gggggggact tcgattatct caaattctcc
44220tttttgtgac acacccctaa agtcactgcc tacttccctc accgccatga agtaaagagc
44280tgtttgcgct tatgtctaca cagtctcggc tcccacttcc tcctcccctc tgcttctgtg
44340ctcatctcct ctgaaaccac tgcagcaagt gacttgtgtt gactgccaca cggaaactct
44400cctcagtagc aggcagcaga gcagagctct gtcttctcgg agcttcttct ctcttgtcgc
44460cattttctcc cacccttaag taccctatct tctctgtctc tgcttgttga tccttggacc
44520cttttccttt ctatgaacaa aatatctcct taaaggatct cttctagttc agggtccccc
44580cgcccccact gtggagaaaa cccagggcct tgcacatgct cagcaggagc tccatccagt
44640ctctagctcc atgacttaaa gcatctctgt gctgtcaaat atacacttcc agcccttacc
44700aaaatattca gtcaactcct tgccattcaa aatggatgac ctcaaagcca gagtcagcgg
44760tgctatgact cccagatcca tccacttggt agcccaggaa tgaactcann nnnnnnnnnn
44820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
44880nnnnnnnnnn nnnnnnnnnn nnnnnnnngg ttactgggat ttgaactcag gacctttgga
44940agagcagcca gtgttcttaa ctgctgagcc atctctccag ccaccaccac caccaccacc
45000accaccacca ccaccaccac caccccacca ccaccccgcc ccacctgctc attcctgatt
45060ggttggttag tttagtctgt gagacaggag ctgtcccttt tctatagtgg aaggtgaata
45120agaaactcct gaaagtgaag gcctacaaaa cagccacact tatttgttgg aaaatactgt
45180aaatgtgaca tgtaaataca tgctaaaata attcgttaag tcagtgaaca accttaaaac
45240acagtctgta gcctgaatta cagacacgac acgagccatg acagaggctg aaataagacg
45300cctttgcaag gagaagggca gaagcttcca tccttgctag caaatctttg ttccaagctt
45360tatcagattt tattgctttc tctttctgtt tttctttatc atatttgttt atttgttggg
45420ggaaagccta atcttcatag cccatgtatg tgagcacttc agcatatgtg tgtgaacacc
45480aacagcacac gtgtatgagc accacagcat aggtgtgtga gtaccacagc acatgtgtgt
45540gagtacgata gcacgtgtat agagttcaga ggagaactga gagagtccgt cttttcctcc
45600tactgtgtag gtctcagggg tggaacttgg gctcagcctt ggtggcaagc tcctttatcc
45660acagagtcat cctgccagcc cagctttctc tttttctctc tgttatgtct atccactctg
45720ttcaaggcta actcactgac tctgagttat cagaactgct tgtgagagca ggagtaactt
45780tggacatctg tgctggtagg aacaccatcc ccactcggct tggatgacga aggggaaaaa
45840aagcatcacc aaggagttcc accacctcaa ccagcaaata tttacctcct atacatggat
45900aggtggggtg ggtgagcctt gtgatttatc gttaggatct catgggagtg attacagctg
45960gtctactcca tgaccaaaat ggtgacggtg gctgaccaaa aagaaacagc tacacctggc
46020tctagttttc tttctttctt ttttcttttt cttttacccc acggtactaa ggattgaacc
46080caggaatgca agagctctgc caagtgagct acattcccag atctgttttt ccatttcttt
46140ctttcctttt agattttatt tcgatttatt tgtctatgtt tgtgtgtatg tgtgtatgct
46200tatgtgtacg tatcagtatg ccatgggtat acagaaacct gagaaggcca gaagagagtg
46260tctgggttac aggagtttcg agctgtcctg tgggttctca aatgcagcag caccaccacc
46320aatcaccccc acccccaccc agccttcgag ttcaattctc atcatcacaa aaacacacac
46380acagacaagg gcctgcaaga tggctcagca ggtaacgaag ctcgtgtcat aagcctgaga
46440acctgagttc actgtctgga acccgcgtaa agggggaagg gaagaatcaa ctctatgatg
46500ttgtcctctg ctctccccat gtgtgccatg gaatgaacag ccctcccaaa cacacatcaa
46560gaataaataa aactaaaatt agcttagtaa cttttatgtt gaaagtggtt tttacatgcg
46620tgggcaacaa taacaccgag agtagaaagg caagcatgta tgtcactgaa cagcattgaa
46680gaaaaaacaa acacatttcc tgtacatcgt tctgggagtc tgagttaggg tttctatgct
46740gggataaaaa caccctgaca aaaattaacc tggggaggaa gctgtttatt tcagctttta
46800tgtctacaac atgacctgtc acccagggaa gtcagggcag aaattcaaac aggtcagaag
46860cctggaggta gaagctgata cagaagccat ggagctgctt ctggcttgct ctagcacaca
46920ggagtactag cctaggggct gtactgccca cagtgggcta ggctctccca cagcaatcat
46980aaatttagaa aatgcactac aggtttgcac acaggccaat ctggtagggc cattttctca
47040attgaggttc cttcttccaa aaggacttta gcttgcatta tgttgacata aaaactagcc
47100agcatattgg gattatagat attctcataa aaaaaagaca tttagattcc cacataacac
47160catattcaga aattaactca atgtgaacca gaagctctga aagtaagagt taaaactatg
47220aaaaattctt acaaccatcc ataacaaaaa tctgatgccc tcttctggag tgtctgaaga
47280cagctacagt gtacacacat ataaataaat aaataaatat ttaaaaaaat atatgaaaaa
47340tcaggctggt gagatggctc agtgggtaag agcacccgac tgctctttcg aaagtccaga
47400gttcaaatcc cagcaaccac atggtggctc agaaccatcc gtaataagat ctgactccct
47460cttctggagt gtctgaagac agctacagtg tacttacata taataaataa ataaatctta
47520aaaaaaaaaa aaactatgaa gaactatgaa ctacaagaag tcaggaatag ggctgggggt
47580gtaacccaac agaaaaacac ttgcctggcc tgcgtttggt ctctagcacc accaacgtag
47640aaagagaaca gcagaggatg agggcatcct gacttgagtc aagtgacaag tgataatcct
47700cgagacacca aaatcacaat gataaaagag atcaacaagt tgggctttat ctgaataaag
47760agctgtgtcg ttaaatacca cgcaggaagt gaagaggagc tgagtctggt aacacaggcc
47820tgaaatccaa gctactgggt ggactgaggg aggacaacag ctagctcaag gcccacctgg
47880acgccagagt taactcagag agcagcttgg gtagctttaa tgagactctg cccaggccag
47940tgcacaggag agatggctca gtggttaaga gcattactcc tcttgcaaag gacctgagtt
48000caattcccag cacccacgtg ggcacttaca atcatccata actttagttt caggggatcc
48060aatgcccttt tcacagtacc aggcatgtac acagtgcaat tacatacata catgcatgca
48120tgcatacata cacaggcaaa acttacataa aatactaagc agataaatct taaaagaagc
48180cgggcgtggt ggcgcatgct tttaatccca gcacttggga ggcagaggca ggtgtatttc
48240tgagttcgag gtcagcatgg tctacagagt gagttccagg acagccagga ctacacagag
48300aaaccctgtc ttgaagaaaa taaaaaaaaa aaagaaaaaa atcttaaaag aaaaggagag
48360gactggagag atggctccac agttaagaac acttgttctg aggtctacag agtgagttcc
48420aggacagcca ggactataca gagaaaccct gtttcgaaaa accaaaacca aaacaacaac
48480aacaacaaca acaaaaccac ttgttcttac agaggacttt ggtttgattc tcagaatcca
48540catgatggtt cacaaccatc agttgcaggg atccaaggtc ctgtcttctg tgggcaccag
48600gcatatatgt ggtgtacata catgtataca ctcatataca taaaataaaa agttttaaaa
48660aggaggctgg gtttgtagcg cagaggtaga ggtaaaaaga ctctagcttg tttaatgttg
48720acatgaaaaa aaaaagacat ttagattcct gcatcacacc atatccaaaa attaactcaa
48780tgtgaatcat aagctctgaa agtaagaata agcctagtat gcactgtaag gctctgggtt
48840cactccccag cactgcaaaa gatcatgaaa ccagaaatgc agatcctctg aaccacagca
48900tgggaatgta actcagccga tgcagtgctc acctgtcgta tacagagcac aggataaatt
48960gattgtggtg gtgcatacct ataagctcac tacgtggaaa gtagaggcag gacgaccaaa
49020ggttcagtga catccttggt cacatagaga atttgaggcc agtctggtct gctggtctat
49080ttggaatgct gtctcaataa ataaaagaaa gaaagaaaaa gaaaagaaga agtcctatga
49140ttgtcttaac ctctgacctc tgtgttcatc aagtctcctc ctcaggaact cactggtcat
49200cttgtgaaaa cctaccccag agtctctgtt cagaggaccc aggctccagc tgtggttacc
49260acataggatt tttatactag aaaaataaaa tgaataagta tgtatttttt aaaaaggtgc
49320agagctggat atggtggtgt ctagttatag catccagaac tgagacagga tagccatgag
49380gttgagaaca gctagactat acggtctcaa caaacaaaag taagggatct gagtagatga
49440ggttttaatt tttttctttg tgtttgttac ctaacgtgta tggttgtttt gaatacatgc
49500atgtctgtgt atcacttgtg tgcctgaaac ccaaggaagc cagaggaggg catcgggtcc
49560cccggaagta ttattacaga aggttgtgag cagccatgtg ggtgctggga atcaaatctg
49620aaagagccac ctcgggctgg agagatggct cagtggttaa gagcactcaa tggctgctct
49680tccagaggtt tggagatcaa atcccagcaa ctacatggtg gctcacaacc atatgtaatg
49740ggatccgatg ccctcttctg gtgtgtctga agacagctaa agtgtactca aataaataga
49800tcaaaaaaga aaaaagaaac agccacctct ccactctccc tttttaaaat cctcttgcct
49860ctgtccctta atgttaataa cacaggtata tgatactatg ccttgtttat gaatagaaaa
49920tacacgtgct aaagcaagtg tgaaccttaa atacattatg ctgagtaaaa ggagtgagtt
49980gcacacaaga cttttctgct caagagtatc tgtatgaagt attgaacatg tgaactctga
50040aatcgggagc tgaggaagat atggggagtt ctaatggcta caacatttct ttttggaatg
50100atgaggatgt tctagaactc aaaaatggtg ataactcagc atatatacta aaactcattg
50160aattgtacac tttaaatgaa tgcaataaaa cttgtctcag taatgtggtt tagaagatgt
50220acagacatgt gtgtgtgtgt gttaaaacat ttcttggcat ggcaataaaa atacagtttt
50280agccaggtgg ttgtggctca aaaaataatg ataataacaa taataaaaat aatgaaaaca
50340gaggctggag agatggctca gcggttaaga acactgactg ctcttccaga ggtcctgagt
50400tcagttccca gtaaccacat ggtggttcac agacatctgt aatgggatct gatgccctct
50460tctgatgtgt gtctggaaac agctacagtg aaagtcattg caaggacttt acaatagtga
50520ccatgataac attgaagcta gacttgctac tactgctgag tgtgtctgct ggctctttct
50580aaggagtaat gttagctttt tgtcctaaat ttgtttcctt cctttcctct ctccctctgc
50640tgttttttct tacccctctt ttactttgct ttcccctctc atctcctctc ttaacagagt
50700tgtcctatgc agcccaaatg ccatcttcct gcctcagcct ccccagtgtt gaaaaatact
50760ctttccacag gttatgttag gagactggag tctgctcagt cggggaggga gcctgggtca
50820agttctgagc tcaattcctt ttctttcttt ctttctctct ttctttcttt ctttctctct
50880ttctttcttt ctttctttct ttctttcttt ctttctttct ttctttcttt taagacaggg
50940tttctttgta taccctggct gtcctggaac tcactttgta gctggcctgg aattcagaaa
51000tctgcctgcc tctgcctccc aagtgctggg attaaaggtg tgcaccacca ctgcccagcc
51060ctgggctcaa ttcttaacat tgtggagaga aaagtattgt agctgttctg gccacctgga
51120attactttgt ttctgatctt ttgctgcagt caaatccttc tcatccatct ttcctcgtca
51180ggctataata tagactctcc ttgcaatact tggaaatgct ctacagtcag ctacatcctc
51240agtcctgctc ctatattttt tcctaagctt ccttctaagg tctttattgg tttatgattt
51300acacagaaca tttttttttc ttgtctatag catgcgttag agtgatcgtt gccagataga
51360ggaaagagaa atgagagaan nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
51420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt
51480cagctactga ttcctcctcc tccctcctcc ttcctccctc ctccccagcc tcatgctctg
51540ctcatcttgg acttctgcgc atgtcctcag cccagacctt ctgctcttgc ttctcctctc
51600cccagcagcc ccccagttct cttcctgaaa cttctgaggt actctccatc acctcctttg
51660gctcctgctc tgattggtgt cacctgctgg ataggcttgc tcctgactcc actgttcgtg
51720tctcaattag ggaccctcac cctctgatat accacacatt tccctagtgt ctccacctcc
51780cacccccacc ctatacgcac atacacactt agctgcatca ggatcctaca ccagggactt
51840cttacccttc taatcctccc caccggacac tgcccaggga cactggggct ccagagggct
51900attgccacac ggacacacag gagatctcat caaggagatg tgcctacccc agagggtagc
51960tctcaccatt cacaagcaca ccacttctgc ctccagcttc tactctctcg caggaagtag
52020ccagcccggt gccaagtatc cccaactaca tccccaaaat tctcagacac tgccagcctc
52080cagctgtcag cctggccccg gctggcgggc gcctgctcct ggcatagcga ctagggtgta
52140attagaaacc cgctagctcc ctaattgcca gttctgagct gtccttgtta ccggctgccc
52200gaggcacaca tagaggaaaa ggctgagagc tgagccaggc tggcatggag gtagccctag
52260tagacctaga gaggactggc atgtggccag ggaccaaacg tggcacagag agggctcagt
52320gcaatctgcc ccgtgggtgc ctcccagcca catccatttg cccagaactg tgacgtcaaa
52380ccagcccggc ccattcattc tttattcagg tggcataaaa atcactacaa aaactttaca
52440aaagagtctt gggagctaaa gggtcccttc cttgcctcag tccccaagat tcctggcagg
52500ggaggacaag agagagaaga aggaggaaga ctcctggcag tgttggcatc tccaaatacc
52560agaggggtga cttgggtgac aggacacagg ttggggacct gaatgtcttc agcaagggac
52620actcttgtag ggtaggtcag cctccaacca tgaagtataa caccaaggcc agtctaagct
52680tgggagacca acacttgtct ctccttttcc cacccagggt gtctggaata tgtctaaaga
52740tggcctctcc agcctctgct tacaaatgtg gagggaccct aagttaggga cttgcctaac
52800ctacctctag ccaaaactgt gtccacaagt gccagcccac aaaagatcac cccctgagcc
52860ccttgggaag aaatgaagat tccccatgcc tgccttcctc caggccccac cccacctgct
52920gcaagagaac agcttctaca ctggtgatgg tccttccggt cccaccctat cccacaaagc
52980tggttagaaa gagtcacagg agctgagagg ctgatccagg tggggactca ggatgctgct
53040gcccagggcc cctcctcact tgggggagct gaactggggg tagtcttcct ccatgcgggg
53100tgcaagtttc aagtcaggac caaaggtctt gcctccatgg aagtcagctt tgtcattctg
53160gcctatgagc ctgttgtcag gggaatctcg ctgttcctgg agctggggca gcgcgctggg
53220gttagggttc ctcacactgc ccacaaagag gggcacgcct atggtgtcct ccatgatgaa
53280gaagaggaag ggtcggttca cagtgaagga ggagagggac attcgattca tggctacgct
53340ggtagctgcg gctgcctcca caccagcctc gctgagctcc atggtagact gatgttgcac
53400gctagacacc accagattct gctcagagat cccacgaagg tctgggccct ggaacaattc
53460ctgcaggcct gcccagaaca gcagatgact ggtcagtgct gccccaaggc tatgtggatc
53520tgtctagcat cctggctaaa gggaacactt gaacccagcg gttgattgga atctgttaga
53580cctcagtcta gacaacactt ctagaaacct tttttttttt tttttttttt ttttaaatca
53640ggatctgcgc taggtacagg acagaaagtc tagaggagca tatcaaatgc tcccatccag
53700gaagcagggc cacctctggc tcaggcacac tggcagctcc cgtactctgc ccagaccacc
53760taggggcacc ctatccccaa gctccttacc cagttggctg agggtggcca ccaggtccag
53820ctgctgttgc agatggagtt taggcagcca caccttggtg ggcctctcct gcagcgaggg
53880atggtacaga gtatcccagg tcaggttggc tagtacctcg gacacgttcc actcaaaata
53940agtgggcatc acgaccacaa agctcatgtt gttcttaaag gggaaatgag ccacctacag
54000ataagaaaag gagagaacat gaggaccaga cagcacctgg acctgtctgg agtctgggcc
54060aaaattactt ctgtactttt gagacaagag ccagaaattc agggttagca tgctttcact
54120taactggtga agtggaataa taccacttac ccctttgcaa ggtgacatgg gaccaaatga
54180gataatgctt ttacacctct ctgtgtgcac acataagcat atatgtttgt atcggtgtga
54240gtgtgtttgc tcatgggtat atggagtcag aagtaggtaa acatcagtcg tcttcctaca
54300ttgctctcca cttttttttt tttttttttg gtgttgccat ctttttgttg ttgttatttc
54360aagacaggct ttctctgtgt agccctggct gtcctggaac tcactctgta aatcaggctg
54420gcctcgaact tgcagagacc cacctgcctc tgcctcctga gtgctgggat ctaagatgtg
54480tgtaactaca catagctccc tcttttttgg acacagggtc tcatggatcc caagctggct
54540ttgaaatgac tgtttggggc tggagagatg gctcagcggc taagaacact gactgctctt
54600ccaaaggtcc tgagttcaaa tcccagcaac cacatggtgg ctcacaacca tccgtaacaa
54660gatctgactc cctcttctgg agtgtctgaa gacagctaga gtgtacttac atgtaataaa
54720taaattaatc ttttttaaaa agagaaagaa atgatggcta catacttctc tctcgtctct
54780ctgccccaag tgctgggatt acagagctgt acaacaagcc caagtttgtt gtgttttaga
54840catgctaatg tatcccaggc tgtcctcaga ctctctatgt aattcagaac gaccttgaac
54900ttcttttaag gtttattttt atcttatgtg tatgggtatt ttgcctgagc atttgtctgt
54960gtaccgtgtc cttgcagtac cctcacagtc cagaggaggg caccatttcc ccctgaactg
55020gttgtgagct gcatggtggg tgctgggaat caaaccctgg tcctctgcaa gagaagccag
55080taagtactct taactgctga gccacttctc caccttgagc ttttcttcct cctatctcga
55140tctaaaagta ctagggatgg cggatgtgcg ttcatgtgcc tggtttatgt gttgctaagg
55200gttgaacaaa gggctttgtg catgccaggc aagcactcaa caactgagct acacatcccg
55260acagactttg actcttctag tagtagtgtc tccactacag cctgagttct ctatctgctg
55320tcagcaagct gtacaaacaa gctatgggcc ttcctgtcct tgcctctcag ttctctccgc
55380aggtggggct actggctttc aaaatgaccc atagaggagc cacagcaaac agtaggaagc
55440ttgcccctcg tctttcaccc tctcccagag agtcagctat aattcgagtt tttttttcct
55500ctctctctct ttaaacagga tctggttatg tggccctaac tatcttcaac ttcagtcttc
55560ctgcttcaac cttctgagtg ctgggattat ggtgtaagcc accacactca gctcacacaa
55620cctttttttt tttttttttt tttaaagaat ccatgcagtt aggacagcat ggaaatgacc
55680aggctcaggc ctccctgggt accagcataa tgcctgcagg cgggtcctct gccagtgggg
55740ggatggaaag atggagccag aggatctttc ctctctgaac ctcaatgtcc cacagtgaga
55800cactcatgtc cactgggaga tactgtagta ttcaaggaag aagcaacagg aaggtgagag
55860ctaagtggag ctgagcaggc tcgtatcctc tcaccacggg ctacagagaa gtctggctgc
55920cccctccaca tggctcctcc ctgcagaact ggcaatgctg ggcccggctt gcccagtcaa
55980actaaccaac agaatggatg agcatgtgtg gtgccacaca cctgggaccc cagcactcag
56040acagctgggg cagaagggtc atgagtccaa agcgaacttg tgtaacattg tcagaccctc
56100gaacaaacaa aactagcccg tcctgttatc tcagccacag atgatgggcc caaggatcag
56160tactctagcc aaggagtcac ggttaggcta gaagcaaggg aagccttagc tgagacagct
56220tggcacggag cttcatccaa tcagaatgtt cagagcaata agctttgaaa cccgacttcc
56280atctatgaag cactgtgtgg gaactcctct cttcccttac gagcagggcc ctggtcctct
56340tgggctccgc taaaacccca gcacagagaa cagttacctg gcacgtgaca aaaactcaat
56400atattttctt tgaggagatg aacctcaaag aagctgtgtc ctggatagac acagcataat
56460aaacccttca ggagctacct acccagggac cagactttac ctcccagtac caggcctcgt
56520ttgccagcca aaggcaaagt ccagactgac ctgtatctca ggttgctcca gcaggaacca
56580tcgaagagga tatgacaccg cgtgcatcat gtccaccgac actgtgaacc gctcatccag
56640gtggaagaaa tctttctggg tgaggctcgg gtcaaacttg gtcctccaga aacctgcagc
56700caggcagagg gcaggagcca tgtaacataa aatcagcctn ctgcctgtct tgcctagaac
56760ctatnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
56820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnaaccaa ggcaggtctt
56880ggaaaaagga atcttaaatt agaagatgcc ttgataagat tggcatgtag gtatgtctca
56940ctaatgattg atgtggaaag tcacgaggga tggtgtcacc ctgggcagat ggcctggggt
57000atataaaaac acaggctgaa caaaccacaa agcagtagtc ctcaatggct tctgctttag
57060tttctgtctc aggttcctac cttgacttcc ctcagtgaag gcatgtcaca tgagagttgt
57120aagaggaaat aaaccctttc ctccccacat agtttttggt tatgatgtta tatgtcaaca
57180acagaaacta taactaatat agttggtttt ctttttttgt ttgttttgtt ttgttttgag
57240acagggtttc tctgtatggc cctggctgtc ctggaactca ctttgtagac caggctggcc
57300tcgaactcag aaatccacct gcctctgcct ctgcctccca agtggtggga ttaaaggcat
57360gcgccaccat tgcctggctg gttttctttt ttttttaata catttataat gcattttaga
57420tttaaaaaaa aaaatggcca tggcatataa tataaaaaga agtgcttaca aatcaccatg
57480tgcccttgcc ataaattatg taaaaatttc catatggaca tcagtctcaa gcttacaatc
57540tcagcactca tgagcctgag gcagaggcag gaggatggtg agctcaaggc cagcttagtc
57600tacataacaa gatcctgtcc aaataataac aacagtaata atttcataca tagaactaga
57660aggggccact gcaaagacag tatgacaaaa ccactggccc tgcctaattg tattttaaat
57720aactgtcctc ctctctgtaa ttttcagttt ctaattttta cataactacc atgtattctt
57780tttgtaattt taattagttt tttaataata gaaacaagct aagtgctaag aatattttca
57840tatgaacatt ttcaaggcac ttgatacata cctcagattt gccctccagg tgagcagtac
57900caattacgtg ccaccagcaa tgttagcttc cttttttccc taccatctga ttctgtttca
57960gtctattcgt agttctgatc ttgttatatc cctttttatt gtttccctgg gttccaacac
58020ctcccagttg agtgttctca ttgaatttca ttagcagctg tttcattaat ggcacagaag
58080aaggattaca gtgttaacta ggatagactt tgacaaagaa ctatgagaac atatcttatt
58140atctttgcat aaattctttt taatcaaagt tcctcaaaag cctctctctg ttcccatctc
58200agggagtagg tctggccact gatgagtgtc caggccacag tacaggtgtg cgtggttctg
58260tccctgtggg aagggcacat ctgtgttgta acaggattcc tgtcttaaca agccttgctc
58320aggctctaag tggtcctgag ctagctaact gcccttggct ttcccttgat taccagataa
58380ctattcactc ttctcatttt gcagagcact taccaggtag ctatgtcctg gaagtacgaa
58440tgagtccttc tattgttttt cttttactta aatcccattt gaaatgcgcc agggacactt
58500caatccaagg tacacttttg ctaaagaatc actcattttt atatgcaaaa tgtcacctat
58560taactgcagc tgatatggta catacatatt ctctcttcct attatccact aataggtgac
58620taatgcgaaa tattgagtaa tttttaaaaa tcaatactca attttttaga aataattaga
58680gagacattca actctgacac cagcacccta ctcagttcct gagccttcct ctgccggagg
58740agaatctata aataactcac gaagctgaca ttactcactg tgttgcagtc atttttttct
58800gagaaaattt tagcaactgt tctaatagag cctgccagtt atcagtagtt gagaatgcaa
58860gtcaactttt aattatgcag acgctgatta ttcagacgac aaattgttgg tgcctgcacg
58920gctccttcct gctgcctacc tttaaccgtt ctcagtgctc attagcacat gttccagaag
58980gtaggctttg gaggggcgga caggcactca aaccagctaa gcacttagag aagctctgat
59040gaaagatgtt aatgcagttt gtagaattat tgactaaaat tgagtcattt ggattccctg
59100tgaattgtat ttacatgccc tgtccctgtc ccccatagca acagataata ggattgtctg
59160cagagagaca acatagttct tatatttaat tttttccttt gtcgaacatt ttcacatgat
59220ggttcgtggt gtttcctttg ttcattacat ttgtatccag actagttact tctgataagc
59280ggttagttag gattcctggc acgcggacag tgacaccaca gttgtctgat cgtttcccac
59340ttttttacaa aaccgtttgc ctttaagagt cagtgttttg cacatttcac ccagattatt
59400ggaaatatta tttccctcct gcttaaaccg aagctgtgat cataatttaa gcctttctag
59460gtagccgatc ttacatgtat catacctatt tctggcatat gtttgtctat tacaaagacc
59520tcgtaggtat gcagttagaa gcctctagtt aaatgaaatg ttgcgtgtgt gatgaacctg
59580gagtggggat ggccttttgt gtgccccaag gctgttgtgt ttcacacagt tgttttctgc
59640ctcctctggt ctatcactat cctgccactg ccagaaaacc ctgctgtgtg ttccccgcgt
59700ggaggatctc tgcttctgaa cttctttggc ctgagaaact ccataaccaa atcagttagc
59760attttgttta aagagcaggt aggctgttag agcttgggtc ttacatgtct cccaggtcca
59820cttgccagcg ccttgaccac tgttaacttt tgttaaccaa ctcatctttt gctgcctgtt
59880ttttgggggg tttttttggt tttgtttaag ccaagatcag ttatatggcc caggctgagc
59940ctctcttccc agcctctcaa atgttagaat tacaagcatg catccctcag catacctttc
60000ctttgctttt tttaaaatag agttttgcca tagcaacaga aatctaacct aactaagcat
60060agccgtgcac atggtatgag gaactcacat atgtgtgaat ggaagttcat agagaccggc
60120atcactgcct agaggcccct ttcttccttc cttgcagttg tcgtgctagc tgactgtact
60180acaaaagagg ttgtctgagg cataagacta ccttcaataa aacatgcaca gacagtttgc
60240ttctctgaga tttcagagca gtgactacct tcaataaaac atggacagac ggtttgctta
60300cctgagactg cagagcagtt tccaaaaatt ttagacaaag ggtaggatga agaaggctgc
60360ggggttttgc acacacttaa ggtgcgtaag taaataaact gagctacact gacaggatgc
60420tcgttctagt agccaaccaa agagcagttg aaccaaagca cctagacttc aaacatcgtg
60480gggagataat cttaggagtg ctatgcttct gcgtcctaca agtattatga aactgtctag
60540aaagcacccc actggtaatc cctttttgat tatttttttt ataaattcta gtcttggggt
60600tttgagtggc acacagacat aatggttagg cttcggtgtg tgctcattca ctttgcttcc
60660tggggaccag agtttgcgat gagtcatgtt ccatctgatt tctgtcggat ccggctgcag
60720agccatgact cagatgggct tcaggcccag ctgctcagtt catcttctgg ggaatagatg
60780acaaggacgg gacaaatgtc ctgacgcaca tttccttctg ttcttgcact tccagggtct
60840aacgagagca tcattaccaa cagcaggcag atacgccttg ccacaggcat cttccctgtt
60900gtcagcctcc tgaaccactc ctgcaggccc aacaccagtg tgtccttcac tggcactgtc
60960gccaccgtcc gggcagcaca gaggatcgca aaaggacagg agattctgca ctgctatggt
61020gagccagcct ttctttccac taccctgctg tgcctcacac ctcacatgaa aaggataagg
61080ggacaggaat cagcagatat gggcccagtg cctctactca tcctctgagt ctttcctgga
61140aagggcaatg catccttggg ccaataaaaa aggtcttctg gctgtaataa aaaagcccgt
61200tgagggcagt gagccatatc cctccatgcc ttgtagacag cctatcctga aaatgagcga
61260ggagcacttt cttggcttct ttcttcctgc cccagcagct tggaaacgta tccactttca
61320cccgtgtttt gttgtttttt ctgagatgat agggcagagt acccaacctc atataggcta
61380ggctagtgtc tatcactgag ccaggacccc aacccagcac caccatgcca gtcacgtgat
61440gactaggcca gcccctcggt agagtaggca ttgactctct tggtgtgact aggaactgtg
61500ggtaatctct ctccagggcc tcacgagagc cggatgggcg ttgctgagag gcagcagagg
61560ctgagttctc agtacttctt tgactgccgc tgtggggcct gtcacgctga gacactgaga
61620gcagctgcag ctcccagatg ggaagccttc tgttgtaaga cttgcagagc gctcatgcag
61680gtaaatctct gctgttccca ggggcagggc tccagctaaa ggttgtcagt cgccaggaga
61740accattcctg cttcccttct tgtaactcct ccctacatgt cgcccggtcc tgcagaaaac
61800acaggttgta tttcctaata ttttccctat aagtgacaca aaatcttaaa ttacacaaag
61860ggaccaaaaa aaaaaaaaaa aaaaaagccc tagaaattta cttgctcaaa taagtcatca
61920aaagttgtgc atcaggccta gcacttgggt actggtaacc ctagcactca ggaggctgag
61980gaagaaggat ctcaagtcgg aggccagtct caagtgacac cccatctaag agatcaccat
62040tccaaggagc tatttcagag atggtttaat ctggggaccc agattgtgga ttttctgtct
62100gttcaattcc atctctctgt gctggcctca tcagacacac tctgtagtaa ctgtgggaaa
62160atccgaccca catagttttc cctcagcctt tgacccagag ggaagagcca cagtggagag
62220catgagagca gacccttggg tgctactgcc aggtaatggt gtagacactg gagtcttcaa
62280cattcatgcc ccaatgcaaa atggtctcca caccagagca tggcattctc attagaaata
62340agtaaatgga attggctgtg ttgaaaattg taaagccaag ggtcaagaat gaagccttcc
62400ccagcatgtt ttgttttgtt ttgtgtttta ggcagcgtct ctctgtgtag ccttggctcc
62460tgccctctgc tacctctccc aggtgtgcca ccatgctggg cctaagcgcc ctgtgcatta
62520gtgctccctc gatcctgctc actcttgaga cagtcttcct tctactctgt atccccagat
62580aacctagagt tcacttcaga gcccaggctg gcctcaaact tgagatcctc gtgtcccagc
62640ttctcaaatg cagtgatatt tacaggccta cacctggctt tccctgatag attcctagta
62700agatgattat cctttgagcc atatctctct tctgcttctt cctctcttcc tgcagggttg
62760atctagaatt tattctaaag ctgactggcc tcagaattgc catccttctg cctttagnnn
62820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
62880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaca tagtgtcaac tttcaaattc
62940tgccttaaga gttctttgtt tatgggaatt tatgggaatg ttccacagaa cccatccagc
63000ggagttctgg ctgttgtttt ttaatcttta ttcatcttgc gtgtgtgtgt gtgtgtgtgt
63060gtgtgtatgc gcgcgtgctc aacttgcaaa attgcaaaat tcagtctcct ctttccaccc
63120tgtaggtcct ggggatcaga ctctgttagg cttggtggta ggtgctttac tgagccatct
63180tacaggcccc ccatggacaa ctttttcttt gaaaacctgt ttctggcttg ggtgtgatag
63240ctcacacctg tgaccctacc accactcatg aggaagaggt aggaggacta acagaattgg
63300aagccagcct ggactacaca gtgagtaaag gctatctata tactcaccac atggcaagac
63360cccgttttaa aacactgggc aaggtgaaac aaaagtcaat taatttcaca taaagtcaat
63420agcttcatta acggcctagt tatctttaaa actgtatgca ggttagtact tggtttcaat
63480tttattactt tttctctgga acatttaaaa gtactttagg ggctggagag tcagttaaga
63540acagtggctg ctgccaaagg actggagttc actcccaagc acccaggtgg caatcacaac
63600tgtctgtcat ctaattctag gggatctgac accctcacag actcacaggc agtggaacac
63660caatgtacat aaaataataa ttaaaaaaat gaaataaaat accaggcaag gtggcacacg
63720cctttaaccc cagcactcag gaggcagagg caggcagatt tctgaattcg aaggcagcct
63780ggtctacaga gtgagttcca ggacagccag ggctatacag agaaaccctg tctcaaaaaa
63840aaaaaaaaaa aaggacttta aattgggctg gagagatgga ttaaaagcat tggctgctct
63900tcccagaggt cctgggttca attcccagca ctcaaatggt ggctcacaac tgtctatatc
63960aacgcaatct aacaccctct tcaggcatgc aggttacatg tagacaaaac atccatatgc
64020ataaaataca taagtaaatg agtcttttaa tgtatactag aagctgggtg gtggtgcatg
64080cctttaatcc cagcacttgg gaggcagagg caggtggatc tctgagttgg aggccagcct
64140ggtctgagta aatagagcct tgtacttcta cttatcacta cagttacatt ttataacttt
64200gggccctagt gcttccattt tccactgttt gcttaaccac tggggcctga agcttttgtg
64260ctgacacttt tgttcgctaa tcatcaggca accaatggtc tctacactcc atcaccatca
64320acacaaacaa aacaaaacac aacactacgg atcctggcat ggtggaacat ctttagcccc
64380agtacgtggg cttgagttca aggccggcct ggtctacata gcaagttcta ggatagtagg
64440gatagtcttt aaaacaaaac actattttat ttatgaacaa aacatgtaaa gaaagaaaaa
64500aaactgcaaa tttatctatg aatgaagtct aagtaatact tcaatattgg aaatagcttt
64560ctaaaatatt tttatttaaa gaaaactcag caaattattc aaacaacctt ataaacgttc
64620gttataaaag taaagaatta tttgcaattg ccttaagggt ccaaggtggc agcctcttaa
64680aattcagaac aatccaagct tcacattcca gttcaacatt tctacagccc taacgtattc
64740aaatacctcc attctgacaa ctgtttcccc tcttcttttc ttctaagctg cttagatgtc
64800tgtcccaggc ttttcatgat tttagtcatt cacacaacta gcaaacatta tctagggact
64860aaaacttgcc agatactggg atatcaccct aaagggggac tgaaagtagc tgcaggctac
64920agtctctaca atctcctgaa tgaaatacaa agtagctaat atttaccaaa taaacatgta
64980cacctgtgat gattgctagc tgtactagca gaagctaaac actaaatcta gaaactcagt
65040cctccaacta gccccttgct cggcttcagc ctcattttta caaacaaggg aaagagtttg
65100gaatgttgcc caaagccata cataagtgaa caaaaaggag ttggagtctc caaatgcatg
65160gatttgggct agttactttg ccaaccaact cagtaacaac tgagctgaac aggaacactg
65220tggtagcaaa agaaactgga actatcaatg gcctctagag caaaaatata tttaaaaaga
65280aaaaaacaaa caaggcctgg caaggagact gtgagaagag tgtgctgact gaaattgact
65340agttcagcca acaaaagact attccagggc tggtgagatg gctcagtggg taagagcacc
65400cgactgctct tccgaaggtc aggagttcaa atcccagcaa ccacatggtg gctcacaacc
65460atccgtaaca agatctgact ccctcttctg gagtgtatga agacagctac agtgtactta
65520catataatca ataaataaat ctttaaaaaa aaaaaagact attccagtgg ggatggaaaa
65580gttaagtgtg gagttaaaat atacttcaac tggtgatgga ctaggtgtcc agagtcgggc
65640aaaaggatgc tctgtggtag aggtgcctgc tgtgtaagcc cagctacctg agctcaatcc
65700acagaatcca cagcggagtg ggaagagaaa caacgtccca gagttgtcct ctggcatccg
65760acgcacattc gccatcccca agatgtcata catatgtgta catactacac actggcgcac
65820gcgcacacac actctttttt aaaattcaga cttagaggga cataaaggat ttgctctgat
65880atatgttcaa ttgaaaatga ctttgaagat agagggcaga tcgaaggaag ctcagcagga
65940aagaattaat aacatgcagg tgaagggcta taaactagtc tgcagagggc cttggctcga
66000caaaaaaatc tatggggttt gccggtaaaa taaggaaaaa gttgtcaaca tgaaacacag
66060aacactagca agagaggagt gttagcagaa agaagccaac aagctcaaac aattaggtcg
66120gctgaaaaat tttaaaatgt cttctgattt ggctactggg aagccactgg tgacttcggt
66180cagcgttttc tctctcgtga ccagagagat gtctagtagc aataatgagt taggaggatg
66240taaaagaagt aaaacagccg aaaacaagtc caaaaagttt ggggtgatgg agaaagggag
66300gaaacagagg ccgccgaaga tagacagcgg catgtttatt tgtcttgttt tcttagatgt
66360aaacaaacta aaaaaactcg tgagttcttc tgccagtacc gggttgcctc cagcatcctc
66420tgatggtctt agagaccccg ggatgctccc ccgcggccgt ataatttcct ccctgacgct
66480ctcccgatcg acagcggctc cctccccggg tcctctttgc accgctccaa ggccgcgctg
66540ctagggccat cgagcccgct cagggtcgtc tccttacctc gatggccccc tcgctcaggt
66600gtcccaccat ggctgcaccg ctaactcccg cgctcgcgct cttgcaccgc ctgagcttct
66660ctgccggggt cccgcgggct gctcaacgat tggctagagc aactgtgcgt gccgatccgc
66720ccccagcgtg agcgcggtgc gaggggcggg cctagacgcc gatagccacc gcattggcta
66780ccgcgcggca ggcagagcac gtgactcttc cgaggccggg ttcgaggcct agtggcggga
66840tggcgggacg tgagggcggg gcgctgggtc gcagtgcgcc tgtgtcagcg cggtgctact
66900gagttgttcc cccgccagct gtcggaactt tgcccgccca gtcctttggc ggacagacag
66960aatggcaacc cagggaacag tcggagctct cccctggtaa ctgctgctaa atatagtcaa
67020agcagtgacc tgggtacttc ttcacgcagt gcgtgcccgg cgccggtgcc aggcccagag
67080cttggcactg tgggataaac aaggtaaatc agactcagtc tccgccctct tgagttccac
67140ctgagagttg tggccgcaag gaacccagcc tcaaggatgg tagacgcgat atgggccaca
67200catgtggagc tccagagtgg gggtcaaaaa tcaatcaggc tttcgagagg cgatgcggtt
67260tgaactgagt taaagtgtgt gtagaaattt gtcaggtgga ttccagtgag gatagtgatg
67320ttcctaaaag cccaaatggc ctatgcaaaa gtattggaga gcctggcgtg ctggctggct
67380ctgatctgtt tgtaatccca gcctttggga tgtagaagca gcaaaagttc aaggtcaccc
67440ttaacaccgt tgagttcgag gtcaacctga actaaatgag accctgaaaa atcaaaattt
67500gggacccagg cgtggtggca ttcgaggtaa aagcaggcag atctctgagt tcgaagccag
67560ccaggctaac ataagatccg gtctcaaaaa aaaaaagtaa taaaaataaa aagggagaga
67620ggctatatga actgaaagaa agacctggag atcaaaacag aaaactgagc cgtctaagaa
67680atgaaaatat ttaacttcat agttgctgga gtaagaagtc tggaaaactt tgggcaacta
67740aggtaaacag gtctagaaag actggaatag tagccatcta ctggtatttt gatctctgtt
67800tgtacaacca caacctacta tagtttctca aacagttcca aagaatatgt ctgggtgaat
67860tggtaccaca ccacagatta actctccttc agcatatcaa cagctataga aaaccccaga
67920agaaatgatt ttggttgcgt gtcacttggt aggatgaaat ctcgattttc tagaactatg
67980cattaataga aagctgaatc ttcatgttct gactttacag agctgcggca gcatggatct
68040accggtggat gaatggaagt cctacctact taagaagtgg gcttcactcc cgaagtctgt
68100gcaggacaca atttctacag cagagacttt gagcgacatc ttccttcctt cttcttccct
68160tcttcagtaa gtgaatggaa acttcaggga aattttggtc tggaaaatgt tctgccttgt
68220catttggtct gaatatctct tttttatagg agagagtagc tttatattct ttatagtatg
68280gggcatttag cagttactgt tggttttcac gtttctccct agtctgtgat tactagaatg
68340ggtaggcact aactgctttc ctcttttggc atgtgttata cttaaggaat gtagtatctt
68400gctgtcgtcc cagtgctgtc actcatagga tctggtgcag gttgtgtagc tgcccctaga
68460agctcattca gtcctaatgg ggagaaagaa ccctggcact tggttagttg agacccanaa
68520cttctcaagt tctnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
68580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnntttagtt
68640tccaagtgcc actttactgc aatgtgtcac cacatccaga gttctgtgtt tgtttatttg
68700tctgtttttg agacagggtt tctctgtgta gccctggctg tcctggaact cactctgtag
68760accaggctac cctcaaattc actgagatct gcctgcctct gcctccagag tgctgctgta
68820cactaccacc acccatccag ctttctatat tggttttcct atggcgtttt aaaatagcca
68880ttatacgtgt gtttatcatc taaagtcctg gtcccaaaag gagatgagag gggctgctaa
68940ggtgaaaagg attacaaacg cttatcaatt ctgttcaaaa attaaacctc agagtgggat
69000tagctgcttc tttcattaga attgctatca gaattcactc aggccttgtt tgcgtgtgtt
69060attgaagaag tctcttcctc atcaggtcag tgactcctta gctcaagtac atgcaatatg
69120cagtattgat aactgttctc gcttaggaat aaaaatagaa ctgacttcca cagggaaatg
69180atgtgctgag ctgtagcaac gaatcttgca caaactctgt cagcagggac cagctagtct
69240ctcgcctgca ggaccttcag caacaggtct gcatggccca gaagcttctc cgaaccggta
69300aaccaggtga gattggctcc ctcgccctag gcctcagccc ttcccttgtt tattttggta
69360tcaccttgcc ttactgagca gtcctcaata aatgactgag gacttgaatt taattatccc
69420agcaccagcc acaagatggc tatgtaggcc agtgagacca gactgtgacc agctgttact
69480ctggtgccct tgaaagtctt cctgatggtt taagctgtgt ctgctgcgcc agatagttct
69540agcagctcga gcaccagaaa ggctgtctga cttccatggg ctttgtgtgg ctccagaggt
69600ccaatgccat catctgattc ccagcttaag gacctaagct ccgagaaggt tgctctgccc
69660tcagcagcag cagcaagtcc tgagtgctgc ctgggctcgt ggtgtgactc aggagtagag
69720ctcggtagct agcctgagct gagagctgag agaaagaaag gactcctctc ttttcagaaa
69780gggatttgca gaactcgatg ttagaccctg acatggtagg aatctgtttt gactattcta
69840gcctagattc tgaagttgac ctttagccta gagtcaagaa aactaatgat tacaggagga
69900atgtagagtt ggttgttaaa tgttggttgg aaaatggatg ttagaagccc agggtaaatg
69960tgaggaagcc tcatctaaca cctcttttac tgaaagagaa aacataagca accaacagct
70020tccctggaat gcccggctgt tgactccgtg agataaagag gcattttcac tttgacctaa
70080ccgatagaga ccttgcaacg tggtctctcg tgtccaggac tagatctgta tctgttgtga
70140ggcatttttc ctttgaatcc atagagcaag ccattcagca gttgttgcgg tgccgggagg
70200ctgctgagag cttcttgtca gcagagcaca ccgtactggg ggaaattgaa gatggcctgg
70260cccaggccca tgctacctta ggtatgctac cttaggtata gccggagttc tccttccctg
70320ccgtgtgttc agtgcggccc ttgccttgtc tgtttggttc tctcttgcca tctgaattga
70380cgctcttctc cctcccattc tgcattcctt gcccccagag ccttaggcta atggtgtttc
70440ttttccggaa tgagacattt ctcttctcac agggaactgg ctaaagtctg ctgcccatgt
70500acagaagagt ctccaggtgg ttgaaactcg ccatgggcca tccagtgttg aaattggcca
70560tgagctcttc aaactggccc aagtcctatt caatgggtag gcctttcttt ttcctagtgt
70620ttggccaggg cacacagtgc tctgtgtttt cctaggtgct tctgtgtatg gctttttgct
70680acagtgcttt aaagcatgtt gaaactcttt tatttcctct ttaggacaga tatttgccct
70740ctgcttcact gatagacttt aagctttgaa ttccttcctg aggatgtgga gaaagccatt
70800aggtctgcat ggagcttccc agggaggatt tggaggcagc ctcacccgcc tctagcattc
70860ctgtctgctt aatcacacct cccttggctg cctcagtccc tgctctctca actccagggc
70920tcggcccttt ccctggtttg cctcttattc cttttaaagc agtggttttc aactagaagg
70980gattgcaaat ggcatttggc agtgtttaga gacagttttg attgttatgg ctgccagcat
71040ctagtaaagg ctaaacctac agtgcacagg accgcctcca cagtggagag acccaagtta
71100gctatgtgaa ggctgagaat ccctgctttg gagattaaaa aaggaagctg agggaaccac
71160tcagttggaa gcacccttgg tggcatgcac aaggccctgg ttctgtccct agctctgcac
71220aaaaaataga atacaaggaa gagtaaccct aatgagctgg tccctcaccc agtgtgccac
71280tgaggtcact tgaagggaag tctagcccca atttagtatt ttttgtggct gccatacctc
71340cagccttgat caaatctcat ggtatacatt ggtaagaaaa agggtttgaa acatagacct
71400gatactcgga catggaaaca gtatgtttgg tcagagagag cgaaggacct gatagacgag
71460ggcaatatca gagagagggc atcagtcggg ttagacacga gcattccaca gtgagcagct
71520ctggataagc ttttataaat gctggttaag gttttgaatt tgcccaattt tgtcaggatc
71580ccagagtcta tcacaaacat acacagtttt ctcaaatctg ctttgcagta tgcccgtgaa
71640tgtctcttat ctatactttc agatggtaag accctgaggg cagaggaact cagacccttt
71700gtgccccctg taagaccctg ggggatgcag tggacccgac tttgtgttct ctgcacagaa
71760aggagtccac tttcgttgag actaaggaag ggaactgaca agcttccctt tctggcttca
71820ggttggcagt gcctgaagct ctgagtgcca tctggaaggc agaaaggatc ctgttggtgc
71880actgtggccc tgagagtgag gaggtccggg agctccggga aatgaggtcc tgcttactgg
71940actcgtcatt cgtccctgtg gggcccttgg tgtagagcaa tcatcctcac cctcaagaag
72000gagctctggt gatgactgag atgttctgtt ggcttggagc tctcatcaga gaggacggga
72060ccttcccacc tgacctgagc ctagtgtctg gcacagagag cacttgaaaa cagattgaga
72120cactcacctg ccatgctggc tgctgcttgc aagagctaac tgccctctga tggaaacccc
72180atgcccagaa aagactaaat ccagtatcta aaggctgctt taaagggttg tcactgcagc
72240cgggcttggt ggcacacgcc tttaatccca gcactcggga ggcaggcgga tttctgagtt
72300caaggccagc ctggtctaca aagtgagttc taggacagcc agggctacag agaaaccctg
72360tcttgaaaaa ccaaaaaaat aaaaaataaa aataagtaaa aaataaataa ataaataaat
72420aaagggttgt cactgatctg caggcagctc atgctagcct aggcttttgg ctcgatttca
72480tctcactaaa cgatgaatct gtttccctgg aacattccta tggtttctag tagtaatgaa
72540gtgctgtgtt ccactccagt gagaacttca attcttagtc ttgtattata attgaaaaat
72600aatatatagc aagaaatcag tatgactgct tacctcaaga gacatacaat tccacttaca
72660atatcctgct tccttaaatt tttcattaag actggtgata tataatttgt gaatggagaa
72720ataaatacgt cttactgttg gcagtttctt cctgggatgg caactctgta ttggtttcct
72780accagtgtcc taattcttac tcagtggctt tcattgagtg ttcttggcac tcactgtcca
72840agcactgatg caaggcaacc ctgtagcatg acttcatagc acaggcctcc ttgttagcac
72900acctgaaagc agaccactct ggctgtttca cttgcagaca gaatcttact ctgtaagcca
72960gtctagcctc aaacaacatc ctcctgcctc agccttccaa gttctaggtt tataggaaaa
73020ggccaccttg cccagcttga gactgcttct tactgccatg tctcttcagg ctcacacatg
73080aagtccaggg cactccagga ggagccgtga gtctgtctgc agggcactcc agggggagcc
73140atgagtctgt ctgcagggca ctccaggagg agccgtgagt ctgtctgcag ggcactccag
73200gggaagccgt gagtctgtct gcagggcact ccagggggag ccatgagtct gtctgcaggg
73260cactccaggg ggagccatga gtctgtctgc aaggcattcc aagagcagcc atgggcgtca
73320ctcattggta gactgtgagg ctacatctcc agatgccccg agtgctgtgg ttgtgagcac
73380tgctgctcat ggtttccaac tgagacagag ggaaggactt tgcccctttc cctaaggatg
73440ggtagtaata gtccagacca caagggacag atagctatgg ggttttctga ctcatcctta
73500gtacattatt gctgatgacc agtttgtttg gatgagttag tgggaaagaa gacccaagtc
73560catacactct gctttttaga acttgctcat cctagccatg cccaaggagc agccgttgac
73620tgtcatggca ttacagtgag gaaataaaca gtcctgaagg tgcctggcag cagcttttca
73680agaagctggt gttaaaagac agtattcaaa catctgcgga ctgggaactg ggcagcattt
73740gagtctcctg ctgtctgtta atttaccctg acaaggaggt gacttgaaag gtttgttttg
73800tttggggtag agctttttca ggaaaaaagt ttagtcctac agacaactct atagttattc
73860tagtccaaac tcatgccttg tgttttattc ctaaaagccc tgtcacactt tgtaaaatag
73920gtgctcttcc tcaaaggata tatttaacgt tttatatatc aggccttatt ctgtgcatgg
73980aagctttttt tagatgcttt gtaagatggc tcagtggtta agagcatgta ctgctcttct
74040ggaagtcctg ggtttgattc tcagcagcta acaccagctg ttattccagt tcctgggatc
74100tgatgccctc ttctggccta tgtgagcact gcatgtgcgt agtgcacaga caaatgcagg
74160caaagcactc atacataaaa ctaaattcaa aaaactcttt cattgtctca tgtgacctag
74220cttgagaata cctgtgctta tattataatc tagtatgagc cagccacggt agcaacacac
74280ctattatctc agcactcaga agattgagac tagatggtca agagctagag tctgggttac
74340aaaacacctg tctcaaaagt aaaagggctg aaaaagtgtc tcagcagcta agagcacaca
74400ctgcttctcc agagggcctc atttcagttc ctaataccca caccgagtga ctcaaccacc
74460tgtaactcca ggtccatgag atccaacacc tctggtcgtc tgcataagct cctacactca
74520attatacaga gagagagaga gagagagaga gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
74580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
74640nnnnnnnnnn ntctagagtg tttcaggttt tttttgtttt tttttttttt gagacaaggt
74700ctctctatta tgctgcctgg aactttctat gtagaccagg ctggactcaa acttatagtg
74760atccactact tctgcctctc agtactggta ttgaaggcat gtgtcaccac accccactac
74820ttcaagatct tagatttcca aagaagccgt agcctagaaa aggttaataa gtactgattt
74880aaaacagaaa gaaatcaggt acacttagag ctgtagaatg tcagcatgtg acatttgtga
74940caagttgtca aaactttgct cttaattcta aagagagaag ctgtcaaaag acttgaactg
75000gggctgtagc caacttggtc gagcccttgc atgaagctgt gtgtttactc cccagcactg
75060tggggtttga attgatttga acccagtaga ttcgtatatt tgaatgttta cctcatgggg
75120aatgacatat tacaaggtgt ggccttgttg gaggaattgt caatttgggg gtgagctttg
75180aggtctctct gctcaagctc tgcccagggt agaaagggag cctcctcctg gctgtctaca
75240gaggacatag tctcctggct gccttcagat caagatgtag aactcttggc tcctccagca
75300ccaagtctgc ctgcacaatg ccatgcttcc taccatgatg ataatgaact gaacctctga
75360aactgtaagc cagccccaat taaatgtttg tctttataag agttgccttg gtcatggtgt
75420ctcttcataa caataaaagc ctaactaaaa cacattcctg ctgggcagtg gtggtgcacg
75480cctttaatcc cagcacttgg gaggcagagg caggaggatt tctgagttcg aggccagcct
75540ggtctacaga gtgagttcca gaacagccag ggctacacag agaaaccctg tctcaaaaaa
75600aaaacaaaaa caaacaagca aacaaatgcc agcatttggg aggtagagtt aagaagattg
75660ggagtacaaa gtcgtctcag ctagtatgtt tgaggccagc atggaccaca tgagacgttc
75720tcaaaacgaa agaaacgaat gaatagataa acatttgagt gtccagtttt ttcctttctt
75780tcttgctttg tttttggcgg tgctgaggat taaacccagg accttgttca tactaggcaa
75840gcattctcca ctgaggaaca ccctggcgag tgcctagtct gtctgtctgc ctgcctgcct
75900gcctgcctgc ctgcttgtta tgtgtatgag tggtaacctg catgtctgtc tgtataccac
75960agacatgcct ggtatctgca gaggccagaa gaggatgttg gatcgcctgg aactgggatt
76020acaaatggtt gtaagctgcc atgtaggtat tcagaattga acctggtgct ctgaaagagc
76080agccagtgct cttgttgttg gttttattgg gggcaggagg tagttatttg gttggttggt
76140tggttggttg gttggttggt tttcttgaga cagggtttct ctatgtagcc ttggctgtcc
76200tggaacttgc tctgtaggct caaactcaga gatctgcctg cccctgcctc ccgagtgctg
76260ggataaagtc atgtgccacc aactccagac aagcagccag tactcttaac cactgagcca
76320tcattccagc ccttctttgt gttttgagat ggtcacaaag tacaactcag actgagctct
76380tgatcaccct ccctcagcct cctgactgct gggggttaca ggtgtgtcac tgtcctcaat
76440tctgagtgtc agatcttgaa aacccattct cgtgaccttg atccttaaaa caaaccctgg
76500gagaatgagt tctgataact atttctcact cctcttcaag aaaaggaaag ccagagaaag
76560gggggggggc aagccccaga aacattgata acttgcccaa agttacacag caaaattcag
76620acagcctgca catctcagtg gccatctgtg ccatatccac cctgcccttc tctgacctcc
76680ccacctccat ccctacagac cttgcagttg agatcagagt ccaagccgta tcgtaagatg
76740gccttaggat ctgacatcat ggggactctc acggtcctgt cctcgtccaa atgaaaatcc
76800tggagggtcg tctttctcga gtcaaacttg gttacccact gccctggaag gaaacagatg
76860gagcatcctg agccactgtc cccagaaagg ccacaggtcc acgctgtgcg tccactggcc
76920aaagcaacct gagctgtcag cagcaagaac acaggagccg ctgggtccca gcatgtgtgg
76980cacagaccat aggctccatg caccacgggt tctggctatc ctcctgtagt aaactcagaa
77040ataagtgggt gttctctctc tgacttggat caccacgctc cttctgttta aagtggcctt
77100taatatgctg gtgtgtggta cgtgcctgct ctcctgtccc ctggggactt ggagtaggaa
77160gccccagggc tttcctctaa gttaggatcc actcttgcta ctactccata agatggtcac
77220aaagcaacgt aaaatggaaa ttaatcaaac cattcctgcc acaagaataa aacagatctc
77280aggggaggcc tgtggaaggg tctcctgagg ccttaccact gtctagaagg aagttgacag
77340cagttcttga gcaggggtgc gactccagga gttgggggct gctctgagag caggacagca
77400tgtattgtag agtgtctggg agggagctgt gttatcctta ccgtgaagat gaggacacgg
77460gctcatgggg gcagagccag gattaaacct ggtctgattc aaaaagccag agatctgtgc
77520ccagccccac gcagccattt cactggtcaa ctaattcaga aacacttggt ctgatatgct
77580catatgctac aagcactgtg gccttcagat ctccctctgg cctggtacct gcattcaggt
77640tcaccaccat caccacacac acacacacac acacacacac acactcggcc agagacaagt
77700ggggaagccc tcacccttga agtaagccac gccaaggaga aggatgctga gggcactggg
77760catttccctc gtggaccggg caatcttccc tttcatctgg gcctgcaccc agttgttaat
77820ctcctgaagg tctactcgag ggttgcccgt gaggatccgg ggcctggtcc cataggactt
77880ctccagaggg gcaacaaagc tggatttgac tcgaagttct tgagaggaaa cagatcaaag
77940atgagagctg aatcagcacc ctcactttga aagcatgcca gaccccagct tcctgctcag
78000catcttcctt tgacttgctg gggcatctgc cggcttgccc agaccctggc tagggaacag
78060tggattccac cgtttgcatt ccccgtccca ggccctcctg ctgtctccca gagcccactt
78120cctctttctg ttcctctgtg gtctcactgg ctctttcctg cccaccagtg ccaggcctcg
78180cctgagcaca cacagcctat tgtttagaca tcatggaagc atacagacaa cccaggccaa
78240tgaagcaact tcacgccagg cataatgggg cgtgcctgcc cttcagaagc agaggcagct
78300ttatgagttg ggggaccagc tgagactcta tagactttga gaggtggggt gggggtgggg
78360ctactgactc ctctcaaaca caattctgga agcactcttg aggttcttct caggggcagt
78420aacagaggca aggagctcct tgtaggtgct gtggatgtca gggttggtga tcaggtcgta
78480gtagagagcc cggtgaatga cagactctgt tcgatgttca gctcctgcca gagagaaaag
78540gatgccaagc ttcataactg cccgtgaggc ccgcatcagg atagggacgt tagacatcaa
78600tccttttgtc ctctgagagc ccgaggaggc cgatattgca gatgttttag ctggacaaga
78660tcttcagggc gtggaaagaa ataatgaccg ccttgctagg aagagctcta agacagggca
78720aggttatcag agctacagag agaagagtgg gatgtggtcc tgaagttctc ccatcgtaac
78780ctaccctgtt ctgaggagga gccagctctg ctcacggcag ctgtacccct agaacctggt
78840taaatgacta aaacacgata ggaggccact taaggaacca aggtcgagtg ccacttacaa
78900agtggtaggg attgtgtgtg tggcccccac cgcccctttc ctgttcctct gacggcggca
78960gcatggaaac tctgagtggg ggaaattcag gtccacctgc agccttcttc agttgacact
79020cacccagaga aagggcagag agggccgtgg ccacgctgag tggagacagc aggacgttgc
79080ccgttgggct ggcactggat ctcaggcggt acagatcgta gccgaagttg gagacagctg
79140ctgccagctt gttcacaggg accttgaaga aggggtcctc ctcctccacg ggctcgcccg
79200tgctgtccgg gactggggag ccctgggtta gaatacaagg accagtaggg aggcacagtg
79260agtacatcac ctcctggttg ggttggtcct ctagtccctg gggccatgag tctgaggtca
79320gaatgagtgt gtgctctctg actccacaac ctgtgtgctg ggaggtgggg agtgggaagg
79380gcaacacaaa agggcttgcc agacctgaac tgtggtctga gaacctgaag cctggcccac
79440tttaaaataa aacttgtagg gctggggaga tagcacagta gataaagtac cagcatgcaa
79500gttcaaggac ctgggttcag tccccagagc tgggcacggg ggtgcatgct tataatccca
79560acactgggga ggcagagatg ggcaggtcct ggggctcatt ggccaatcag cctgaactaa
79620tcagcgtatt ccatctcagt gaggggtcct gtttcagagg gcctgaggaa tgactctggg
79680ttgactacta gcctactctg tgtctgtttc tgtctgtctg tctgtctgtc tgtctgtctc
79740cacccctctc tgtccccttc cctctgcagg gaacttcctc accaccacca acccccaaag
79800aaacccaccc tcagaccagt cttccctatt cagcttgctg gctggtccta gtctgcctag
79860gttctgctgt gacgccctcc ctgtctttcc tgacaagcca tcccctctga ctagacccga
79920gaggaatttg tcgttttctg acctgttttc agtgtcagcc tcttccttat gagactttct
79980gcttttttgt tttgttccca gggctttgga tcaaagctgg gctcttacat acgttaggca
80040aatgcttggc cacccagctg tacctcccgt ccctgttgct tttcggtttg gaggactttt
80100tttttagttt tctgtttggt ttttggtttt gattttttgt tgtttgtttg tttgttttga
80160gacaggattt cgctatgtga ctctagctgt cctgggactc actatgtaga ccaggctggc
80220cttagattca gagatccacc tgcctctgcc tcctaagtgc tgggattttt agattttaat
80280ctgtacctac caacctcaaa ggaagtgtcc atggatagag ttcagtacta catcatgtgt
80340gtaacatgtg tgagggcctg ggcttcaccc ccaacagaga agggggagtt agtaggtgag
80400gaataagtga ctggctagtg gcaagacagt attgtctaag gtcactaagc cttaagccac
80460acttaaagcc cacaatccag gtctaatatg cccatctgcc ttgtccttgt gtgacatgac
80520cccaccccta cttcctccgt atagtggcag ctcctctgga tcctgaaagg agagggaaga
80580tattcttgtc tcgatgttaa agtaaccaag gcctagaaga gtgaaggcca aagcccaccc
80640tggatccagg gctgcctccc tgcactgtct cttctgctgt cccacctacc caccctactg
80700acctcagagc tgctggggac gttctggctg ctgccgtgcc cgagcagggc tccagtccag
80760aggagtagca ccagggcctg catcccggaa ctacaagaga aacaagagag caaacgactc
80820ccctcaccca caccctcccc tgccactgca cattgcacac tgcacaggga caagagtcag
80880gcagagtgag cccttcccct ccctccagct ctcagcccca agtggaccct tgacttgagg
80940tcttccgtcc ctgacctgcc cctgcacttc tccttgagct gtgcccccat gttggttcct
81000atcaggagac ccaccttccc atctaagctc cagcacaggg aagacccagc agcaggctct
81060tcaggcccca agacaatgct ctagcacaaa cacacaccaa ggcttttccg tggaggcaca
81120cggccagctc ctttggtagg atttggaacc ctgtctcagg atgggagcag agcccaggtc
81180atagacttac agaacatctg gtctggtcct gctatccacc aatagttctc tgaccaaagc
81240ctatgttaaa gacacacaca cactttcttc taggtaggtt cttgtgtatg tagcccaggc
81300tagccttgaa gttgcagcta ccctacttca tttgcctcct gagtactaca atgccaggtg
81360tgagccatca tgcctgactt gactcccttc ttctatttca agagcaattc ttagttaaga
81420gggtatgaac cagggccaca ctgccnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
81480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
81540nnnnntctta tttttagctt gtttgttttt cttacttgag acctggggag gggaggtgta
81600tgtgtcttcc atttgcttct tctacctaat aaagtttctt ggtgatgttt gggggggggg
81660gagggggtag gactcaagaa gggattctcc cataagctgt tccgtttggg tagtactatg
81720taaggaagtt acaggtgggc agagctcggc tctgcctgac tggcgtgctc tgaggtaaag
81780gtgagatggt gcaagatttg ggccctcagg agttggctct gttggccctg taccttctgg
81840tctgtgggta aggatgacca gtaggtgaga gatgagggaa ccagaacaga aggtgaaagt
81900tagtggggcg gagccccaga ctagtcaggt gggggtaaac tagatgactt tctggaaccc
81960caaggggctc ggagactagt ggtgttggag aagacctcta atgtgttgta aggcctctga
82020actcagtagc cgaacttgat gccagaaagc cccaaactgc taaacccaag caggagcggg
82080acgccatccg ttccatggct tcacccgagg tggccccatg gctgcgccaa tcaatgagca
82140gccgagagat aggggcgtgg acaagccagg aaaagttaca gcacgctgga aagataatac
82200aggccaggaa gccccaggca cagcagggtg gaaaagctag atcccgattc tgccggaggg
82260gggccccttc gaggtcccgg gcaccgggtg ccaggatcag agaaactgac tgaaacctag
82320ctgacctgcc cagaccatgg catcctgggg actccttgtg gctggcgctt ccttcacggc
82380gtttcgggga ctgcactggg ggctgcagct gctgcccacc ccgaaatctg ttcgggaccg
82440ctggatgtgg cggaacattt tcgtttcgct gatacacagc ctactctctg gagtaggggc
82500gctggtcggg tgcggaactt ggggactgac aaagcactga ggggcggggg tggaaaagag
82560ggcctggaag actgaagttg gaaccttttg gaatggaact ggtttgggtt gtggatgggt
82620gggagtaccc agtgggagaa tggatctagg tctgggagaa attgacctta gctctttgtc
82680ttctccaggc tgtggcagtt tcctcaaatg gtcaccgacc caattaatga tcacccaccg
82740tgggcacggg tcctagtagc agtgtcagtg ggtgagtgta cagaaaaggc tgaatcggga
82800aaggccttgt tggaccggga attctaggtt cctcccccat ctttggaatg gagcagatgt
82860tgctggaggt ttgctgtgag gaattaagga cctgagaaaa gtgggacttg agatatctag
82920gctgtgcatc agctctgagc gaggagcctc atagtcttct ccggtgcctt caggttattt
82980cgctgcagat ggagttgata tgctgtggaa ccagacattg gcccaggcct gggaccttct
83040ctgtcaccat ttggcggtaa gactctgaag ggagaggcca ggtagtaagg gagcatgtcc
83100aactcaaggg cccaacctct ctcttcagtg ttctgtcctc tgacttttcc acaaagcccc
83160ctgaaaacct atcctctcag acttggattg agttggaggg aggttttgac tggctagcca
83220ctcctgggca ctgcccaagg agtttggttc tccccacaaa cctccagctg atcataaaaa
83280aaaaaaaaaa aaagccagga atgaaagcta gggtatgcta tgcaaatagt gtggcttggg
83340gtaagagaac ctctggtcca gggctgctca tgccccctag ataagggtca gcagaaaggt
83400caggattgga ggcagtccta aaaaatgctt gggtaatata aagtgaataa ataaaaaata
83460aataaatact aatttttaaa aagctgatac ctggaaggat gaggcagaga gtagaaaaaa
83520catgcgtggg tgtccctagg ataaggagct gggacttgtt gggcacaggt catgcaaagc
83580ctgaaccttg aaccttgcct gcaggtagtg agctgcctca gcaccgctgt tgtgtctggc
83640cactatgtgg gcttctctat ggtatccctg cttctggagc tgaactccat ctgtttgcat
83700ctacggaagc tactgctgct ctcccataag gccccatcct tggccttcag agtaagcagt
83760tgggccagcc tggccaccct ggtcctcttc cgccttctgc ctctgggatg gatgagtctg
83820tggttgtccc ggcagcacta ccagctgtct cttgctctgg ttctgctttg tgtggctggg
83880ctggtcaccg tgggcagcat aagcatctcc acagggatcc gaattctgac caaggatatc
83940ttgcagtctc agccctaccc gtttatcctc atgcacaagg aaaccaagac acgtgagcct
84000gttgccagga acacttccac tctcagtctg aaaggtgtgg aagttttctc ttctgtcagc
84060ccccagggag gtggggctgg gaagaggaga tggtagccca ctgcatagtc tactatgtag
84120caaggactag actgtatcat cagagagaga gagagagaga gagagagaga gagagagaga
84180gagagagaga gaacattgta tgagatctcc attacagtca ggaaatcagg agatctaaat
84240aactttaaaa gtcccacagt ctttacatat tcttaaaatt tcaatctctt taaaatatcc
84300atctctttta aaattcaaag tctttttaca attaaaagtc tcaactgtgg gctccactaa
84360aacagtttct tccttcaaga gggaaaatat cagggcacag tcacaatcaa aagcaaaagt
84420caatctccaa ccgtccaatg tctgggatac aactcacgat cttctgggct cctccaaggg
84480cttgggtcac ttctccagcc aggccctttg tagcacacgc gtcatcctct aggctccaga
84540tacctgtact ccactgctgc tgctgctctt ggtggtcatc tcatggtact ggcatctcca
84600aaacgctgca tgaccccttc agtcctgggc cttcaagaga gaagactaga gcctggcaaa
84660gtggcacatg ctgataatgc tagcacttgg gaatgacaag cagaaggatc agaagttcaa
84720ggccagcctg ggctacaaga gactctgttt caacaaacaa acaaacaaac aaaccaaaga
84780agagaaagaa aaaactggac atgacagccg gaacattatc tgacattcat aaggtcctga
84840gttcaatgcc aagttggcag tgcctagttt gataagggtc tagccactct ggtaatacca
84900tggctgactg aacaccttac ccagcaactt gctgatagac tctgccttcc agcaaaaggg
84960aggagcttcg ctgaggagag aacattgaac cctattgtat atgaataaat tgctgtgcaa
85020atgatttcat cagtctcttg tgaatgtgat tgctttgagt catttttctt ggctccagtg
85080ttatcctggt ctgcagtgtg gtgtggagtt gtggaagctt tgagttggga gggtttcctg
85140ttaaggtttc tctggctctt ttctttcctc ccggtttttg ttttgtttgc ctggtggggt
85200tctctggtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tagaagttgg
85260cggggggtgg agggggctgg agagatggct cagcggttaa gagcgccaac tgctcttcca
85320aaggtcctga gttcaaatcc caacaaccac atggtggctc acaaccatcc gtaacaaaaa
85380aatctgatgc cctcttctgg agtgtctgaa aacagctaca gtgtgcttac atataataaa
85440taaataaata ttaaaaaaaa aagaagttgg catggatgat gtagtgaaga ctggcattag
85500atatctctgg atccccctgc ctctacctct tagacactgt gagtatggaa gtgtaccacc
85560gcaccaggcc aggctagaac attctctgat ctacaaatac ctagagtatt attcctctat
85620gatcagaaaa cagacccagg gggccacaga aatgtcttag taggtaaaaa cacttgcttt
85680caggcctgat aacctgcggt tttttgtttg ttctgggggg cgggagaggc tggctggctg
85740gctggcctgg aattcacaga gatccacctg cctctgcctc ctgagtgtca ggtaccagga
85800tcacaggtgt gtgccaccac acttggccta actgcctgag tttgagcatc agtactcaca
85860tggtactgag gatagaatag actctcacca gctcttctga cttccacatg tgccctgcag
85920catgggctct ccttccccaa aggaaaaata aatgtaagaa ttaaaaaaaa aaaaaaaaag
85980caaacccagg tcttgtgtga tggctcagca tcaaagctac ctcccgccac agctgaccac
86040ctggtgataa cttatagcct tgttatgctc tcctttgacc tccacgggca tgctgtacac
86100gtatgtgtgc ccacacaaac acacaatcaa gaaataaatg cagccaggcg aggtggcaca
86160cccctttaat cccagcactt gggaggcaga ggcaggtgga attctgagtt cgaggccaac
86220ctggtctaca aagtgagttc caggacagcc agagctacac agagaaaccc tgtttcgaaa
86280taaccaaaaa aaaaccactt taaatattat ttttattttg ttttgtttat cctggaactt
86340ggtctgcaga ccaggctggc cttgaactca cagagatcca actgcttctg cttcccaagc
86400acattaaagg atgtcccacc actgcctggc taaagattta ttttttcttt ctttttgttt
86460tgttttgttt tgttttttct aaaaaatttt tttaaaaaga accatccctc ctagcactca
86520ggagactctg aagtcagggc cagccaggtc tactgagtga gctctagggc agccagggct
86580ccacaaagaa accctctctc aacaaacaaa caaaagagaa cagacccaac cagacctgag
86640gacacacact tgtaatctaa gcccttgaga ggctgagaag ttcaaggcta gccacaagtg
86700tgtggtgcat tcaagagcag cctgggtggg ctacagaaaa agaaagaggg agagagagaa
86760tggttaatga agatgactct ggaaaagtga aactcaagag aaagcccctc agatttgctt
86820aagacgagtt gagggtggag aaccgccaaa gcggacgagc cagacagaga ctgccaacaa
86880agttcaatcg gttcaggtac attacttcca aaacgccatt gccacatcag gatgcttcaa
86940tcagccaaac caacgcagcg actattgact tctgcatttc agagacttcc gtctctgtcc
87000agggcaatgt cactttagct ttcctttgca gaaaggaaaa gtccctgcct ctgatgtggt
87060agatcctcac acaccttctg ccagatccag acactggtat gactcagcct cggggagctc
87120tatctacaga gataagggta caaggcgtgt gtgtttaaag tatgtgttta aaagtacaaa
87180gtgagagtcc ctggaaaggg ctccctgccc tcaccatcac cgaaagcaca aaccttaggg
87240taatatctga cattcctgga aatgtatgta tgtattcatt atgtagccct gactgtcctg
87300gaatggggta taaaccagga tggcttcaca tctcagagac ccatttgcct ctgcctccca
87360agaactaaga ttagaggcat gcactaccat acttggctca tgatttactt aactttattt
87420tatgttcacg aatgttagcc tgcatgtatg tgtgtgcacc atgtgcatgc ctggtgcccc
87480agaggccaga agaaggtgtt ggttggattt cctggagatg aagtcccaaa caactgtaag
87540cagtccaatg tgtgtgctgg agatgaaact tggttcatcc acaagagcag tatgtgctct
87600taactgtgga ggcatatctc cagcctcaga tttcccagtt aatgtttgct ttcgcaccca
87660ggcccatctg cgcatgcgct ggagacctcc tttaccgcct tgagcctcat tggccaattg
87720tggctgggag acttgcagat cccaagtggt acaagagaag aataaactgg tgtgctatga
87780actcacctct tctctgtagc cattggctga gcatactttg cctcaaccta ccgcccttcc
87840ttcccctaat cctaaatctt tgccctctcc aaatgtgctc ctcccccgca gtaatccagt
87900ggtcgctggg gctctagaga gatggggggg gggggagcaa cgggtacagc ttaaggcagc
87960tgcagcagaa cttttttgct gtatattgag tcttaaaaat tcatataaac tttgtgttct
88020gtttctaaat ataaccccat ctgtttcaac acaaaatgca acaacaaaat gtttcaaatt
88080gctatttgga ataattaaaa aatttcaata cttgatttaa aaatgcttta actttttaaa
88140taaattttaa atgttattat ttttaaaaag ttacaagttt aaaaaaaaga aagatagaaa
88200tcacataatg aaattaacca tacgcaagtg aggctcggtg cactggtaca cagttacagt
88260agccatttgg agtggaggcc atggcgcttc acattgaatt ttatactttc tttatagaat
88320attttttatg cacctatcta ctactgataa caaaacaccc atgagagagt tagaattaga
88380catcaattag ctttgatcct ctgtcataac tcgtgtccac tccctgcctt agtcctacct
88440catccctgtc ctcttttcta catcttatac tgaatccaca cactcagttg tttacacaaa
88500cacatacatc actgtccann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
88560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnag
88620tgcctggctc tgttttgctg ttgttgtttt tgtttgtgtg tgtgtttaga tttggtttgg
88680tttggtttga ttttgttttt ttttagagaa tcttactatg tagctcaggc tgtccttgaa
88740ctcacagaga tctcttgtct ctgccttcca agtgctgaga ttaaaggtat acaccacctt
88800acctggcccc tttcatctat ctatctatct atctatctat ctatctatct atctatcatc
88860tatctatcta tctaaaattt atctgtgtgt gtctgtgtgt acattcccca gagcctgtgt
88920ggtagtcaat aagtaaccct cagaagttgg ctttctctaa tccttggatc aaacttgaat
88980tgttaggctt ggtagcaagc atgtttaccc actgagccat ttatgacccc atggcccagc
89040atcttccatg ggttctgggg acacaaatgt gtactttgat gtttacagga caagcgctta
89100accaaccaag tcattttccc agccccatcc tgactcccat taagtgttct ttcccccaac
89160ccaggaccaa atctagagga gtgtccatgc ccaacaaaca ctctgccaag cctctcccct
89220tactgctctt ctcccttccc ttccttcatt tcttcgttcc ttcttttctt tctttttgaa
89280acaggtcttt tctctgcatc ccaagctagc cttgaacttg tgatgtagct caggctggct
89340ttgaactcac agctgtcctc ctacttcagc ttcccaaaca ctgggattat agacctatgc
89400taccacacct ggctcatttt tcaaataaat aaaaagaaaa tcaaaaagtt cctagaacag
89460tcacaggatt cacaaaaact ttggaaggag actaaaaatg gatttttaaa aaatgcttga
89520agcacaaaga gttgttgaaa gaagagagaa gaggaaaagt tagcttagta ggtagaagtc
89580aatcaagcct cacaccctga gttcaattcc tgaccctatg gtagaaggag aagagcaatg
89640ccggaaacat tatcctctga cctccagacc cgctgtggca cgtgcatgca cacacacaag
89700caggagccct tggagggaag tcctagaaat gaatcttact gaagcaggtc tgccaggccc
89760tgtgctcagc cattttattt ttcctttgtg tacccgacac gcttccattc tcaaagttgt
89820gagtctgaga ggaagtactc actgtgtccc cagtgagctt ctgtcttacc ctgggtcact
89880tagatggggt cacttagtgg tagccttggt gtggagaaag agaacacagg tccgagtagc
89940cagtagacct gagtctttat atctgcaaag ggtgttgggg cataatcaaa tctccccccc
90000tccccggggt cctgatacca ggttgtatta agtgtatgtg catggtcctt ccaagtcttg
90060acacatcatc caactccaag tggctttctc atttttcctt gccagtagcc tcttggtgag
90120gaaatggctg aggaaaacag agttgcagaa agacagggcc atggcctggc tgcaggcttt
90180ctctgagtct gaagagggtc agcgactctg agaaatgaag ctatttctga gtgagagggg
90240ccaaagaagg aacacggcag agggagagcc cccgaggaga tggagacaga agccggagag
90300ggaccctgtg cgaggctgga ggggaggaag aggggggagg agtgagaccc actgtcatct
90360gttgggcaga gaggggctac attcatctgc agtatggtgt agaggggaca gagagtgatg
90420gtaacaggaa aaatttgggg ttgagggggg cagcctgtag ggctgggccc cagcagtgta
90480cagctaggta gagtacacag taactcccag aattctctgg ctccactaaa tccctgttcc
90540gctccgtgca gagtaaaacc cacacagggt ggatttcagt ctcctttgca cccccctcca
90600ccccccctcc acccccagct ctggtcacag ccagtcagag ttgggggtgg ggcagatctt
90660gtaaaagagg ctggtgagga catcggaaag tctgtaccct ccactagcaa agtgccagac
90720gctccgtgac actttaaatg cctcagataa aacagtgaga gactctcctg gtggcaggca
90780agatgatggg tcagggacct cagcgcctct gaggctcaga caccaggata aagaataaaa
90840acaccacgga gacccctgtg accccctcgt gcagagggag aatgccaatg tggcccagct
90900agctgctgga gcgcaagcct caaggcctgt gctagttatg agtctactgc tgctgccttg
90960tcccaataaa cccctttccg cccaggatta gtggacacgc cttgctcaag ccgagtccct
91020gatctgccac cttatcacac acatacaaaa atcccttgag gatggttacc atcctgggac
91080aaagcctcat tctctgctta acccaagtga cacctatatg gcagatccct gtgtccttct
91140cctgatgata acaacccttg caatccaata gaggggaact cgggtttctg tcagcttcct
91200ttatgctgat agaaatgtac tctgcatgtg gggagcctgc cttgctcacc ctgagacccc
91260atggggctgg ctggggcttt gcacatcatt gggactcaga gatgttgact acatgaacgt
91320cccacacttg gttgcacaag gcagaatgac aggatgttat gcctggtgtg tgagtgtgtg
91380tgtgtctctg tgtgtgtaaa acgcctctct ctggagccct cctgtctgtc tgcctcttgt
91440tcaatggctg cacaattgtc ctttctcttt ccaaggacct ctgtatgggt gtgtccttca
91500ttcagtgcct ttcctctgtg ggtttgtcct gctagccccc tgtcactgag aaagtcttct
91560gtctgtcctt gggttgtctg gctagaacac agacatcatt gtcttttttt tttttttttt
91620ttttttttaa agatttattt atatgtaagt acactgtagc tgtcttcaga cactccagaa
91680gagggagtca gatctcgtta ggatggttgt gagccaccat gtggttgctg ggatttgaac
91740tccagacctt cggaagagca gtcgggtgct cttactcact gagccatctc accagccccg
91800acatcattgt cttgcccacg actgctctcc agaatggtgg gcaggaggat gtgacccccc
91860acccccaggc accgggacac aacatcttct acacgtgtag gtcttgtgca ctggctttgc
91920tttcttcttc caagcaggtc tcccaggaaa tggcacttac agagattgaa gagtttaata
91980catgtctcgc tgcctctctt ttcgggaacc ccccagaggg agcagcagaa accagggctg
92040gcaggggctc taagctgcct gggcaaagga gcagggggta gcatggagcc ttagccaatt
92100tggaaagcac tgtgacccaa gcacattttg cagcagtaat gtcaaattct gccgttcagg
92160catgccattg atgtgcacgc tgccacacag aaaccagtga cacaaaggca cagccttctc
92220caccctcctg gtgcttagga actaacggct ctaatgagaa atgagagctg aaaggagaga
92280gacgggggcg ggccacagca gcgcaggctg gcactgcgtg ttggaggagg ctgacccact
92340tctcgtagag gtaaggggcc cactgaaatg tcacttaaat tagccaccac tcccaacact
92400agatctcctt tgtccccata cctcagcccc acgcttcttt ctttttttct tctttttctt
92460ctcctctggg gcagcctcaa gcccagcacc cactttttag agctgtaaac caccctggtc
92520ctagaagccc tcttacgtta ggggatgaca ggaggtagag atcaggaagg agggagggag
92580gggaggagga aaggaaaagg gaggggagag agggaaagag atcgagagag catgcattca
92640tcacaaagag ccctcttttc tggctttttg actgcactgt gagttattta gccaacaata
92700gatgtttatg tattttttta gaacccgtat ttattaacag cctgaaagga gagagacgga
92760gatttatata ggaagtgcag tgagttaagg ggggcaatta agagagcaga aagagatacg
92820gaacacagac ttgtaaaggg ttttgtaaca tccaatcaaa ggtgcttcag gtattttcca
92880aggaagcaga aggtaaaaaa aaaaaaaaat tgtcccatta gaagctgaca ctggatggag
92940caatggccca ggcggaactc ctgcttgaaa gaaggtgaga agggagggac acagaccagg
93000atccgatgag ccagagtgtg gccatagctg ggtcatgagg cccagggttg gaaggacccc
93060actaaagtgt gcactggcct ttccttgaca aaggatgcac ctatagctag gcgtggtggc
93120aagtggttgt tattctagta cttaggaggc tgaggcagga ggatcaccat gagtgtatgc
93180ccagcctgga ctgcatagca acacccagtt tcaaaataac aacaaaagga agtgggggtg
93240gggagggcaa catttggaat gtaaataaat aaaacatttt ttttaaaaaa agaaaggggc
93300tagtgagtta gttcagcggt taagagcgct gactgctctt ccgaaggttc tgagttcaaa
93360tcccaacaac cacatggtgg ctcacaacca tccataagga gatctacgcc ctcttctggt
93420gtgtttaaag tcagctacaa tgtacttaca tataataata aataaattct ggagtgaggg
93480ggccagagca agtagaggtc ctgagtttaa ttcccagcaa ccacatgatg gctcacaacc
93540atctgtacaa ttacagtgca ctcatataca taaaataaat aaataaatct ttaaaaaaag
93600aagaaagagg gtggggcagg ggagggaaga agaagaaagg taagaagcta aataaaaggc
93660acagagatga gcttcatgtg gaaacacagg cctgtagtcc tggcactcag gtggggttgg
93720ggggggctac agtgagagta tcatgagttc aaggtcaact tgggtgagac cttgtctcaa
93780aaaatacata ngcnaaaaaa aaaaaaaaaa acatagccag gcatgatggt atacatttat
93840agtcccagca cttagaggac tgaggcaggg cagaaagaaa aggaattcaa gatcaggctg
93900agctgtatgc agtcctgatc ctatcccctc cccccccccc ccagagacag acagacagac
93960agacagacag agagaaacac aaagaaaggg gccttcagat ggctcagcaa ttaaaggcgc
94020ttgctattca gaccccatga cctgagctca aagcctggga cccaaggtag aaggcaagag
94080ccaactccac agagctgttc tatgatctct atatgaatgc tggggcatgt gcctacacta
94140tgttgtgcac acatgcacag attagaaaaa gaggaggaag aaaaacataa gattgtttca
94200agaaaagaaa ggctggcttc ttccacgtca gtgtgagagg agggtctggc ccctttgtag
94260ccaggtcctt cccagtccag tgggggctga actgaggcag cggaggaggc aataacggag
94320ctttcccaac gcagtgtcca gcaaactcaa ctctacagcc tgtcctgatc cacagagaag
94380ccttcctggc tccctcacca atgcgggggc attggctccc aggctcctgg gccccccccc
94440acacctgtgg agtgctaggt gatttgctaa tgttgggcaa catttgccca cgtggggttc
94500ttggctcttt ggtaatagac atgcctagca ggagggcgga gcttggaggg gggagtcctg
94560gggttgcccg tggctccctg cagctggggt gtctggccag ctgaagaagg agccatggca
94620cgcaaatggg agagcatgga acagaggctg tggatgctaa gcaatatggg aggcagtcta
94680agcttggaag cagcaggtgt ctgggaacgg gcctgtggcc caggcagatt tccagtgagc
94740actccagttt tttggcacaa ggaacaagct ggctgagccc aagaggcaag tggtgataat
94800gaaacccgca gttgaggaac agcgggtaag ggtgccatgg gagcccatgt gctcatgaag
94860aggctggggt gtgaagaaga gcccatgcag ggaagccaca catcccctcg agttccaggc
94920agaggcagag tccctgagtg gggctccctg ggtctcccct tacctaacca gtctcccggc
94980accccagcaa acaaaatccc atccataatt tgaggtttat agagacctca aaggctgagc
95040tactgtgtgc cactaaccat cagcctaacc ctcccccact gtcttctcta gctgcccctc
95100tttcttctga gactgtgata gtggcgggga cgggttggga gtgtgtgtga agccctctcc
95160gactctccaa ccccagctga gccccttgtt ctgcagctca gtaacacagt aacacaggct
95220cagttctaca ctggttgaga acactcacgg ctctctcagc tccttagaga gcctgttttc
95280tcattttcct gtccccaaag cctagacaat ggctggtcca tttgtaagct tatctgagga
95340tgccaggggc caccccatgt ctccactagg ctggcaatgt tctctgtcac tgtagtacag
95400aagactgcct ggtgggaggt gagataagga aagggatggt ctcccctggg gttcccacac
95460agtgctgagc ggaaaatggc agaatgggct gggaggtaac tctgttgcta gagtacttgc
95520ctagcatgtg caaggaaggg cctgggttcc atccccagca ctacagaacc caggcgtggt
95580ggttcatgct ggtattctca acattcagga ggtacagtca ggaagagcag aagttcaagg
95640ccatcctcag ctacatagct agcttgagac cagcctgggc tatgtgagac tttgtctcca
95700acaaacaaca acaaagcagc agaaggccaa ctggcaagag gagtattacg taaagtaaat
95760ccatctcaaa aagcaagtag catgtatctt ctttcatttt tttttacatt ctataaaggc
95820ctatcaagtc atgtatatat gcatgtatgt ttgtatgata tgaaagtagg gggctggata
95880gatggctcag cagttgagag cacttggatg ctctttcaaa gaacctgggt tcaattccta
95940gcacccacat ggcagctcac aactgtctgt aattccagtc tcaggggatc tggcaccctc
96000acacagatat ccacgcacat aaacaccaat gcacataaaa taaataattt ttaaaaaaag
96060aaattggaag taaaactctc taaggagaca aaagggactg aggggaagtg ggaggggcat
96120gaagggggag ggcataggtg tgtggtgtgt ttaacatgca gaatacactt ctataaaagc
96180tttggggttc attatgcaat gtatacatgt gtgggtgcaa gatgtaagct gtgcatatgt
96240gtgggggcca aaggtctcct cctcaatccc tctctgcctt attttcattt aaattataat
96300tattactatt agtgtgtggt gtgatgtgtg tgggtgtgtt aagccctcac ggcaatcaga
96360ggatgtctgt ggtctgagga tgctctctta ccatgttcgt gtgggttctg tggatggaac
96420tctggtagtc aggtttgcaa agctagtgtc tttatctgcc gagccacctt gctggccttc
96480aaccttattt tttgcattga acatggaact tcctgagttg cctggacagc aagtccccaa
96540gaccctcctg ttcctgcctc ccccntgtcn nnntcacang aggacacacn gcttantggg
96600tntccggatt gctgcncacc tccccgccnc ccnagcctcc tgcctccccg cccctcgccc
96660ccgctggncc ctcccccccc cccccccccc cccccccccc cttccccccc ccccnnnnnn
96720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
96780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncagaca cacacactca aattaaatat
96840agctctaact gctgttaaat tcacactcct tcacatcccc acgctaggac tctaaggagg
96900caccagcaag gcccaggtcc agcttgactt agagcaaagc atcctccccc ctccacacaa
96960tggaaacgga cggaaagggg catggaagca gaaccagaca acagcagcct agccaagccc
97020aggactctgc tccttccccc catgcctgcc gtgcaactgg ggaggcaaag ccccagccgg
97080tgctttctga ccgcttagcg gaagacaagg ggagcctgtg attatgattt ctgctgattt
97140gcaatgaaac actaatgcag tgggcttttc attaagccag atttattcaa tctaaagatt
97200ttatttcctt tatgtagaaa gtgcatcttt atatgttgtt ggaggagcag agatgtgata
97260aaaagaaatt tctcttatga actaatagca ctgatacata gtggtagcta tgcctaggcc
97320tctctctctc tctctctctc tgtctcctgt gcatgtgtgt gtgtgtgtgt gtgtgtgtgt
97380atgaatgcac acaaagtagc ccccccccat attatttctt ctgtgggatc tccagactca
97440gcaaatggtg gtgactggga agtctggcca tgcaattctt gccttttctc ttgccagccc
97500aatccctttg cattcaaacc cgggctgctt gctgtggcca gccctttcac ctggagtcct
97560tcctcctcct tacctgtctt cccatccttt gcagacaatt atcctcaata actagccaat
97620tacccttaag gacaattata ctcttccatc agcaaacacg ggtgttcttt ccttgagtct
97680tttgatgaag tcgatattaa agagatgctt tatttacata aagtcaaata gctccctttt
97740agaagggttt gggttcgatg tcaaagtttt aaaatcttaa ctagaggatg ggtgtagagg
97800gcttttggct agggtagaaa agagatggag atacttattc tgatgttgct ttaaaaggta
97860ggatgcccag agaaggtgga aggatggggg agggagggtc cctcctcaag ctaatgaatc
97920taaaagcagg gatgagctgg gcgctaggag tggaaccagt cagaagtgtc tgcctttgac
97980tgaccacagc tcctgccctc ccctccccca gtctctctgt gaaccgccag cattaggagc
98040taatcgcttc agaaagccag attggaatgt gttgctcacc ctccactgct cagaaaacct
98100ttattccagg caaggactga cccaaaccga tcatggcatc tgccaatcag gaggccaaag
98160gtgccggcag ggcgggacct agctgtgcag aaacagctcc gttatggcgc gcagaaaaag
98220ctggggggaa aggctaccgt tttatctctt ggcagatggc ttctctcttt gatgctttgg
98280gccttacctg ttactgcctg cacttgactt gacctaggca aaaatagcag cgagatacag
98340gttctcgaag ttagaaggaa aaaaaaaaag ccccaaacca caacacaacc cggaagtgtg
98400cccccgctgt gtttctaaag agctgttttc ttcccaagct ctacagcgtg gtggctctaa
98460tcggaaattt ctttttaatc atagcaggag tcccaattag cgtgttgggt aatctttcaa
98520gtagagtggg agttccgtgg ccacagagag cagaggcaat attcagcata aagccctaga
98580gaaagaggtg ttgtgggcct gtgcacacat gtgtgtcaac gcacatgtgg cttgtggagg
98640ctggcttccc actctcaaga tgaggtgtgt gcaccccagg ccttttgatt ctcaaagctt
98700tattaggacc agagggactg tgtgtgtgga ggggtgttgc tcacagtgca gaaacccaaa
98760cctggcttct ccaggagccc acatgccaac aaacaggctg cacactcttg ctagtacatc
98820ccctaaaggt atggggatga gggaccaagt gctttgcaag acagcaggca cagagttctg
98880ggacgctcct gtaccccaga ctcagccgcc acccagggcc agctctgatc tggcttgacc
98940tactttcttc tgttgttgtt tttggaagtg ctgatgtcaa tgcagaattc agcagagtgg
99000ctgagtgaga aaaaagagga gagggaggaa aagggggggg ggacgggacg ggccgaggcc
99060aacaggaaag ggcaggcaac aagacaatga ccacaaggtc cctgtaacta cactaactgc
99120ttacctttcc tgacccccag ggcttagcca atatagctga gacccagtct tggtgctgtg
99180gcttcaggct aagtaaacag ggaagagttg gacatgggtc tccattctct ctcctcatcc
99240aacaagggga ggaggcagtg gccaggcagc catgcccacc gatgccatcc ttctgggagg
99300agccagacat ttcaggcacc tctccttccc tgggtgccta gaggtgctgt gtctgcatcc
99360atctgccatg cctgccatct gagagaggcc actgggactt ggtagagagg ttctccacac
99420atgctggcct ggaggaaatt ggtctttagg gacactgaag gcagtttcct ctgttcagtg
99480gctccttgga aacccacgtg acagagctcg catgacaact tgccggctct caactcccat
99540tcttagctgc ctcaagcact gtaaggttta ggagagcccc agatgtaagt atggatggga
99600agaccctcca gggagtcatt gcctaccctt ctgaactcta acatggtcca gcttttccat
99660tccacaattg aggagacgcc agacctggca ggggagcaag cctttgtttc tgacccattt
99720gcaaacccca gccactgagg aacttgcata caagaaactg cctctgggcc tctcctggac
99780tgagccctgc ctcccagggg acaactgggc aacagatcct tccaggtggc tgcagtgaca
99840gatccatgct tttatgacat agaaaggcct cagtctcagg atttcacaca ctgtatttcc
99900ctcatcctgg ggaccaggga aggcgagcat cttctgctcc ccccaaacaa gtgtgggaat
99960gattaaaatc attttttttt tctgctccat gaactcatac agttttcaga taccgaggag
100020acaaagccct cctgtgctga aattagaccc cgaaaaatag gttagctgac aattacttgt
100080ttctaagtgg agtgtgatgt agtggcagga gcgcaggatg ggctgccagg gctgcagtct
100140cccccccccc aaacttactg tctcttaacc tctcgagtcc ctgggtttct tgccgggatg
100200ataattctcc ccatctccct cctctggtgg gctggtggaa agcgtaatga atcaacgctt
100260gaagcacgct gaagaggcca gactcgggat gccatgtaag tacacagcat cgccagccac
100320ctctcaagtc tacacggagc tgatttattt acctcccgtg aaagagacaa caatcatcat
100380atttacactt catgccgcag cttcctgcgt ggcacggcag caccccctcc ctctccgctg
100440ctgaggactc catcaagcac gctgccttgc caggatgaca gcagcccact ctcagcctct
100500ccctggcctc cttacagatc atgacctcct gccccgtgag gtctgtcacc cgaaaaccac
100560ggtacaccgg gggctgcagc ctctctatgg gggaggctga ggaaatgaat tccgtaggta
100620aaaggcttcc taggaaatca gacgctgcta gtaattaagg agcgaagcat aggtgcgtga
100680aaggtaaatg gatgttattt aaatgttgcg tcatttaaag agtgtcctgg tgcttcagtt
100740ccttgttacc atgcagggct gtggacgggt ggcaattagg ctggcacggg tagagctcac
100800ctgctgagct gagggagggt ggggacacac cttccggtaa ttgctgctgg gcagctctgg
100860gtctccccac ccccgccccc gccctcactc cccacccccc acttctttcc tgacagctct
100920ttcatttgca gcagcttaca gggcttgttg cccttaccca gaaaatcacg ttggaagaaa
100980tataagaaaa agaggaatga aagagaaagc cagaaaagtt catattaggt tcggatctgc
101040ggccaaacct ggccgagaga atccatgacg gtccgcgcgc atataaccct gtggcaacag
101100ggcccggcac aacagggccc gccacaagag cttcttgagt tgccacctgc caggagacag
101160gatgaatgaa tggatcatct gtccttagag cacaagccag gcctgattct ccaatattga
101220tgtgtgaggg agatgtcaac agaggttccc taaagaatga tgcttctatt tccatgctaa
101280tcctggggcg tcagcttcag tcggaacagc cggaccgtta ccttagctct gctgttctcc
101340tgtctgtaac ccgcagaggg aagggcgggg tcacccagca ttgccactcc ccccaccctc
101400acgtggtcca gacccctctt gggttgatct gctcctgaaa aacagtgttg gctcaagttt
101460gcctctgaag gtatgtcacc gctggctcag ccagcttatc tccccggtgc tttcaagatc
101520aaaacaccca aacgaaagaa aaactttgtt tcaagagcag agtgtggtgc caactctgat
101580caaagtgttt ttcagcatga caactcactg cccgtgacaa ccagtacttg gctgttgtgg
101640ctcagagtga gatgcggagg gaagtggatg acaacagctg tatccaggtc caaacagagt
101700agattcacgg ctggcagaaa atggctgaga gccttgggct gcatccctcc tcccctcctg
101760cctctctctc ttttcaaggt ggtttttgga aatgtccttc ctgtgggttg tgtgcctttt
101820ccatgtagga cctggggcct gtgcagatgg ccctgtgttc ctggtgctgc tgttgagatg
101880tgaacgagtg ataggaaccc aggcactaaa cacacaatgt ggttgtatct gactagaagc
101940aaggcaagag caggaggcat ttgagggtaa aggagtgtaa ggactgtgta aagagatgag
102000ggttctatct gggaggcagg agtcccaatg ccagcaaata caatggactc tcctggtcga
102060cccaaccaga gagaattcaa gatggcagag ggacaggctg tctgagtttc ctatggctgc
102120accgataaat ggtcataagc agagtagagg aaaaccacag acagaaattc atgccattga
102180gactagaaat ctagctcaag gttgtgtgtg gcagggttgg ttcctgggtg ttcaaccttt
102240tcacactgtg acatgatgct gtggtctgca gatgtgttgg gctgcatcca tagctaccct
102300gggacacatt catggaccgc aggttacaca tgctatttaa aaactccaag ggaagggcta
102360gagaaatggc ctggtagtta agtatgcttg ctgatcttcc agaagacctg agctctgttc
102420ctagcagcca tgttgggcag cttacaacta actatgactt ctgagctcca aagctctctt
102480ctaatacata catacataca tacatacata catacataca tacatacata cgtacacaca
102540cacacacaca cacacacaca cacacacact ttaaagaaaa aaattctggg ttggagaggt
102600ggctcagcaa ttaagagcac tgactgctct tacagaggtg ctgagttcaa ctctcaacca
102660catggtggct cacaaccatc tgtaatggga tctgatgccc tcttctggtg tgcgctgaag
102720acagatacaa tgtactcata tacattaaat aaataaataa gaaagaaaga aagaaagaaa
102780gaaagaaaga aagaaagtgt aaacgaggaa aattcctaat taaaaaagaa agaaagaaag
102840aaagaaagaa agaaagaaag aaggaaagga attctgaggg agaatctgcc ccttttccta
102900acttccaggg ctataggcaa cctgtggcct ggggaagctg tagacaacct gtggcctggg
102960gaagctgtag acaacctgtg gcctggggaa gctgtagaca acctgtggcc tggggaagct
103020gtagacaacc tgtggcctgg ggaagctgta gacaacctgt ggcctgtggc agcatcatgt
103080caacgcctca ccctctgtgc ccaatttcct gttctctaag gacacatgcc atcaaatgca
103140taggacactc tacatcaaga tgatcttgtc tcaagatgtt taacaaaatt acatctgcaa
103200agacctatct ttacatgtga ggtcactcca caggttctag acatattttt gaggagccac
103260catccaactc actatgtgac agagtcatct agagatttgt gtccaggaca gactggctgt
103320atctgctctg agagtcccct gcctgcccgt gggaactccc cagtggtcct taagggccct
103380gaggactttg gatctgcaaa gccacatctt ccaaaaccat tttcctcttt tggagagcta
103440ctctaccctg aaaccctttt ctctgaggtg gcttttagag aggcaggtct cagcagggca
103500ctgtgcccac aagaagtccc ggggagaagg gacccaaggg ccagtgctga actatcgctg
103560agactgagaa cattgtgtct cacctaaaat cggtggtcgc aaggaccaag caggctctat
103620aaatgtctta ctgcctttat tccttttcct ccgctccatc ttactcctca tttttgtttg
103680tttgtgtgtt tgtttgtttt cttctgagat gtagcccagg ctggccttca gctcactatg
103740taactaagga tgactttaaa cttctgatcc tttcttccct ccacttccag agtcctgggg
103800caggtgtgtg ccaccgtacc ccagctttat ttgagactat gattcaggct ccatacttca
103860tgcatattag gtaagcatgc taccaacttg gctatattcc cagcctttct ttctttcttc
103920tttgagacaa tgtctttttt tttttaatta tatgagtaca ctgtatctgt tttcagacac
103980accagaagaa ggcattggat cctattagag atggttgtga gccaccatgt ggttgttggg
104040atttgaactc aggacctctg gaagagcagt cagtgctttt aaccgctgag ccatctcgcc
104100agtccttgag acaatgtctt gctatatggc acatattggc ctcaaactca gaatccttcc
104160gcttcagcct cctaaatact gggattacat gtgagccatg gtgtttggct tctagccttt
104220cttccttccc tttcccttcc cttttccctt ccctttccct tttccctttc ctttccttcc
104280cttcccttcc cttcccttcc cttcccctcc cctcccttcc cttcccttcc cttcccttcc
104340cttcctttcc ctctctctct ctctccccct ctttcttttc tttcagagag tttctctgtg
104400taatcctggc tgtcttggaa cttgctctgt agaccaggct ggcttgnnnn nnnnnnnnnn
104460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
104520nnnnnnnnnn nnnnnnnnnn nnnnnnaaga agaagaagaa gaagaagaag acaacaacga
104580cgacaacacc ggcgccgctg cctccactgc catccacctg agacaggact caaatccaga
104640ccaattttta aaaccagtgt ttcaagccgg tacactgaag tagtagtccc acttgggatt
104700atagctcctt actttgtttt gctttgacgt tttgtgacat ggtgtgatgt agtcttggct
104760gtcctagaac tcaatgtgta aattaggctg gccttgaact tgcttctgcc tcctgctggg
104820atgatagact gatggtgtaa aactccactt aggaggcaga ggtgggagga tcagaaattc
104880aaagtcatcc ttggctatgt tgtgagtttg aggaccaacc ttggctacat gatatcctat
104940ctcaaaaaga aataaatgta ttgccgggca tggtggcaca cgcctttaat cccagcactt
105000gggaggcaga ggcaggcaga ttttctgagt tcgaagccag cctggtctac agagtgagtt
105060ccaggacagc cagggctaca cagagaaacc ctgtctccaa aaacaaaaaa caaacaaaaa
105120agtgaacccc aacagtactg ccggacagtc tggtgtcttt cctaagtctc ctttcaactc
105180tgtttaccca ggtgtaccca caaggtgtgt gagcagctct atacccagag gtgatacggt
105240tgtttgaatg agagaaaagt ttcccatcag ctcgggtgtg tgaatactcg gtccccagtt
105300ggcagtattg gctggagagg tgatggggag gtgtagcctt ccggtggaga tgggctttgg
105360gagtttaaag cttcctccac gaagaccact gggctgatct tgttaccaac agaattggat
105420tggcctgctc ttggctccgg cctcagttta tctaaaattt acatgttacc tgatcaaaaa
105480ctgtttcctc ccccacccct ctccctgtct gtattccctg ccctctagtg gtgctggctg
105540tatactacac tggtgatctt gactgtattt cagtttacct cttgttctct ctctgctgac
105600tctagagatg tcctttcatg gctgggacct ggctcagaaa tttctaaggc actgagcctt
105660cctccatctg aactttagga aacttcttgg cttaaaggtg tatttctgac ttagtatgca
105720atgagacctt ggagcctgca ctttgttaag caccctgggt ggtggtggtg gtggtggtgg
105780tggtggtggc agtagtagaa ctctgatgaa cagttagtta ttcaaggccc atctagaaaa
105840agaaaggctt tgggtgcaca tggctataac tcagtggcga gagacgtgct tcccatgaat
105900aataatgatg acgatgaaaa taattctctg tgactgttct ccccacttcc ctctctctca
105960ccctagctct tatctaccga atccctgcac aagcacccat ggggtttaca gaatctgggg
106020cggaacgtta gtcacttccc ttcgcctact tcagtattgt gtttccagaa gtacccattt
106080tggctagtca ctgaggaaaa cggcagctgc ctgtgggcca ccagcccatg ccaagtgagg
106140tcagcaagaa agaagctgac agcaaatgtg ccaactgtgg gtctgctgga tttctactgt
106200gctaagtggt ttcaagaagt ttcttcttaa ccccctacaa gaaaccacaa attttattat
106260ctacactgtt ttgtagatga agaaaacacc attccgaagc tcactgccag tcagcactgg
106320aactggaatt tgtttggcta attcagtggt tctcaaccag ggtggatttg gactccccag
106380gggatatttg gggacacttc tggtcgtcat aattgggatc atgtgctact ggcacctagg
106440gtagaggcca gggtggtact gaccttccta ctatgaacag ggcagccatg tacataaatt
106500ctctcgatca aaacatcaac agtgctaagg ttgagaaatc tcaggctgaa accctgtcat
106560ttggccttga ggtgggtggg aggaggttag agagtggaat aaaatcagaa gggccaccac
106620agaggcctcg agtgaggagg gaacagggct cctatgctag ggataatgga gaatagggca
106680gcttgttgaa acttttcttt cttccaagct tggctagagc cctgctcaat ttcccccaac
106740tctgccaagt cagtcccggg acttcgcact aagtttgtcc tggagtgacc ctgactccag
106800cttggagctg gggcaacaca tttacctggg tcttcccagg agtgggttaa aagtcaaaga
106860taagtggcat ctgagaagtt aaaagtgggg tgagtgggta aaggcaggag gaccccatca
106920atcagctgac ctaggaagga agagagcaac tgaagcagca aagagctggt ccaggagact
106980ggattctgac ccactaggct tatcttccac agcctttctt gtttaggctt gggctcagtt
107040tccctgcatc atccgcagga gcccctggag cacccacatt cagccggccg ccaggacagg
107100ctccccagca gtggcctccc cactaactga cagtggtgac aggaaatatc tcccattcca
107160atctcctcag aagtctgaat aagtaaggga cagatgttgg ggagaggcgt cactcttggg
107220ttgatgaaga aaagatcatg agaagcatac attttacccg ctatggttgg ggttccatta
107280ccacccatgg cggggttgag ggggaagggc agaaaaaagg agatggagaa ggacagacac
107340gtaagaagga ggatgtggtt agggctgatt cgtcccctgg ggtcgaacaa tcagctgtta
107400ctggggcgga aggcaggaag tctccttaaa gacacaatat tctgaacgtt gaactcagga
107460tttgaagcaa gcccagcagt caccttagtg gagcccatac ttaatttaac acagaagcgg
107520ctatctcagg cttccctctc atttctttgt ctcagatgct ctcacttaga aagcttagat
107580gctcttagaa atgactcaaa agtcaagaac cccaggccac aagtttctct ttgggggtgg
107640ggagggagtg aagaggtggt cccagtcttg tccctttaaa taagcaattc agcagctttt
107700gccaagtcat tgggttcatt tcggtttttg cccatccccc gcctttcaga ctctgattgg
107760cccctaggga aggagccgcc tcttcattgg tctccacctt tgaaatcact tccctaagta
107820ggcctgagtc agagaagcgt ttcggagggc gggactgaat gggtgttaat cttagaaccg
107880ggtttctggt tgatactact ttggtaaaga tcttccccta atttttaaaa agacgcttcc
107940tctctaaaag tgagggcgaa tcctttgtta agaacgtgcc ccttgagaag ccgtgggctc
108000ttcagcgact aagacgagac attcactaga aaagatttca ctaaacccac gagggataga
108060ctagacctcc agtgaagatt gggcctgtgc gggtgacatt tgtccctata ccccgaagac
108120ctcgagctag ctctccagtg aagactgggg ccgtgcgagt gacagtggtc cctatacccc
108180gaaaaaaaaa aagtcctatt tgtggaaaaa aaaaaagact tcgggtgttc tgctgcatcg
108240gtggctggct tccatcttta gttctactca ctcctgttgc ttcgcgtgct ccaccttcgc
108300ttagctcagg cctcctgtga atcagttttg aggctaaaag aagttccaag aaggaggggc
108360tgtagccctt taaggacttc cccgcgaccg agtcagagat cagtttaaaa atgccaactc
108420acagagcgcg ctgcattctg ggaagctgag tgtcaccgta agaacttcat tgaccggaat
108480gcactgcaaa aatacacgcc tatacttcct tctgctcttt aaactgtagt ttgacgtaaa
108540gctggtctaa gcaagtcgcc taggccgagg gttagccaca ccttttcagc cattggccag
108600ttggttagtt ggtaggcgtg gcttagagaa gctcctccag gcaagggggt ggcctccttg
108660ccaatcagag cccagacgcc tgaatgggcg ggagtaagca gaggtgctgg cgcccccgag
108720tgggtgtggt cacgttgccc agcaatgggc ggtgattggc cctgggtggt tcattcgcag
108780ctcgtgcgtc acgacgccgc cagctgatcg gagactggag ccggtgtgtg ctgggcgctg
108840ggaagagaca gagcggtcgg ccgtgcggac aggtcgcagt gattttgctc ctctgtccac
108900agcaaccccc gcacccagca tcaggtgggt gtgatctggg gacccggtca tcccgggggg
108960aaccgcggta accgggtgat ggggaaagta gggtcctgac ggccacaccc tgcccttctg
109020ggggagggga gagggggcgg cggggacagg ggcgctcttg ggagaggagc ctggactctc
109080ccgagtagtg tgtctggacg tttaaagaga gagtcccgga caggagtcgt ggcagaaggt
109140ttggagaagt aactggggag gaatatgaga ggccagaggg ccgggggcgt ctaaccccga
109200cgccctttgg tttgaggatg cccgagctga ccatttagcc tagggaggat ctggacgagc
109260gaggggtgcg gaggtgcatt gcctctaccg gcgctgactg ggtcagggcc agttcaagtc
109320cctggcaggg aaggggtcgc tgggcggtcc ggcccctcct ctcgttccct cccggggatg
109380ttatgtaagg ggggagggga aaggagtagg gggcggcggt gcggaggcct tatgcaaccc
109440aaaggttagg gtttcaccgc gggttgggcg gaggttgggg ggggcggaca ggaggagtgc
109500ctggaaactc tacccgcacc ccccctccca gcctaactgg ctgtcttgga cagagagaag
109560gtcacctttg cacctccccc ctagtatgtc cggtagagag gcccctagcc cgggcttggc
109620ctgactgcct gggaagccgg ctggctgggt ggggcgcctg ggttagtcat cgctgggctc
109680cctctctccc cacctcctgg ccaactcttg gcccctcccc acggcctccg gttaggctaa
109740cgttcccacc tccctctggc cctagtttca gtctccaact catttggcct gtcaccctgg
109800ctgttagagt aggctagaag ctgtcatggt gccagagagt tgatggagca gctggtcaga
109860gggtcagtgc cctgggccca ccccgccccg cagccaaggg cacctgcttg gcacaaactc
109920tcagcagcca gtgaaccctg tggcctgaac agagctatcc tgggcagaga gaagtggaca
109980gagactgatc acctaggaga aggaagatcc gacaaagttt atacttccca agaggctttt
110040ggaatttgaa agttgcccac cctagtgtaa tctttccact ctctgaaaat agaaatccca
110100aggcaaagtc tccttggccc ttctatctgg cagtggccat gtccttggac tgactgtgca
110160gaaccaccct ctcgggctcc cagccctcta gcctgccacg cccccagccc cctccctgag
110220ccatgctgta gggccccggc ttttactgct gattcatgcg ttggaactgt gggggcgggg
110280cttggaactt ggaacaaagt tcagacgtgg aggggccggc agacagcctg gaattcatac
110340cagatgtacc cggaatgtgc aagcggaatg cctggcatct ctagtcctga ggaagctgcc
110400cagccaccct acccatacct ccctcccctc ctgcctttgg tcagctgtcc tccctcagac
110460tcctgagagc ccctgctgac cttccaactc tagtgcccct cccatttcta accctacaca
110520aaccctcctt gctgctgaat tccctaagaa caagtcattt gagttgatca cagagctcat
110580atttctgaag tacatttttt tttttaactt gggacttggg ttctacaccc tgccctttga
110640atgccgaaga tgctgggctc cttagcaggt tgccaagagt tgccagctcc tagtctgtaa
110700aggggcacaa agcaagtgca tttagaagcc tcttgcttct tattcaagaa cccctcatta
110760gaaggtactg aaagtcagct agagccaggt ttggatggcc tctgggtcgc tggccctgtc
110820acccagcttt cctgtttttt tttttcctcc ccttcctttt aggaacctgt gcctcccaca
110880ccctcacctg gctgagccgc agtagttctt cagtggcaag ctttatgtcc tgacccagct
110940aaagctgcca gttgaagaac tgttgccctc tgcccctggc ttcgtggagg aagaggagaa
111000gcagcagctt tgcctatcat ccggaaggtg acagaactgg ggtgggaagg tctggacagc
111060tggggtgatg gctttatggg agggaaaccc tggtcctctg gggagccctt acccccactg
111120gcccagtgaa agatttaggt taaaggcact gtctataaat tggggaatag gtgactccac
111180ctccccaaga ttagttgatg tctgtgtggc agtgggaaga aatagaagga aaagtctgtc
111240tgtttactga gacttccttg taggcctgcc tttcttatct tcatcatcac catgccaaca
111300cacacacaca cacacacaca cacacacaca catacacaca catacacaca cacacacaca
111360cacacacttt cctttccatg aggtccaaaa gtaaatgtac tcaggaaggg ggacattgaa
111420actccgttct aagtagtcat ttgtgtattt actttttttg tttatttgtt tgattgactt
111480tcgagacagg gtttctctgt atagccctgg ctgtcctgga actcactttg tagactaggc
111540tggcctcgaa ctcagaaatc tgcctgcctc tgcctcccaa gtgctgggat taaaggcgtg
111600tgccaccacc gcccggctgt atttacattt ctttatttat ttttagtctg gcccagattt
111660tgggtttagg ggtacttacc cttacacctg tggatttttc cacctgtata atggggaatc
111720ccatagataa gtaggcagga gggcattaaa agtccaccag tggtgactca gagcctgggc
111780tcttcttctt ctcgtggatg gaaacgaaac agctcttcac atgaactgtt gtccttcccc
111840caccccctga ctactcaccc agctcagggg gattaggatg gaaggaaagg ctatggttaa
111900gtcccaggca agctcgtggg aggctagtcc tctactggct tctcaccatg catgggtggt
111960ccaaggcttt ccctccacct aaagcaaaac tgtagctctt ggttgggttc tagcaaccac
112020tgccatttat tttctgcctt tgctttccag gatagtgaga ctctgctcaa tactgtgcag
112080gcaagaaatt gtcaggggag atgggttgta tgatatgagt cccttctgct gcctctagct
112140cctgattcat tctcacgtat gggcttggtc tctgattgtg gttcaccttt ggcccagtct
112200tcctaacaga agatgggttc agggggtaca ggaggctgtt tgttgtattt gacaggagga
112260ggagttctag cctgttcccc atttgtgaga aactgaaagt cataggggag actagatcat
112320ctaatccagc cccactgcag tctaagctga gggataggat gtgtaaggga ctgtagcaga
112380cgggctgggg aggctgagtc ggctcacaca ttgcgacaaa gattgccctt ccctcgacct
112440cgcttgcttt ctttcctcct cccttccctg gccacagtgt gtccctccag cactgggtac
112500atggctctgc tgtcctcatc caacatggag cctcagaggt gagaaagggc agcctggaag
112560caacagaggc aggcacaaga cagtggagga cctggcctgg aaccacaagg gcctatccgg
112620acattggtca gagaggcacg tagaagcctg gagaacacca ggaaagagag cagccagcca
112680gcctcagtga aagacacgtg cttccagcca tctcctctca ggacctgcct tcctgggaga
112740tgaagggcct ccaggaagta tggtcccatc tctaccctgc agtttctata aacagcctca
112800aggagcatga gccacctctg aaaggaaata cacagcaaat tcaaaaagag attcaaatgt
112860gtaacactgt gggaaaacat atctatgact ggggttgtag ctcagttggt aggtttgctt
112920aacatgcacc aagccctggt tctgtcttct gcattgcata aaactgaaca ggttggccca
112980ggtctgcaat cccggcactc tggaggtggt ggcaaaggag cctacattca aggtaatcct
113040ctgctataca atgagttctg agccagcctg ggctatatga gactgtctca aaaaataaaa
113100caaaataaaa taaagcattg gttagtaatt caaagaaagc agatgtggct gaaaccgttt
113160tccctgatca taatacaaca agcaaatgaa agccagaaga aggctcctgt gccttgtgtg
113220tggcagtacc aaccattgtg agagatgcct ttggacctgg tagtttgctg tcttagaaat
113280gtatcctaaa ataaggattt ggttataaaa tgttcatctc agggttgtaa tagagaaaaa
113340tggaacgcag ctgtttgttt ggaagtccat tccttttctg ctgtcatgaa aatgtatagc
113400tagggcttgc ctaagtaaat tatattcatc tgatggtggt gttctgtgca gccatccaaa
113460gtcttacaga agaaaaattg agtggaaata taaatattga atactaaaaa gattataaaa
113520gtatgagttt gtgactgttt ttaaaatatg aacacatact tgtaatatat ttttttaaaa
113580accatccaat tgagtggaaa tataaatact gaatactaaa aagattatga aaagtatgag
113640tttgtgactg tttttaaaat atgaatgcat acttgtaata tattttttaa aaaaacactg
113700aaagtggatt caaaatgtta agaatggttg tttttgtatg gtgggatagt acaattgtga
113760attttcccct tgttttttct gtctttctaa tttttaaata ttgtgcattg ctttcatatg
113820ttaaataaaa tacaaaagac aaataaatgt tttaaaattt ttactctttt atgagtgttt
113880tgcctgtgag tggcaggaat ccaacattgt cttctgaaag cagctagcgc taacttctga
113940gccgtctctt cactccctct gtaattttta aaaaatatat ttgtatgtta tattatgtgt
114000ctttgtgcac cagagtgtgg gtgcacattc tgcagaggcc agaagagggc atcagattcc
114060ctggagctgc acgctgtttg gatcttctga tgtggatgct cagaatcgca ctcaggtcct
114120ctagaagagc agcaaatgct cctagccact aaagccatct ctccctctag tcctcattgt
114180catgtttaga ttttggagaa tttgcttgta ggaggatggg ctacaccaag tgccaggtga
114240aagaaaatgt ttgcttggga tacctattgc ttcttgagtg tgtgtgtgca tgcttgtgtg
114300tgtgtgtgta tgtgtgtgtg tacactggag ctaggaatca tatccagggg ccttttcaag
114360ctccaccaca ctaaagtcaa ttctatgaac ttcattaatt gtctgaatcc acctactctc
114420tacacacagg aagcattcct ctgactttct gactgtcagc cagctaagga ggtgtggctt
114480agaataagaa agaagggaaa tgctcaaaac ctgtcactct ntggggnnnn nnnnnnnnnn
114540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
114600nnnnnnnnnn nnnnnnnnnn nnnnnncccc tattttacca agcattgcag gataaataag
114660aacatagctt aaagaattct agagaaaaaa aaaaaagtcc actaatgttc aatgttttaa
114720ttaattttta atgggaaaaa gtaatatcaa agaaatccat gtctacggca tattaatgtg
114780gttaatgcaa acaggacaga ttatcttggc caaattaaac cacagtaaca cgttttgaac
114840taaaagtcat ctatttatac gagatgaaat gtgaattagc gtgcgcgcgc aggtgcacac
114900atgcacacat acaaacagag tctttttatt cttaagccta ccaaataact ctttaagcag
114960taaataattt taagctctaa aatttaaaaa atagtgaaac cccttcaggc tatgacagaa
115020tgctgctttt gccattcttt ctgataaaag tcccaaaggg tgtcataatc tgtatccttc
115080ttagaaaagt aaggagcaca tcctatgagc tggtgacctg agttttacac ccaagtcaca
115140cgtcagcaca cagcaatgtc ctggaccatt tgtgaggagc cccacgctgg tcctgagcaa
115200caactcactt ggactggtac ggggtgaggc tggcgatggg caccactttg gactgtgtcc
115260cacctgaagg ctgcagcagg ccagttccgg caggttttcc aaatggcttt gaggcaccat
115320aagccttagc tgcagtggaa cctatgcaaa cacacaggaa aagggcagtc attcccgttt
115380tatttctcga agtgaatgta caggcaccat ggcacgcatc tggaggtcag atgataatct
115440caaaaaaatc caacttttgt gagacaggct cccttcattg tcttcccacc acacttgcca
115500ggttaggggc acaagctccc aaggagtatc tgctctccta agggcactgc catcagagac
115560actctgacta ctacacctgg cttttaggtg ggttctgagg atttgaacgc agatcatcat
115620ggttgtatag caaattttta ttcaccaagc cagacaaccg ttttttaact ttttttaaaa
115680aaagatttat ttttatgagt acagtgtggc tgtcttcaga cacaccagaa gaagacatca
115740gatcccatta cagatggttg tgagccacca tgtggttgct gggaattgaa ctcaggacct
115800ttggaagagc agtcagtgct cttaaccgct gagccatctc tccagccctt atttaacttt
115860taaaagttta aaattaattc tcattatgca cgtgccacag ggaagtgtaa cataagcaca
115920tgtgtttgaa gtatatatgg ggatgtagtt atgtgacggc cagaggacaa ctctgtgaag
115980ttgattcttt cctttcactg tgggtcctcg gagcgtcaat agggtcagca ggcattttac
116040cagctgagct atcttgtggg ccaccaaact tatttttaaa aagctagaag ttggttaaag
116100agaaggaatt catgtaattg aaatagaaaa gcagagttga agagggagga gaggaggtga
116160gtatgatatg gtaagaaaaa ccaagaaaaa acctctacaa gtaaatctag tcgtgttcca
116220cagatcacta tgttcaagga tggtgtccca gaaaagggtc ctaaaacaaa gcccaggaag
116280taatcaaaat gatcccatcc agacctgcta cagagaagct ggggggaagg agggagaggc
116340cctgtaacct caagtccccg ggcagcacag cagagctgtg cccaatgatc tccgtgatac
116400aggacaacag gatatctgag aaggagtccc agtgagcgtg tagcagaagc cagaggcctc
116460gaacaaaacc aacaacttca ccatgatgaa catcatgaag atgtgtggac aaaagggtgc
116520actctgggac acagtgtcac acactacagc ttccacctgg gacaaaaagt cacacactac
116580agcttccacc tgggacacaa tgtcacacac tacagcttcc acagtcagat tatttttctt
116640ctagcggaga ggctgcaagg gtggagggca ggtacaaggg aacaggggtg agtgggatcg
116700gggtgcatga tgtgaaactt acaaagaacc aataagttag gaaagagaga tgaagacata
116760gaagccatgg ccaacaaccc cagcactctg gaggcaggca ggagggtctc catgccaaag
116820gtaatgaatg ctatgtctaa agagatgata aagtcccatg agagttacaa agtcagagga
116880gaaacttgga cagaaaagcc aaatgtaaca aatgcatggt aggtggggaa aggtggggat
116940ttaaaagaaa tttcacaaaa aggcacagaa tgtaaggtga aagcgagcga ctaccagata
117000aagcatcaac taagaggagt caggaacagt gtattttaac tacctttata aaaaaaatac
117060tcgggctggt gagatggctc agcggttaag agcaccgact gctcttccaa tggtcctgag
117120ttcaaatccc aacaaccaca tggtggctca caaccatcca taacaaaatc tgatacggtc
117180ttctggagtg tctgaaggca gctacaagtg tacttacata taataagtaa ataaataaat
117240aagtattaaa aaatatgcaa taattggaat tattctttgc aattctacta tgaatggagt
117300ttttgttcct gttgtttgtt ttccgagcct gtttcatgta atccaaactg gcctggaacc
117360tgctaaatag tggaagatga ccttgaactt ctgattcccc tgtctccacc tcccaagtgc
117420tggcctaggc tactctgcct gactcaggct cagtgaattt tcaaacactg cttcaacctt
117480gacccctaac ttccatctct tgggtcccat cttacccatt cccagactcc cattctgtgg
117540ctggggcttg ctggctggtg gtgtcgctgc agaggctggg gaagggactg cttgctgctg
117600ctgctgctgc tgctgctgct gctgctgccc atatcctgga gatcaaaaca ggccaattca
117660gtgcaaaccc agtcaagaga tttcaacacc agcagtaaat accttaggaa aacccacctt
117720tggctcttag aggagcagac tgcaccacgc tgcacaccct gtgtctgcaa agggctcact
117780tttttgtctg cgcatagtaa agactggttc catttccctt tcaactgttt agaattgaca
117840gctttcagtt ttaatgaggt tctccctgaa gccttgccca tttctccttc aagaacctgc
117900agtaaagtca ctgggctcaa tctgtgatac taccactatt tccatagcaa caaatcgatg
117960tcatcaacag gacggtgtgt tcgtggcagg atccattaga agagaaagca gggtggtaag
118020gaaaacctaa aagcagttca attgtctctc aagtgctttg gcttcaaagg aaaaggagac
118080gtttacaaag ggttctaccc tttcccccaa acaaagcaac cttattttgc aactgacagc
118140tggtggaaaa tatgagttga aagagtctca ggtaccgtga agtgctagtg aactgtcaca
118200gcagggagag atgagaacag agttcagaag cagccgtgtc aaggcaacag ggagataaaa
118260aggaggggga ccgagcacgc ttctaaagca gcaaataaga cgcgccaccc tgtcggagtg
118320tgtatttgcc cttcaccctt tatacaaatg tttaccaagt tgaagaatgt taacattgta
118380aattgcctac cactatttct aaaataacga gtttatgggg tttgatttta cttctgtgat
118440tggctctgaa ggctgaagcc agcctcgttt aggctgctca cctggagtgc ttgtagatga
118500caaaggcagc taaaaaaaaa aaaatgtccc agagctcctg aaacactaaa actggtgcgc
118560acaggcagga agtctccctt gccactgagg ctctcccttc tccaactgta agttctaact
118620cctgctgggc ctcctgaggt cccaactcac cgggcatgct acatgcccat ggaattcatt
118680tttggcaaaa ttactcttaa gtagctcaag gaatgagacg taattgtgtt gctggagaca
118740aatacattca gcacaaagtt gagaagatta aatagaattg attatgcttt agttcatcct
118800acagagagaa aaagtgagac acctatttac ataggagagg ggcccaggct actgacaatg
118860agcccttcct ttacagctca gccttcccat tatcactcaa ctcagacacc cagcccagtt
118920cagtctatgt gaagcatttt taaaatcagc aagagaaagg tgagttgctc agtagtacac
118980tgaaggaaag tagaaatgag cacacagtga cctgctgcat aagacacagt ttaaaaggtg
119040acctatcttc cagcgaagtt cttgccttct ttaaaaagaa tgtggtattt gctggtatgt
119100gcacctcacg aaggtgcttg cagctatgtg gagggcagag accatcctga gggaatcggg
119160tctccttcca ccagagtcct agggatggca caaaggcacc gggcttgaca gatgccttta
119220cctgctgagc cctttcccca gcctctcctt ttctacagtc tccaaattac ctgaggtagg
119280agcctcattc tcagaaccat ttctgctgcg catgtctgac tgaactggat atggtcactc
119340gactcaattt ctataaaaag ataactgagg agccggcttg gttggtagag aaaacataaa
119400gacccgagtt ttgttcccac atggggcgtg cgtgctagag aggtgaggac ggactaggca
119460gggagcagag gtaggcagat cccaggcctc aatagccagg cagcccaacc tgagctctaa
119520gttcagtaag ggagcctgta accacagaag acactgtgcc gtgtaaccac ggcaagcggc
119580ccgctgcagt ggcatctgct catagccaca gctacccagg aggctgagcc acaagactca
119640ttgaagctgg aggtcaaggc cagcataggt accataggta gacccccatc tcaaagttga
119700acagtaaaca tatattttac tacaattaaa aaaacaaggc cgggtgtggt ggcgcatacc
119760tttaatccca gcactcggga ggcagaggca ggcggatttc tgagttcgag gccagcctgg
119820tctacaaagt gagttccagg acagccaggg ctacacagag aaaccctgtc tcgaaaaacc
119880aaaaaaaaaa aaaaaaaaaa aaaaaaccaa aaacccaaca catgtaccta ttctaacata
119940aaattttcat tttttataaa aattacagtt ataattttta gttgaacata atacaatgat
120000aagtctccaa cttgataatc tgaggctggg ggtgtagctc atttagtaga gtacctgcct
120060agcagtcgct aagtccgggt agtcccttgc ctgtatccca gtgcttgggg aacaggcaga
120120aagaggacca gaagttcaag gtgctcctcc tcttcaggta atgaggagtc tgagccagcc
120180tgggatgcgt gagacacacc agtaacagca actactgcct ggagtgctca ctctggacca
120240ggaacaagag ttaagtgcgt tactcccgta actgtctgca caatgatgga gacagtacgt
120300catctttaac atctttggct gagaagagaa aacctggtat tcctcagctc gttctggcta
120360agttcatgta cttcatcatc atgcagttaa tttgtaaaca accaatcctg gcagcaataa
120420ctctaattat atagaacata agatgtggta attaggaaaa gctactaatc cacttaatag
120480agtaaccttt atctcttgca aatctggtac aagagacagt cccaaatcaa atgatggcaa
120540gaattccaga gcattgtaaa tagcagcaat tgcccttcaa ttaacgcatt gtaacgcagc
120600agctgcccac aagacctcaa atcaatcagt ctatagctaa ggaaaaatct ttctaaagcc
120660aaaaccattc tacaaagcag taacgtaggc tccgtttata ataacctgtt ttgggccacc
120720tgcaaatcaa gctatcccag gaagccagat cgtaattctt aggctctgct ggctacacac
120780tggtcccaag ccatgaggga actagattac agcaggctcc gccctcggtg acctgctcat
120840agctatcatt cttacagtct attatggcaa gtgagctctg ggcagagaaa aattcacaaa
120900caaacccacc aacttcccaa gcaagcattt tcttaacaaa cacaaagaat aaataaatag
120960agcctgccat ggtagtgcac acctgtaatc tcagcatgtg ggaggcagag gcaggcagat
121020ctctgccagg agttccatgc cagcctggtc tagacagttg caagatcaac aacgctatat
121080ggtgaggccc tgtctcaact cccaaaccac tgaaaacaag taagacgata tggatcaatg
121140caatatttct cagttcctac tgacagaaaa tggacacaat taggctgggt ataattccaa
121200tatgaataat aagtatatta tacagtacct agtttaagtc tgagcaagat attatcgcca
121260acagcacaga tacaggacac acacacagct gcaagcgtga aggatttaaa ggcacccatc
121320cctacagtat atgcagcatt gactgtctag ttttatttcg cacactttga atcatccatc
121380catttttgct caatatggca gcagtaataa aatgtatatt tgcattttga tgcatggtgg
121440aattcttact agcctgggct gtgtttgctc actaactcca gtggacttct gacgtaagag
121500gcgctggact agcttccaga ggatgtaaat ctaactttgg ttctcggcct cccctgaagc
121560ctttgctgtg gtgaaaggtg ctgtttctga agccacgaca gtcccatggt ggtttgagta
121620acaatactcc tggcttggta acaatgccaa aaaataccaa aaaaaccaaa accaaaacca
121680aaaaaagcac agagctcaca tctgagccaa aaaaaccgac actccctatt ttttgaagaa
121740ctcacagaaa tcaagaagaa aaacaagcaa acaaacagca cataacaggc taacaacaac
121800aacaagtcca cttcagaggc caaaaaccaa aaggcaaagg ggctcttaaa catttacaag
121860gacagacctc actcagaaca caagctatag ccatgatact gctcttcatc catcaattct
121920taaagacaca gaagatgggc tgctggcatg actgggaaga gaccagtgtg ctcactcatt
121980gctggggtac gctgtaacaa gcagaggaga atgtctaact gtgtctgcca atcacaggca
122040cattcccagt taacccagca atcacttctg gcaaaaaaag cccactctgc ccgcacactg
122100gtgtacccag gaggtaattc actgcggctt ctggtggatc ttttttcctt cttgcagtgc
122160ttgggttcaa attaggatca agcacacttt tatagtcaga gcacggagac aaaaggatct
122220aaccacagag ttggttaaat aaacaacaga acagccacac cagaactgct ggggggcggg
122280gggagggggg ggagggaagg gggagaagct agaatcccag cattcaggat gcagaggttg
122340gtaaacaagt ttcaggctca gtcggggtat aaggtaagac tttatttcac aaaaataaat
122400aattttttta aagaggacag aaaaaagaca gcgtagagaa ctagctatct tttatgtaaa
122460ccatgtgggg tacaggagca gcatttgcat ttggttactt ggcgaatagc cctggtagga
122520taaagaaacc ttaaactgtg ttatctataa agggcagcag tgggtgcaca atgcagtggg
122580taggagccca cccattgcac gccatagttt tgattacaaa gtcacgcagc tctactcaaa
122640aattaaaaca aacattaatg cttgttaaag aaacagggct gcagagatga tggctcagtg
122700gttaagagca cagcgcccat atggaggctc tcagccatct gcttctccaa ttccagggga
122760agctaacgcc ctcttttggc cttcaagagc actgcatgca catggtacgc ttacctacat
122820gcccgcaaac attcaaagaa aaatacaaac tgctataaaa cccatatatg accatcttaa
122880gattctttca tttttttgag acagggtttc tctgtgtagc cctggctgtt ccagaacttg
122940ctctgcagac caggctggcc tccaacccag agatctgcct gcctctgcct ccacagtgct
123000ggaattaaag gtatttaaca cacacattat acatatcttc cttcttcttc ttcttttttt
123060aaagcgtttt gttgtttttt gttaagtttc aattaaaaaa ctacatagtt ttataggcaa
123120acataattaa aaatgccaat gtgaaataaa taatatatac atatataaca ttctgtaata
123180gattcactca cacaacttat atacttaaat acaattttca caataatgaa aagctttgaa
123240atgaagactt ctggatacat tagaaacgta ccctgaaaat cgcaaatgac ggttttcatt
123300tctttgtgtc agacattagt gtgagtgtct aaacttgcat aaaggctctc ttctctatca
123360cttcctacct attgcagtgg ttctcgctaa acctggaacc tggggctcct gttccttggc
123420tggactacaa ggcagcaagt cccagcaatc ctcctgtctc acctttcttg gaaccaatgt
123480tataagtgtg tgtgggcact agccttgtta catggctgct gggactagaa ctctggtctt
123540caatattagg catcaagagc tcttaactgc taagccatct ttctaccctg attagaattt
123600cttgaagcaa aagaaactca cagatggtca gagtttacac acacacacac acacacacac
123660acatacacac acacacacac tcacacacca aggcttagtg accactgtga aaagggaagt
123720gcggtgagga actgtaaaaa taaggtgtca ggaaagctct gcacaaaatg gtgtcctctg
123780gacaagccag ggcctctgcc ctcatcagct cttagtaact atggttgcct acagcaaacc
123840atgccaggga ccactctaac atggagctgg gatgggctcg agaggccctg ttattaaagg
123900agaagctgta gagagttgat ttgatggatt ctaaagtagg gggaatcgat ttcctttatc
123960gatatgggtc agccatgctc tagtgagtgg ccccacaccc acccatgagt atgtgtggac
124020agcacacacc ggacctggca agtcaataaa acaaaaacaa aaacaaacaa ataaacgttc
124080tggtccacca tagtggctgc tcgtggttgg gagctgtccc gcacttatgg caggaagaca
124140gtggctgcaa agaggacaaa agtctctgga actgatcaac tctagagtct gcttgttatg
124200agaactggga agtacccgct gggacagaag cagactctga aggtgatcag gacagagatc
124260acagaaggag agactggtta tcggaggaaa tctgaaacat aactcgacgc atactggtcc
124320aaactggtgc ccatcactac aacagcagta attgaattgg gcacaacatt cagaaaacag
124380aaaaagacta cagagtacgc accctggcta tcatcaaccc aggcgattct ggcactattg
124440gaagcaagcc agactggaga aaaggaaaca aaaagttatt taacaaaact tcccagagca
124500tgttaaaaaa aaaaaaaaga aagccagaca tgggggtgca cgtttttaac cctagcactc
124560aggaggcaga ggcaggtgag gcaggaggat ccatgagttc gaggccagcc tggtctgtac
124620agtgggttcc aggaaagcca ggcaacaaag aaaccctgtc tcaaaaatca ctgactgggg
124680agagaggaag tggatccaag agcaagagag agagagcaga gagagtgggg gtttggatgt
124740cagcgttatt aaatgacagc agaaaagatg ggcccgacca atgacatccc agaaatggca
124800aagatgaaaa aataaacaca agttctaaat atcattttaa taatggctgt gtgtctgctg
124860gctcattctt gttatccaaa caaactaaag caggggtggc cgggatttac ggccagccag
124920gactagagtg agacactgct ttaaaaaagc aatagatgca cgctaaccat taatcagcgt
124980aactggagtt tgagggaggg agtgggcccg ggaagcctgt gcctataaac ccaacctgcg
125040aggcctgaag cccgaaggtt gaactgagaa catctcaaga caaagcacag gcacaatctc
125100ttacaaacag tttaaacaca ataccagcaa taaattgtca gctttatgac agataggctg
125160acaggcatac cacaaagatc gggaagaaga aacgggattc ttgcacaaca ttttacaaat
125220cgacacagct gagcctagtg acaggccgtg ataagctcaa aacatgcaca ctgcaaacac
125280cacagcatgg ccagagtgac agggtcacag caatacagca acagagacag taagaaacat
125340tggaaaaggg aagaaggcaa agagcaccac gagagaaaag ttaacccgcg attccatatg
125400agggcccagc atccctctcc cacctgacag agaaaaccag gaggacgcag ctgaaacact
125460gtacaactat aaatactcct tcctagtgta gacagagttt accaaagggt atcgtaatct
125520gaagcacaaa cataacactg taagttacca gaagtttaga cacgttgaga atttaataga
125580agttgagaca tcaaacacac gtttgatcct aggggaatta aattattggt aacagaaaag
125640ctctttacaa agtctccaca tttataaacc aaatacactt gtatcagaat tgttgtctga
125700gccagaggcg gcggcacaat ggtggctgag gcagcaggat caagtctgga gaagcccctg
125760ggtccaggga gcactagtga attcaagtaa acattaaaca ttaaataaga aagacggatt
125820ctacaggagc ctagacaccc tgtaaggtca gtactactaa gataccaaaa ccaaactgac
125880tcagggggct gatgagatgg ctcagtggct aagggcatgc actgctcttg cagaggacct
125940gagttcagtt cccagaaccc gtatcaggca gttcgcccac ctgtaatgta ccagagaccc
126000taaacatttc tatcctccaa gcacagacac acataaacat aattaaaaat aaatcttaaa
126060aaaaaatctc tcgtggacag acagcaaaaa atactgaacg aaatctaatc aggtagactc
126120agcaaagcaa atccaaatat gcgaaaagga taatacaatg caaatccaga cttatcccag
126180aaaccccagg tcgctttagc atccaaaaaa ttcaatcatc ataattcacc gtattagcta
126240accgaaacag aaaaggcatt tgattattaa taaatacagg gaaagcattt gacaacattg
126300accattcact cttaataaaa atgttaccaa acaggagaaa gaagtaaaat ctcctcaacc
126360cgatgaacag gaacacaaac tggctgggca cagtggcaca tcctgaaacc ccagcaccca
126420gatagctgcc agactgggct ggcatgagaa ccaagggtaa gaggcaccca aacacttaat
126480gatccaaggt gctgcctccc ccccccccct gtgtcactta aatccgtaat taaatttctg
126540ggaggagggg tttgacacag ggtctcactc tgtggcacag gctagcctga atggagtcaa
126600tgctggcctc aaaactcttc tgcctcagtt ccagagtgag gggaaacaat catgagccac
126660ctcacccagt tttaatgcca tttaacagta caaggttaaa cgctgcctga aaagaacaag
126720acaaagaaag atcacacaga caaacatcac acaggtgctg tttgagacta ggtctctagt
126780gatgtaggct ggtctcaaac ttgcacaaga aaagaatact tttgccatca agatcaaggt
126840tgctatcacc catggtggca tttcggaagc agaggcagga agatcagtag tacaaggtca
126900tcctcagcta ccatgggtgt gaggccagcc aaggcagcac gtgagccagc tgtggaagca
126960cacatctgta accccaccac tcaggaggct gaagcaggaa gttcaagcca cacaagagcc
127020acttaaaaat aaataaaaac ataaataagg ttggagagag ggnnnnnnnn nnnnnnnnnn
127080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
127140nnnnnnnnnn nnnnnnnnnn nnggaaaaca cccattagac tgctgaagag actgagaaat
127200tgtcatcttg aagggttgaa atcactgagg aatggctaat gacaaaactg cttaaggaga
127260attttattaa aaaacaaaac tccttctgga ggagtactgt gctcccttca gcattaacta
127320ctgtgccagg gaacccactg ccagcagaca cccaacttca aggataatca agcttgctac
127380gtaacatggc tgcttacgca gacaccacac acacttcctg tatgctgggc atcacctcta
127440gcttacttat aatatcaaac ataagtacca tgtgagcgct acagggcctg gccaatgaac
127500agcgatggga aaaataacct gcacatggtc agtaatgggc aatccactcc ccagacactt
127560ctaacctcag ctgcagactt gtgggtactg agaacggacg tgactaagtc aattcacaga
127620agcagagccc tgtgcctgtg ccagggaacg gaggacgctg ctcaatgggc agagcttcag
127680ggcgggaaga gatggaaagt ggagctaggg cccgatgacg tgaatatggc taatgccata
127740ttgtgtgtgt gtgtgtgtgt gtgtgtgtat gtatgtgtat gtggtgtgta tgtatgtgtg
127800tgcatgtgtg tgtgtgtgta tgtatgtatg tgtgtgtatc caaaatggtt ataaaaatcc
127860catacaatgg ctatgcttat gtgtctttta ccacaagtaa aaaattttaa gtaatcttag
127920gaacacattt ctaaaatttg gaatacttgt tggggaagct atctgagccc cagcatgaat
127980caggaaatgg gtaggagagg caggagagat ggcttagtgg ttaacacaca catggtggcc
128040ctttcctggc accaactagg tcactcacaa ccaggatctg acagcctctc ctggcctcct
128100caggcaccag gcaggaaagc ggccccacat ccctgcaggt aaaacatttg tactgaaact
128160aaataaaaac ggagcagaag ctgggcctgg aagtccttta cctccagcac cgggaagcag
128220gtggatattt tgtgagttcc gggtctacat agtaaaaact tgtcgccaag taaaacaaaa
128280caaaaactgg gggctggtga gatggctcag tgggtaagag cacccgactg ctcttccaaa
128340ggtccaaagt tcaaatccca gcaaccacat ggtggctcac aaccatccgc aacaagatcc
128400tcttctggag tgtctgaaga cagcaacagt gtacttacat atattaataa ataaatcttt
128460aaaaaaaata cctttaaaaa aacaaaacaa aaactcccac taaaataatt ggaaaagtcc
128520agggaaagcc acactcgatg gcgcaagcgt gtgatcacag tattctgtag gtaaggcaag
128580aggatcacag cgggacggag gccagcctca gctacacagg ccggtctggg ctacagtgtg
128640agaccccggt ctcaaaacaa aacaaaacaa aaaaagcgtt cacattatca tatactcaag
128700gccacaaaac accttcttct tcccaatcct tgaatgctat cgaatttggt tacttttttt
128760tggtaatttt ttgtttgctt ttcaaggcag ggtttctctg tgtagccctg gctatcctgg
128820aactcactct gtagaccagg ctggcctcta actcacagag atgtaattct ttttcaaagc
128880tggattttga tttgggggtg tgtgggtgat accacaggga cacttgggcc ccaaaccaaa
128940ccaaagaaaa aacacccccc ccccaaagta tgaacatcaa ctgtatataa aactaacagt
129000tcatataagc taagtagcct gcaggtacat ggttatggaa acagctttat catacagact
129060tctttcagta gatgccattt gagaaaaaaa aaaaaacaaa acattctttg gattttcaac
129120atgtaaacga aaatatgtta aatcttaaaa aaccttaaag cagacgccac tgctctttgg
129180ggtcagggaa gcacggctgc aggcccccag agcagcacat tccctgaggg aagccttgtg
129240cgtcctcacc agcaccaaga acagccaact ataataagct cataaaaaat cctgaaaggc
129300tgcccaagcc tgaaaatctg atatgaaaac agagggcaag acagataaaa ggcaaactat
129360actttaaaag tcaattccag ctatctgtgt ggaaggactt ctcgggacgg actatctgct
129420aagaccttgt gggtgattta aacagcgtgg agggcagaaa aagaaaggaa agtgcaggaa
129480acaatggcaa aagctcctcc tccgtggcct ctacacgcat ccaagcgata tggagggagg
129540agggaaggac actatacaca ggtaccaatc cccacgaact agaaaacaca gcttttacta
129600actagtctat tttttttttt accttagcta gtgtctttct tatgtttggc ataatttctg
129660ctctgcatta aataaaccta gtgtataaga aggcaaatga gtaaaggtaa agtcagttaa
129720tgttcatttg ttttgactgt gtggtgtgca tgccaggcgc attcgtggag gtcagaggac
129780aaccgcatgg agcaggtctt ctccttccac caccccaggg gtcgcagggg tcccagggat
129840ggaactcggg tggtcagatt tgcttggcaa gggcttcact cactgaggcc ccccaagggt
129900cccaattcac ctgtttactc taatgtatat atttttgaga cagggtcttg ctatgtagtc
129960caggctagct ttgaactcac tgtggacact agctggcctg gaggtctgag tgaacctcct
130020gtttccaccc aagtgctggg acaacagaca gcaaccatca aggcaaaatg aagtcttccc
130080ttcacaccaa agtggcttcc tatgaccttc tcctgacaac cccaaacagc aagtgcccgg
130140atatgcactt gcgtttctct ttttttactt caagctttga ctccactttc ctaaaaggtg
130200tttactaggc tgaagcacac ttcaggagac accctgtagc tctggagtga ccggaaacac
130260accagctctg atgcaaaaac aaaaaacagc gatggggatg gggcaaaggt ggcttgtgct
130320tctgtgcaat ggcattccct gacgcattac ctcacaccag atacatacag aaaagacaaa
130380ggtaagtcct cactcagtgc gcgcatccac cacacccaaa gcaagcattc aacagctcag
130440tggcataagg ccagcattat cacagcgcgg acaaacacaa agccaacctt ctctttgagt
130500tccccaaggt acatagtgtt ctgtccaagg atggcctctg cagactcctg gaaacacgtc
130560acccactggt tctcttgaaa atctgcaata tttgcctaaa aaacacaact ctattatttc
130620agcgaataaa taacaaaagg aagacacccc aaggacgagg gaagattatc ttttcctact
130680taaagtaact tccaagggag aaatgacatt gccgccacac acagcccccg actctttggt
130740taacggttcc cccttatgag gaagtattca cttctggtca ccccgtgttt cacttttagt
130800acagtatcta atgaattaca ggagccactc aataccttta tcataaagtg ggctttgtgt
130860taggtgattc tgaccaagcg taggctatgc aaatgctctg agcacactta ggggaggctg
130920ggccgtgcta gccctgctac atgcaggtaa tgctacgcca gtcttacgcc acaggaagaa
130980gcttaaaatg tggtcgaact caatctgcta gaggtgtgag tttagacctc gggagacggg
131040ccaagtaaac ttccgcagat gtcggagcat cagacagagc tgtcccctcc tgatgcaaaa
131100ggcttcgcac ggcaagtttt taatgagcct ccatggtaat agtgggttcc tctctctcct
131160cctcccttat caatatgctt gacatctttt agttttttga gacagcaaat aacgtagccc
131220aatctgcccg catacttact gggcagccac agctggccct gaactcctcc tgcgtctcct
131280ttaccccgta acaagtgctg gggttacaga catatgccgc catgttcagt gtaagtgacc
131340gctgcccagc agggcaaagt cttccttatt tacaaagcag cagccaagcc ctgcagccca
131400ggcctatctg atttcctcag cacaccccca agggtctcac cgatggcagt cagtccatga
131460acaccgtagg cttctcacaa tccacactac tcaccgataa gatcatgcgg tatttgaaat
131520tgggaaattc ccggtcacac ttctcacagc ggtacaaccc attctgctgg tcaatcactt
131580tcttattgca gtcctgggtt gggcaggcct ggtacataca gttctctttg cggagaaaca
131640ccaccgctgc cacagtgctg aaatagtccg cctgcaggcg aaaggaagac cgccatcagc
131700aagcaacaca ggtctggaac aggcaactca aagctgctct tccttgcaag tggtgagcgc
131760gtgtacatcc tagctcccac gctcacgagc gatatgcaga atcactaact ctggtttcag
131820aaatcacacg tgctacacgc agaacccaaa gaagtaacaa accggcactc acgcacctca
131880ctttttccat tttggagacg gcggctcact agctagcctt gaactcagag tttggtctgc
131940ctctgtctct atagagtgcc tggctaccac acctgggcac tttgttttcg agacagggtt
132000tctccggaac tcactctgta gaccaggctg gcctcaaact cagaaatcca cctgcctctg
132060cctcccaagc gctaggatta aaggcatgtg ccatcagcgc ctgacccaat tctttttatt
132120tatagttatt atttgttaca tatgtgtatc agtgtgtatg acgtccatat gtatatgact
132180gtgtgcagtg tgcacatgtg tgcaggtcag aggacaactc tcaggagtca gttctctcct
132240cctactgtgg cgtttgggga actcaggttc caagatagca ggaaaagtgc ctttaacagc
132300tgagttatct cgacactgac atgtaatgat tcagtgtgca cacagtggtt ttgcatgtag
132360tcatataaac cacagcgtgc atgtgcaggc taggaaacaa cctgtgggag ttggtgatct
132420cccctcacca tgtgagtgag ggagaggaac tcaggctgtc aggctcggtg gcagtgcctt
132480tactcactga gttcccttgc tggccaagca ttatttataa gatggtgatg tctaccctta
132540tctttaggga tcagtaagtt tttccccaag acaagccaga aaaacctttt aggcccaatg
132600agccattcag tcagtctcta ctgccactcc tcaacctgtc tgtggggcag gacaagccac
132660agacaacctg aagacggaag gtgagccaat aaaatcttac tgacagtaac agccagccag
132720ctcacaggct tgacaggcaa ttcttggact gaatgcgttt aagagaatgc agaattaccc
132780atgactaaga tcttctaaat ggaaaaatgt ctggttaagt aacccagcaa ggagctaagt
132840cacgcaagcg gtggatacct gctttgcctc tgaaccctgc acaggttttg gtttatgttc
132900aatcatgtca agtacctaca aaatccagat ctagcctgaa ttcaaagata ggctacctac
132960cctgcccccg gacccccacc cggggtctca ctgtgtagtc ctggctgtcg tagcgctctc
133020tatacagacc agctgttatc aaattcagag acccacttgc ctcccaagtg ttgggattaa
133080aggcatttgc cactatgcct ggctctcact ggttgtacca gaggagcaaa acaatgtggc
133140catttaaaga gacgaagcta gaaaccagtt tagacagctt tggggctgtg ggtgtagctc
133200agtggaagag cttgcttagc atgcacaagc tgttggtttt aaccctcagc gtgacagaac
133260cgaatacata agaaggctta gaggaggggg tggtggtgga gagatgggtt agaggttaag
133320agctggttgc ttaatttccc agtccccaca tggtggctca caacatccat aactgcagtt
133380ccaggggatc tgatgtcctc ttctgacctc cttggggaca tgcgactcat ttggctcaca
133440tgcaggatgc ctcgggccac tatgctggct caggggtgaa ggtgcttgct gccaagctgg
133500gtggatttga gtttggtccc tgggacccac aaggaagagt ttgacttcta tacactgagg
133560tggaacatgc atgctctacc cgcaaattaa aaacttaaaa tttaaagagg aagctgtaga
133620gaaatagctt ttaggaggat gcctaaggaa cttctctgcg ttttcaggtg agattcagac
133680tcaaagccca atttaaaagt ttgagtgctg tcacgtgttc tgtatgccca gttctggctg
133740tccgttgtct gtctttagtt taagagagca actgggtgag aagtaactga gagtctagcc
133800gatgtttaat tctcaagatg tcctgtgata gcattataag ttgctgtgga tgacagtgat
133860ggatcgagca cacagtgaaa tgagacagtg aggaagaaaa cctatattgt atacgaggac
133920aataatggag cagtggcgag cgatattttg ggaatacaat agaaataaat gattcaaata
133980tccaagagaa cgcaggttag accaaggtag gaagaggatc actgggctgg agagatggct
134040cagtggttaa gagcactgac tgctcttctg aaggtcatga gttcaaatcc cagcaaccac
134100gtggtggctc acaaccatcc gtaataatat ctgatgccct cttctggagt gtctgaagac
134160ggctacagtg tacttacata taataaataa ataaatcttt aattaaaaaa aaaaaaaaaa
134220aggaagagga ccactgagca cacattgaaa tggaagatgc aactgaaatg caataccaga
134280gccagtctgt ggcatggcag caggaattac agtctgttca aaacccagca cggacctgag
134340gctacattat gtccctctac actggccgtg actgaaaagc agcaacgtgg taccctggag
134400caggcctgag gtcccaggta aggacacagc cctgacctgg cactaggaaa caggtgtaaa
134460aactcaagcc cctgccgttc tctgggtgtc tggagcgagg ggtgcgaggt accttgtctc
134520cctggcccag gttctcagat ttagcctcat gcaaagtttt ccagttggtg ttgccccctc
134580cggcccctcc actcctgtgg tcagagatgg aaacaccatc taaggcttgt ccttctgagt
134640caaacctagg ggagaaagaa acaaacgtac tgctgacagg aacttcacat ccttctcaaa
134700tgtgctgtga atgcaccagc cccgagctgc gccccctcgg ctctcaccta ggtctcagca
134760cacctagtcc actcaagaaa tgcaatgccg ttgctccttc catctcgtgt ccttcaatgc
134820ccctcctcct ccacccgcaa ggtcagacaa actgccaaag taccacagac cctttcccta
134880aactgggctc acattgacag cagccatgaa caaaggcagc aacagcaagc cacactggca
134940cccctccccc caccctcggc ctcaagcctt caccccaatc accaaaatga accagatgaa
135000aagggacatt tttgtttcat gagtctgcca aaaatgtttt catgggagaa aatcttttaa
135060gcccaatcag ttatagaact caatcagaag gcattatctg ctgtgttaga gatttgcatt
135120ctcagtgaga atttgtttat ggaaatatca aggttaaatt catttatatg aaattattat
135180taaaattgac tatacttctt gaccatctat tggctaatct gaaggaaaag aggccaggga
135240gaaggtagca aggacaggct agtggcagag gacagtgagc ctgagatgaa aacacctcaa
135300ctacacgatt ggtggtaact aaagctgccc ccacacagga ctgactccct aaggagactc
135360ccaacagagt agcggtggcc caaggcaagc agtcacatcc gtcagaggac aaggtccata
135420aatccgtctc accactacta gccgctcaag tttaacttca cacaggcatc ggcaaccaaa
135480gacggctcat caggagtgaa cagagccagg agacaggcag cctctcttat agtaaaaggt
135540ccactcttag agccaagatc taaagatcgc taatccttgc ctgggtaggg ggggtatact
135600gaacttgtag atcaatgaca gtattttgtt cgggggacag acatgcctgt ttgtgatttc
135660tgtgatcaca gttactgaca gtgaaatatg aacaccttaa tgtagacaca caattaacct
135720ggggttgtgc tcagagtctg ggagaaaaac caaccaacaa accagaccaa tgccatggct
135780gtctctggtc agacttctgt cctacaatgc aacgctagct atcagcacag ttacacagcc
135840agacccacac cactgaggat gccttaagtg acagacaccc cagatggatg ctctaagtag
135900taaatattgt tattggttta catcagactc aaaacaaaaa tgaggagcgt gaaatatgag
135960ctgtttttct tgagtctctt aaattctacg acacagctgg aaaccacaca tgcccaccct
136020ggtactacag ctattcaatt tccttaagct tgggctaggt aaagtatttt ctttgaccac
136080ctcaatcctt ctacacaaca ccctcaggat gaagtgctct tccaaggcag agtttaatgc
136140cttaaaaagc tagatatggt gtggaggttc taacaagctc tcctcatctc ctgagtgctt
136200tgggaatgac aatgtacagg caacaaagag tccattgttc tcactgtact ttaccgagtc
136260cagtgacaac acaacagaag atgtgaccag ctccagagac tcagcagagt aagaaagtac
136320attctacccc cacatcagct gcccacagtg caaactgaag atgctctcag cactgttctt
136380cagcgcgcag ctgttaaagc cttgccgtac ctggcgggat caaggtcagc acttagattc
136440taattgcttt ctctggctct ctatgttcat caggggatcg tgggtttgtt ggtttaagag
136500ggctttcagg ccaccatctc agaagacata atgcctcgaa gaagtatagg aaccactcca
136560ctcaatagca aaggcttggc aaaacagctt ggcagcagtc cacatgctga cagtgtccag
136620ctctggttta ccagagcccg agcacagata acccctgagc tgagttgcac aaaacctacc
136680agccacgaag cttataggcc tctgggatgt caggattcac aatgacagtg ctggatgaga
136740ggaccgagag gctccgtcca ccgaagtcag agactcgggc tcctttgatg gccatcacgg
136800gctgccgaga gccgtcaaac ttgtcagcct gcaaatcaaa cgcacgcaca catcactgac
136860aagaagaatg tacaggatgg ttttatggag aagagtcagt caatgtctgt ggactctaag
136920acagacctcc cactcgggaa ggagcattgc actacaagaa gctgcaataa ccgatcatct
136980cacacagcga aggtcttcaa atacttactg gatagcacaa ctccctgagc taaagcccca
137040ccctcaggac tcggctcagt gcaaggactc acatcttctc cccacagagt tgtggtcacc
137100accttccctg acatgtccat caaatagata tttctcttag caacttctct gttgttcgac
137160ttcactgtga ttttaatcga atcttcatag ctcttgcaga ttccaatgat gtctgaaaca
137220aagaacactt tgtaagagct cccatcaagg ctcctcttta aaacacggca gggacgaaag
137280gcaagcacgc tagtcacgca tcaccccaaa cactggagac aaaaatcacc actctttgcc
137340ctcaaccctg gacgcaccta ctagtgcgtc tttagccttg ctctctaggt caccgatccc
137400tgtgaaatca aactgaactg tgggtaagtg atggccatct tcacagggaa ggacagaagt
137460ctcattattg aaggtcatct catagtcatt tttaacagcg gagaactgtt tgttagcgat
137520cttcagggcg ccctttgaga agtaatacac ctgcaaaaga ggccaagtca gggcaagcta
137580ctcagtccat caaagcccgc cccacaatgc agccttccaa actgtgctca tggctctcac
137640agttcagcct gtcagctctg actcaacacc gtcttcttac cttgttcact tcaataaggg
137700gaaagaactt gtccacttgc tcattgaaag cagtagctct gatttcaccc tgcagaagca
137760agggagagca cattagaact gcgctgctag gccctcgctc tcaacagcga gagcaggcca
137820ttttgacagt tagacaccat cctcggagca acagtccaag tgtaaagaga ccctcacagg
137880gctgcagaca ttgcagaatc ccacatatac agttacattt tcaagctatc agctctggta
137940aaacaaaagc agctcagcca cccgagccta cagttgtaag ttaacacata aaacgaggga
138000gcctcgagat gatctggaag acaaaggtgt ttcctgtgcc aacctggcca ccctagttta
138060atcccggaac cctcgggatg gagaagacaa tcgccccacc aagttattct ccagcctttg
138120cacacgtgtg catgcataca cacgaaaata attcttacat cttatcaaaa catttttaga
138180aggaatagct taatattccg ataatgaaac ctaatattct gggtcccgag tgatgggctg
138240gaagcccaca ggaaggcgtg ctgaactcca atgattcagc actcttcctc acgcccgcaa
138300cctaagtgac ggcttacttc acagtcagga gctggtgcca aaaatgttcc aacaagtcat
138360gctttcaagc tgactgcact gttacttttc tgtcaggcaa tatgttacag atggataaga
138420aacttaacta aatggctaaa accttaccca gacaagctgc aggaaattag tttctgatac
138480tgaatctgcc atcacagtta agatatctgc tgtgctacac cgtgacaaga ggaagaagag
138540ggggagagag ccccaaagct ttgcccttcc cggtatcatg gcttatttta cgtgtgtggg
138600tgttttacca gcatgtatgt gtgagcatca ctgagtgtct gatgcctgag gtgaccagga
138660gaggtactgg atcactgaca cgggagtcac agatggttat gtgctactct gttggtgctg
138720agaattgagc ccaagtcctc tcaaagaaca gcgagtgctc ttaaatgctg agcagtctct
138780ctggcccctg cattttgctt taagtaaagc ctagtgagta taaatgatca gaagccctcc
138840ccgcagacta gttaaagctg agtagctgct gctccttctg ctggaggcaa acccgccctg
138900ctcggcaggt atcaccccag ggcttcaact gtgcccctag caatgtaatt agagcgctgc
138960agtctctgca gacggagact atttacagtc caccaatctg tattgctaca gacgcgtccc
139020tttaggagca ctttatctca tgtctgaccc tgtgccccag atagcatcaa aggtctccaa
139080cagaaggaaa gccacaatga ccgccatgtt cctcggagct gagccccacc cgnnnnnnnn
139140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
139200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntttttatt aattatgtat agatatagat
139260atctctatat atgaatgagt atcctgtagt tatcttcaga cacaccagaa gagggcatca
139320aatcccattt acaggtggtg gtgagccatc atgtaattgc tgggaattaa actcaggacc
139380tctagaagag caatcagtgc tcttaacccc tgagccccct cccttttttt tttttttttt
139440ggcagcttat ttttaattgt tttaaatcac gtatatgttt ctgtatgtag ctatgttcac
139500ataagcgcag gcactcatag aggtcagaga tgtaagatgc ctttggagct ggacttaaac
139560atggttgtga gccatctgat atgagcacca agtggacttg ggtcctctgg accgccttgc
139620tgccttccac ctagaattac agtatatgca attagctgct cagccacccc ctcagtcctc
139680cttagtactt tcaacatagt gtaaatcgga gagcactcct tggtgcaaag attctgtgtg
139740ttctatcatt ctctacaccc aggcttcatt gggacgtgtg atgccagcga tgattcattg
139800gcgtgaagta tgagttagag tcagacaatc agtgctctct gctctgagtg ggtctccctg
139860cccctgtgtc tctgagacag gggaggattc tcctgttgga tcacatagtg catagagccc
139920tgtgtgcaca gcgctctcaa ccatttgttg ccatttcaaa tacagtgact tcagagtctt
139980ccttgaaata acagatttct ccattttgtt tgttcccttc tttcgtcaca aagacctgag
140040gatgagatgt ttttgaagga actttctagt agttactcgg tggaaaagga caatgatgct
140100cccctcttct acagagaaga aggaaacagg aaattccaag aaaaggagta cacagatgct
140160gcagtgctgt actctaaggt aacgtgcgtc caaccagcag ttgaacaagg cggaagatag
140220aggcaaagtg cagactcata accagtccat tgttttgctt tggttagttg ggttggttgg
140280tttgtttgtt tgtttgtttc ttcttcttta ttagtcggat tggatttggg aaaaatgtag
140340tagaattcta ttgttgatta tattcacaaa caaaagactg tttatatgtc ctgaattttg
140400gcaagattct tcccatattc cttcagaccc atagcagcag caagcttagt ggccctttgc
140460caggtattct cactgagctg tacagcattt gtctgaagaa ctgatgaaca ttttagctta
140520tgctcctatg ttagtaaata gctaggtttt tagctagcca ccttgctagc ttacaactag
140580aatgcctctg gctgagtttg gtggcacttg cctttagttc cagcactcag gaggcagaag
140640caggtgaatc tctgagttca aggcagcctg gtctacataa caagttccag cctggccagg
140700gatatgtagt aagactctgt ctcaataaag taaacagcca gaatgactta aatattgcta
140760aaaaagaaag aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaagaaagaa
140820agaaagaaga aaatgagtgg atttgccata gcctagtcaa actagatttt cttggttata
140880tgttaacata ctactattaa caacaacaaa tactttacac taaccatgac aagctatatt
140940tttaaattat tattttatat gtatggtgtg ggaagctgtt gcagagtggc agttggctac
141000tgctggccac cacacataca taggcagtga aggttctttt gccaagacaa gttaaccaat
141060cagatgtgag acacgcctct cctaggccta tgtaagcagc accagttctg ggctcagggt
141120ctcttcgcct ctacaatcaa gctctcccaa taaacgtgtg cagaaggatc ctgttgcagc
141180gtcgttcttc ctggccagtt gagcgcgcac aagagtatgg gttttcacct acatttatgt
141240gaactgcatg tgcacttggt acccagggag cccagaagag ggcatcatat cccctggaac
141300tagagtcaca ggttatgtat gggttctagg aatcaaaccc atgtcctctg gaagaacagc
141360cagtgttctg gatttaactg ctgagtcatc tctccagccc caccatgatg gatttaactg
141420ctgagtcatc tctccagccc caccaagctg gatttaactg ctgagtcatc tctccagccc
141480caccacgctg gatttaactg ctgagtcatc tctccagccc caccacgctg gatttaactg
141540ctgagtcatc tctccagccc caccacgctg gatttggaag cagaactgag gctttgaaca
141600ctgtgttatt ctaactcttg cttccttgga accctgagaa aattccttct attggctttt
141660cagagatctg tatgggctta aaacaagaat atgtccaact cttgatctct gattttatat
141720attaaaagaa tagagtgagc ctaggagttt ggcacactcc tatgttccaa gcttcaggaa
141780gggaagaagg tcaggagttg aaggctagtc agtcttagct ttgtgacaag tttgaggcta
141840acgtgagctc tatgagaccc tgtctaggag aggagaaaga aaggggtaaa gtgaggtgtg
141900gctccacgct accacttgcc taacacgcat gacgcccaag gtttccgttc ccagccctgg
141960ggttcagagt attgtgcaca ccctgtatct ttaaaagtca atagtcaacc cttggaatta
142020actttccgaa aaatattaaa gctacatcat ccactctaca aacttgtaaa agcccttctt
142080tgtaatggtg taagaaagta tttggcttca gttttatggc ctttgcatcc taaatgttac
142140cccatggaaa gttgcttaat gaaaacaggt aaaaataaga caggagggac tgcaaagatg
142200ctcaggctca cgagtgctta ctgcccttgc agagaccatg gcaggcagtg ggtctcccta
142260ctgcagggaa tccaaaaccc tcttttggcc tctgcaggca accacattaa cacatgcaca
142320catacacata attttaaaaa taaaataaat cttttaaaat gagctctaga acgagtttga
142380catcagtcta ggctacacaa gaccttgtct caagaagaaa gaaatgaagg tttgctgtgg
142440atggagaggg agatgcactt cctattccat gaagctgcta ttttggtgat tatggtactt
142500tgcaatttta tagagagctg ctattttctt tcttttaaag taatgcttgt tgttttgact
142560gtaaaagtaa taaatgttac tttggaaaat atagagaagt ataaagagta aaaaaaaaag
142620tcataaccag tgaagtaccg ttaacatttc tgcttctatc cggccagcca gagtttttcc
142680tgtgagaatg tgtttttctt ttacaaaatt gggataatgc tgcacttact gttttgtagc
142740ccactctttc ccttcacgat ttattgtacc cattttctca cagtattaaa ttttcagctc
142800caagtaattt tccatagcta actctgtgtt ctgttacaga gaagaaatgt acttaattta
142860agatctaata ttggcataat atttggcatt atgatgctat aataagcatc tttctgtata
142920aatattttta tgtacagcca gtgttttgtt tgggatgaat tagcacaaga gtaaagtgtg
142980gggtcaagcc tctgagtgac gctgaattgt tctcaggaaa ggactagtta acatccattc
143040tgaaagaatg tgggaatgct catttcttaa gcaacactgg ttattattac atactattat
143100tttgttagta ttatacattt atttaggggg ataggaaata tgctcatata gttcaatttc
143160aagagttgca aaaatatatg gtgtggtttc ttttcctgtc ccctagttta gtctcacttc
143220cagccccata atctaccttg ttaaatatac tgtatataag gcatatgtag gtgaataaaa
143280aacagcctag tatggtggcg ttaacctcgg gttacttgag aactgctctt gcagagaaca
143340tgactttggc tccgaaagcc ttcctggaat tccagctcca agggatgcag tgcctctggc
143400atccttgggt acgtcactca tgtgcacaca tacacatttg gtttttaatc ttaggaactc
143460caagtgggcc aatgagatgg ctccatgtat aaaggcagtt atgcaaaggt ctggatgaca
143520tgagttcagt cttcagattc tgcaagataa caggagagga ccaacccctg cgagttgtcc
143580tctgacctca gtacacatgt catggtacgt gtgtagtatg cacatgcaca gaagtcccag
143640cactcgggag gcagaggcag gaggatctct gagttttagg ccagcctggt ctacaaaacg
143700atttacagtt atataaagaa actctgtttt gaaaaacaaa acaggggttg gggatttagc
143760tcagtggtag agcgcttgcc tagcaagcgc aaggccctgg gttcagtcct caactctgga
143820agagagagag agagagggag agggaaaggg agagggagag ggaggagagg gaaaggaagg
143880aaggaaggaa ggaaggaagg aaggaaggaa gaaagaaaga aagaaagaaa gggaaagaaa
143940gaaagaaaga aagagagaaa gaaagaaagg gaaagaaaga aaggggaagg aagaaagaaa
144000gaaagacaaa gcaaagcaaa actaaataaa atacatacaa tatattaaat tttaagactt
144060gagggcatag atcagtgtta taatgcttac ctagcatgcg taaaactctt ggcttctaaa
144120cctagcaccc taccgcaaaa tatttgctct gtcttgctaa attatattgc tagttgtcag
144180actactgtgt actttcacta gcaacataat gagaatgttc actatcccac tcctctgtca
144240agtaatctgt tcttggtttt atttttcttt gccaaattga tgggtgaaca agtatttcag
144300ctagcctaga acacacagag aactctcttt atgaactcaa gtttcttatc cttttatgat
144360ctccagaggt ttgtttttgt gggtttatta gtgttttttg tttggttggt tgtttgtttg
144420tttggttggt tggttggttt tgctttactt tttcttattc atttttttat ttctttattt
144480ttttgttttt aatttaatgg actggttcca tgtggccaag gatagcctca actttgtagc
144540agaaactggc tttgaacttc tggtcttcct tcatctacct cccaagtgat gggattaagg
144600cacgtgccac cacatctaac aatatctggg tttctttatt ggagtttgaa agggattcct
144660ccagcattac tttgactctt catagtttct tccagagtta ttacactttc atttgttaca
144720ttaagagttt gatccagggc tggagagatg gctcagtggc taagagcacc aactgctctt
144780ccagaggtcc tgagttcaat tcccagcaac cacatggtgg ctcacaatcg tctgtaatgg
144840gatctgatgc cctcttctgg tgtgtctgaa gacagctaca gtgtaatcat ataaataaaa
144900taaataattc tttaaaaaaa aagagtttga tccatttaca ctggacttct tgaggcagca
144960ggatcataaa ttcaaggtga gcctgggtga actggcagaa gtggcagaag ctgtgtctca
145020cactgctgta ttcatttcct cattgatctt cagaggtttg ctaacgggaa gtaagtggaa
145080cagaaggttc agtattcttt ttttcccaat tctattcagt ctttagtagt agatccctca
145140ttatctgaga tgcagagtcc cctttattcc tgtgaccatc tcgttgtttt tcagggagtg
145200tctcattcaa ggcctaacac tgaggacatt tcactgtgct atgccaatcg ctctgcagcg
145260ctcttccatc tgggtcagta tgaagtgagt attgaagaac ctggtgtcct gcctgtggct
145320gcagtggaaa atgagctcct ctctgttctt ctgcacacat tgaaatcaac tagcttgcaa
145380acactgacat ccacccagac ccattctctc ttctgactca tgtcacctct cataggtgac
145440cacaaacaat atgtagttga caagaagtag ctatgtcatt gtccacagtg catggatttg
145500ttccaatagg ccagcacttc tgtgtccata tcagctagat gtgctgctga tagtatttta
145560gattccaaaa tgtgtccaga tattacctcc ttcgtttgtt tcttctaaat aagccaggca
145620caaagacttg aaagatggct tgatggctct tccagaggct gggtttgatt cccagcccca
145680acatagcagc tcacaatagg ctataacgtc atttccaagg ggtctgactt cctgttctgg
145740cctctacagg cagaaagcac agacatacat gcaggcaaaa cacataaaca taattgaaag
145800aagatattaa ataatagccc acgcttgagc ttattcctct gatgatacag ctctcctgaa
145860gtcatcatgg gcagtgtaaa agtaaaggtg ccccgccctg cccaggggca tcagtgaggt
145920agctgactgt gagtggcttt cttctcatcc cctaactgct gcaacatcat caactgtgga
145980gcattatatc cgtggatttt aagttaggaa atgacaaaga ttagatctat ggccaggcac
146040tagtgcacgc ctttaatccc agcactcagg aagcagaagt aggtggatct ctgtcagttt
146100gagtttacag agagtgtcta ggcagccagg gctatataga gaaattctat ctggaaagaa
146160taaacaaatc agatctgtat ttcaggaaga tgcctatgag ctgtttgaca tgtgtgatag
146220aggtccttaa ggacaggaaa gtattccaca tgtgtgcatt ttacagaaat tggttataca
146280ctggagttaa ggactgttgg aatagttgaa tgttcacact cagtgttctt caaatcaaat
146340agaagaacaa gaatctatct gggcatggtg atgcacaact gtattcctaa catgtagaag
146400actgaggcat gctatgtgtt tgtggctaac ctgggctaca tagtcaatat tggacagtca
146460gagctgctac taaaacctaa aaaacaaaaa tctaataatc tgggatttta tattttcctt
146520tttttaaaaa aagagtgggc agaatgtctc tgattttgtt cagatggcca cagaacctag
146580aaaaactgct gctgctgctg ctgctgcata gcacacagct aatatttgac tatacgtata
146640taaattttgt tgtatacttt agctgtgctg tcaactttgg aaaaaaagta tcccagttta
146700tcattttaaa ttggcactgt acagaaatta acagccatat tagtctagac acattaaact
146760tcatttttcc atttatacag aagcaatgta ctgtattaaa tattcagtct tatctacagg
146820ggtttgatta cagaaactat caaagtattc tctaaatgat gaaaaaagat tcaagaatct
146880gactgtagat ccaaaggaca agtggagaaa aacttaggaa gaattttccc tttatcccct
146940ccctatattg atcatctctt ttacttctaa taatagtggc catttattga acatacccag
147000gagttccttt catcacttta gatatataat ttatctcatc ctaaaatgac ctgttgatga
147060gtcatctctc tttcagatga gaaacaaaga cattgaaaaa tctaacttgc ctgcataaga
147120tcacacctag cctcttactc actccatgaa tattcctttt ttttttttcc tttgagacag
147180aatctcacta tgtagttctg gttgtcctag aactcaatat atagaccagg ctagcctcaa
147240actcacagag atctgatagc ctctgcctgc cgagtgctag ggttaaatgg atgtgtcacc
147300aagcccagca aaatttacct tcttaatttt ctaagacgtt tctcctctta aaaaatggaa
147360ctattgagcc agtcatggtg agacaggctt aatctttaat ctcagcactt aggaggcaaa
147420gacaggccta tgggttcggg gcagcctgat ctatagagag agttctatgg gttaaagttt
147480agggttaaag ttttgagaca aaactttgtg tcaaaaacaa acaaacaaag ccagactgct
147540taataagaca aatcagacat aatattataa acaagtatta gtgtcactca attaaaaagt
147600cactcaggag gtgagatcaa tccccagcat aatgagggga ggaggggaga gaaggaaatg
147660aatgggaggg gaggaggaag gaagagagaa aaaggaaaga tctcaaagca gaacacagga
147720tgtaatttaa ggcctaagct ctcgactgaa gttgtccact tttaatgacc cttttcatgc
147780tcatggtttt ctgtcttcgg tacatagtga agagtggaaa ccaagggtca tcctggaatt
147840tccttttgtt ttcaggcatg tcttaaagac atagtggaag caggtatgca tgggtatcct
147900gaaagactgc agcccaagat gatggtgcgt aagacagaat gcctggtgaa cctggggaga
147960ctccaggagg caagacagac catcagtgat ctcgaaagca gcctcactgc caagccaacc
148020ctggtgcttt cctcttacca gattctgcaa aggaatgtcc agcatctgaa aataaagatc
148080caagaaaagg agactctccc agaacccatc cctgcagctc tcaccaatgc cttcgaggat
148140atagccctgg gggaagagaa cacacagatt tctggggcct ccctctctgt cagcttatgc
148200acacaccctt tgaaaggccg ccatctagtt gccacaaaag acattctccc aggagaactg
148260ctggtgaagg aagatgcttt tgtaagtgtc cttatcccag gagaaatgcc acgacctcat
148320cattgccttg agaacaagtg ggataccaga gttaccagtg gagacctcta ctgtcaccga
148380tgtctgaagc acactttggc cacagtacct tgtggcagct gcagctatgc caagtattgc
148440agccaggaat gtatgcagca ggcatgggac ctctaccata gcacagagtg ttctcttggg
148500gggctgctcc tcacactcgg ggtcttctgc catgttgccc tgagaatgac tcttttagcc
148560agatttgaag atgttgatag agttgtaagg atgctttgtg acgaggttgg tagcacagac
148620acctgtttac ctgaaagcaa gaatctggtc aaggcatttg attacacaag tcagggagag
148680agtgaagaga agagcaagat aggtgaaccc ccaattcctg gatgcaatgt caatggaaag
148740tatggaagta attataatgc tatcttcagc cttttgcccc atactgaaaa gcatagccca
148800gaacacagat tcatctgtgc catcagtgtc tccgcactgt gcagacaact caaagctgac
148860agcgtgcagg cccaaacctt aaagtcccct aagctgaaag cagtgacccc agggctgtgt
148920gcagatttga ctgtttgggg agcagccatg ctgcgacaca tgctacagct gcagtgtaat
148980gcccaggcaa taacatccat atgtcacaca ggtaagtcag aaatggtttt tacttacatt
149040attggtattt caagagctaa tgtttaagga gaaaaacact ataaaggaag cctggcatca
149100aataaatcag tgacctaaaa ggaaaacaca gccgtcttat atatcattat gctattgaga
149160agctttgagc acatttctgt gaacccagag cttgggaggt ggagatagga tgattaggag
149220tctaagacag ctttagctat acagcacgtt tgaggtcagc ctgaactaca tgagaacttg
149280tctcttaaaa acttgagcca gagccaggtg gtggtggcac atgcatttaa ttctagtact
149340caagaggcaa aggcaggcag atccctgaat ccagcctcgt ctatatagtg agatccccac
149400caggctacat agtaagatcc tgtttcaaat aaataaatat aacaaaaaca gcaataataa
149460caatagcaac aaattaattt tttagatgta tttatttatt ttatgtatga gtacaccatt
149520gcttttttca gacacaccag aagagggcat tggatcccat tacagatggt tgtgagccac
149580catgtatgtg gttgctggga attgaactca acacctctgg aagagcagtc ggtgctctta
149640gccactgagc catctctcca gtccattaat taaaaattta aaactagagt atttttaaac
149700atttattcat tttgtgtgtg gtatacatac tataatacag gttcataagt caattctctt
149760ctaccatgtg tgtcttggag atcaaactca ggttcttagg catgggagca agtatttact
149820tcctgaacca tctccctagc catttctagt attcttttct tttgtcttga aagatttatt
149880tattatatgt aagtacactg tagctgcctt caaataccgg aaaggggaat caggtcttgt
149940tagagatgat tgtgagtcac catgtggttg ctgggatttg aactcaggcc ctccagaaga
150000gcagtcagtg ctcttaactg ctgagctatc tccagcccca tttttagtat tcttattaag
150060tggtttccat tttatccaaa gatgccttta agggcctggg aagatggctc agtgggcatt
150120gaacttggtg tgtgagcatg aagaccagag ttcagatccc tagcacccag gcagatgctg
150180aatgatggtg gcctgcctga gattccagga caacggagac agacaggggc cctagctaac
150240catactacac actagctgag ctgtgtgctc aagagagcag ccctggctta ctgtgcagga
150300tggagagtga tcatctccac aggcaagcac acacctgagc acacagacat gcacaaagga
150360aagaaaagtc cttttaaggt ggtggtggtg ttgttgtttg ggggtctttt gttttgtttt
150420tttctccccc tccctcattg tgatggcaca ttcctttaat ctcacatctg ggacaaagag
150480gccggaggat ctttgtgaac tggaggtcag cctgttctac atagcaagcc catttcagcc
150540aggacgacat agatataccc tgtctcaaac agacaaaaat tatttatttt atatatttga
150600atgttttgcc tgcatgtatg tctatgcaca ttatgtctgg tgcccatgaa agccagaaga
150660gggcatcaga tctctcagaa ctggaatttc agacacttat caagtactgc ctgagtgcta
150720ggaatcaaac caaggtcttc tggaagagca gcaagtagtc tttttttttt ttaatatttt
150780tttattacat attttcctca attacatttc caatgctatc ccaaaagtcc cccataccct
150840cccccccccc ccccgagcag caagtattct tcattgctgg gccatctccc catctccttt
150900tctagttaat taagctgaaa gggagggagg tagatgttgc ccaaacttag gatttattga
150960cagattaata ctctgttagc ctaactacac tatagaagct tattctttag actttcacat
151020tacactgtcc agattttgcc atcctttttg ngtgtatatg tctacagatc ttaattcagc
151080tgccaattta tacagtgttt ataggtattc tttgtgacgt ggatctttta cccatcttaa
151140agcagtagga tttgaaagct gacatttatg tggcctatgg tcctgttaaa tcacatttca
151200agttagtctc tgtggtacac attttggggt ctatctgcgg ttccgcatct cacacttttc
151260cctctcaggg tgtccagaag ctgctgcaca ctgggctgga aggatgaagt ggagtccaga
151320gtgagtggaa ttctgcagca tcccggtcca gctgggagtg aatgctgggg tcaggaggag
151380atgggtgaga gggccttctc caagggcctt cttagtgtta cagctctagg caaaggcctt
151440ctctgacaat cttagcctgt gcatagtttt ttattcgaga tgagcttgta tgcatacact
151500ttattggcag taaatcagag gttatccact cttagggaag gagataggaa tacccaaggt
151560ggacagaggt cattggctga aggataacgt actgagatgc tcattagcac ggggaggcat
151620cccaggaatc tcaggtgctt gctgactggg tttcttaggg ggttgagagg gtagcagtga
151680tttcaccaag gttatgtatg gcagagggta taggggtttc aggctcccca gacaaagaag
151740gagaaggaga agccctgctg ataagggaag tcccccattt tgagccactt cagaaggcta
151800tcaagacact gatagacttt gtccttaatt agcagggccc aacaagtgtc tgttttcttt
151860tctgccttca ttggctcttt gagccactgc tacaaaatat ccaaatctgg gccaggaagc
151920tggctcggct agtaaaggtg tttgcctcta agcctgaagg cctgagtttt cacttgattt
151980ggtttggttt ttgttttttt gagacaaggt ttctcagcat agccctgggt attctggaac
152040tcactctgta gatcaggctc aacttgaatt cagagatctg cctgcttcta catcccgagt
152100gcttagatta aagttgtgcg ccaacactgc ccacctaaaa aaaatatgag gggctggtga
152160gatggctcag tgggtaagag cacccgactg ctcttccgaa ggtccgaagt tcaaatccca
152220gcaaccacat ggtggctcac aaccacctgt gatgagatct gatgccctct tctggtgcat
152280ctgaagacag ctacgggtgt acttacatat aataataaat aaatcttaaa aaaaaaaaaa
152340aagaacacta ttcacaaggt acaacaacag tgtactagga aactttaaaa tagcccatca
152400ttcattctag gggaaaaatc tttttaaact ttagtgtgta agaaagagag aggggctgta
152460gaaaggccac agcacgtgta tccaggttag gagtcaactt ttcagaagcg gagtctcccc
152520ttctacctgt ttttgaggca gtctcttgtt tctgccctac actttgtaca tgaacttcaa
152580gatggttgtt ctatttctgc ctctcatgtt gccctatgca tgctgagctt actgatgcca
152640gccaccacat aagcatggca ccagcactga gccaagggca ggcatctggc tcacatggag
152700agttacccat caagccatct tgctagcccc agaaaatgta tttttgacag gtgtggtggt
152760gcacatattt aatcccagca ctcaggaggc agaggcaggc agatctctgt gtctgaagcc
152820agcccagttt acaaatcaag tcccagaata gccaaggcta catagagaaa ccctgttttg
152880aaaaacaaac atccttctgt gttccagtca caatgactgc tgtaacaata atatgaggat
152940ttgggcgtgt caaaaatcac aagtagcaag gtgtaatggg caggccttta gtctcagcac
153000ttgggaggca gaggcaggag gatctctgtg agtttagcac agccagggct gttacacaga
153060gaaaccctgt ctcaaaaaaa ccaagcaaaa atagaattac aagttaacca gggtattggt
153120gttaatatga aatagcagaa ctcaagatag ctaatgaaac aaaggattct attttataaa
153180ggagacattc catactgaaa tatatgcaga gcctgatgct tgcctagcaa ctgaactaca
153240cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
153300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngcctgggca agagtaagca
153360aaccgtcagg ctcagaggca ggatggcagt gatgtgtttt ggggcacaag gccccctttc
153420attaaagcac caaatcttgt actaaaaaca gccctctgct ctcgatctgc agagatacga
153480gagaggcaca catagcttcc gggcccttgc cctctgctct cccggtcagt tcctggactc
153540ctgggagaag cttgaagctc aagaactggc cgctgttgcc tttgtgctga cgccagaacc
153600agcagctatt cagcagctgc ggctcttggg caagcttgga agtagctcgt tccttctcct
153660tctgcagggg aaagacaagg agatggcact gagctgggta gccaggatgt gggaagtaaa
153720ttgtgtctat gtgggtgagg gaagtgccgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
153780gtgtgtgtgt gtgtgacaga gacagaaagg gagagtgcat gttcttgtgt gttcctgtga
153840gtcagtgctc cctttggctc ctcctgccaa aaagcatgct gtgttctcag agtcaagctt
153900ggccaggctc gcccacccca ccaaagccca ggatctgcca ccccattaag aatgcggggt
153960taggaaataa aaaacagggt tcttgtccaa gagaattttt attattattt tttctctctt
154020aattggattc agcatttctt actcctcagt atcctctctg gtagggaatt caggtctgtc
154080tatgcagagc acaagagact gtgcttgcca gaaacccttg ggccaacagc cctattccct
154140gactgggctt gcctgcaggc tgccttctgg gctgacccct gagtctggcc ctctgacctc
154200tgcccgtcct ggggtcatcc aggaggagca ggaccactgt gatacagggt tcctgagatg
154260gctactgaaa caccctcatc tgctaaggcc actagatttt tttcccactg ccagactact
154320cagggccctc agtaggtcac tggccaaaga gcctagatgt taaaactgca gctaaagcct
154380ctcctgaggc cagagctcag agcctccctg gcctgcacaa agtgctaaga aggacattgt
154440ccatccaacc cattggacta acactgtaga cgctgccttg tctgccagct tgaaaacccc
154500agaaacctct tccccagctc cgcctagctt gcctccagca ccctgacatc cacttctctc
154560gctaatgtct gcagcttcta cagtaggggt gacggggtgc tccggggtgc cagacaggcg
154620tgtgttgcat ataaaaacga ggtgatgttc taagtatcta agaatgttgg tcccctgaag
154680tgattcttgc tgcttctctt ccttcccgac tcttcccact gacacctttg cccccagcaa
154740gcccagggac tattgttgct ggctggggtt ctgaacaaaa ttgccgcagt tttttgtttt
154800tttttttctt gctagaagtt accgatacag tccttaattg agcaaatata ggttcccgta
154860tagtttataa acatgataag acatacagtt tggtaaggag tgggttgggg gacctgtgca
154920tttatatatt tatatatata tatatattat gtgggtgtgt gggcagagtg aggatatata
154980taagtggaca taggtataaa actgcacctt ctgtgtgact ctcattgcga gtacagttct
155040aaatgtcatc cactggcgat ctctcctttg gtgattggtt cttggacccc agcaggtctg
155100ccgggggctg cctgagacgt caggaatgag aggcattacc cccatggata gggactgagg
155160gtggcatagg gttggacagg gcaggttaac taagtgatct cagacaagag ccaagagtgc
155220tctgagattg ctggttgccc cagctggctc tgggagagcc ttgttctgag tcctgctcct
155280tccaaaacca gcagggtcct tcagcccttc tctccaaatg accaggcttc cgcagagccc
155340agcttcttca aggggcgcat gtcccgacac cactaatgac tcactttgcg tgcctttgac
155400cactgtgctg gagtggatac ggtccagagg cgctcggtca ggacagccga gtgagacgtg
155460atacccttcc cgtctacggc tgtacatttt gggcttataa tccaccagga agggtccaga
155520cggtggctga aggcttcagc agccttttcc tgaaacccag cagatcttcc acttaggaaa
155580aaaaaaagaa agaaagaaag aaaagaagaa aaaaattctg ttccttgctg gacatgtggc
155640agtgctgggt gacggagccc tgggtcctca gcggagagtg actgccagcc ccagtatcca
155700ggccaggagt ggggccccaa gggccgtgcc tgaggatgcc tgctgcaccc cactgggggc
155760acggatgggg gtcctgcgag cacacttgcc cttcctcttg ggtcgtgcgg tgggcatgat
155820gtcaaagctg aacttgtgct gatagtcggg ggcatagtcc tgcagttcgg taagctcttt
155880cccagagctc accttagaga tctggttccg gttcctgtgg ctggtgcagt tcttgcctgc
155940cttcttgtaa cctgacctgg agccaggcgg atggccatgt gggtggcctt tgtccctgga
156000ggccccatgg gacggatggt gctccttgcg ggcagccctg tcagaggtgg taagcgtgtg
156060agacttgatc tggtgaggag acactggtcc tgtgcagttc cggaagtcct ccaccctcag
156120cagcttcaga tcctggcctt gccgcagctc gggggtcgcg caggggacag cagagctaga
156180gccacggaac cttcgcagcc attcccacag ggaacgtgcc cggcagccac agtcccaagc
156240attcccattg aggcgaagga actccaaggc caccaggggg gccagacagt caccctgcag
156300ctcagtgagg ctgttgttga agagaaagag ggtggttagc ctgtggaggt catggaaagc
156360cttgtggtga acccactgta gctggttctc atgcagcagc aaccggtcca ggttcaccag
156420gccccggaag atgccttggc ccaggctcca tagcttgtta ccatggagaa acaagtgact
156480gagattgacc aggtccacaa agatgtcatc ttggaggtac tcgatatggt tgtcctgcaa
156540gtagagatac tgcaggctgt gcaggccacc aaagatgcct gcgggcaggg cgctcagtcc
156600acacttatag aggtagaggg cgtgaagctt caccaggcct tggaaggtct cgggtgccag
156660cgttcgcagc tgtcggttgt ctccaaggtc tagctcctcc agatgcacaa agccctcgaa
156720ggtgttggga gcaatgaaag tgatgttgtt ggagtagatc cagagggtga ccatggcggg
156780gctgaagtgg ccctgctgga ggaaggtgat gcgattgttc tgcaggaaga tgcgctcact
156840gtcctctggg atgccctccg ggatggcagc aaagttgtgt gcctggcagc tgacagtcat
156900gggcgcaggg tagcacacac agtctcgagg acaaccacca cccagaggta gctctccagc
156960gagcagcaac agcagcaatt ccacacagca ccctggtggg gagagacaga acagcagtga
157020ggggctgccc agaggaggtg gagatagatg gggaacagag ggtggaatgg gggactggca
157080aatgactctg ttggctcaca gaggttctgt cctctgtatt gtatgcaggg gtcccttgga
157140cagggcattt ggggccaagg cccacattat ctcctcacct ctttagctct gtccctaaag
157200tctctaattc catccgaaca cttcttcaga ctgtcagccc caccgcaggg tggaacacac
157260ttgtgaacac aggcgagtcg gcctccggct ctgggtccgg ctctgccact cgctcactgt
157320tagctgcctt agcaaggaat gactctaaca aagcaaatct ggagtcctga atgatcactt
157380tatttaaata attcctcaaa ataaagaaag cattgagtcc atggtaccaa agcatgcctc
157440aataagcgcc tctttcacac tgtggtacaa aaacttcaaa cctacaactc ctccatggct
157500gtttcctcat gagttaaaca cagttcacag ggctgtgtgt acagacaagg cacatttctg
157560tgaggggctg tgagtgacac cagggctgac gcacgaggct tcccttgggg ttcacagtac
157620tgccagatcg aggctgcatg ctcctctccc ccattcacac ccccccctcc tgtgctggag
157680attgccaggc tgtggctgta aaaccgggcc ttgcctcttg actgtccaga gcatttcctc
157740tgtagcttcc ctctaattgg gcattaatta ggcattcgtt aatggatcct taaaataatt
157800attttcggat gtgtccagcc tgtggtcggg taataggcct atgctcatta tggaagccgc
157860ctcattatgg acgattgtca ttacctgcct ttttccaggg tcacagctgc cccaagtggc
157920ccagcaggcg cgtcaggaag atggggacag gctccaggcc tacgggcgcc caccctgaag
157980ggccaggcag ccacgaccta tgtcgcctca gttggcctct tgccccttct tttccagctt
158040gttcagctgg gactcttggg agagccaggg cccctggggg aatatgagct gagctgaatc
158100ttcttgctgc tagctgtgct cagagcaagt ggaaggagca gggaccttct gaccaggctt
158160cccacttggg gtcccaggcc cagggactgc ccaggccccg gcagagtagg ttgccacctt
158220gacttctgac gccccccccc cattcccaac agaaacagca tcgtaagttg acagctccca
158280gctgttggga gttatgggct cccagagagt ggcagctgct tctcgtccct gtaatcaccc
158340ggcttcagct agaatgtttc tagcacataa aaatcatcgc atataattta gtttttgcat
158400aattgggttc agttgtgatt tcagagcaat tatgacctca gcagcagggg tggcacagca
158460taggacccct ttctgggccg ggccatgccc tgcaggccct ctgcagtgct ttctgcccac
158520cggcccttag acagcatgca ggctataacc atctctgcct caatttcctg ctccaaaacg
158580tgtctagatg ttactctgtc gatcttcctc ctcagcatcc tgggtgtggc ctccagcctg
158640ccagcctctg tcctagggat cctgtgctgc agagggaggc acagtcggag ggaggggagc
158700ctgccctgtg ccaccagcac tcacactggc tgcacagtcc acagacccac agctccaacc
158760tccctgcttg gcttgcaccc tctcttccag gaaggccatt cttgccagaa cctttcccaa
158820cggtcccctg ggaaagcctg gactctaggt tcaaggacat tcatgatgct tgccccacat
158880tttatgctgg atgagacaca gcagagcctt cttcactggg gggtcctgtg aaaatgaaag
158940cttttcttcc ccggcctgca gctgcaggca ggtaggggtt gcagtgggct tatcactaat
159000accattcgac atttgtacag ctcatcggag tttacagagg gcttttgttg taccctcaac
159060ttcccctgtg ttctcccacc tactgtggct gctctgtctc tgtgcacccc aaaagaatct
159120ggaagtccct tggggagatt acccccctta cataggggcc tccaaggaat acaggctaca
159180gctctattta ggaaaaaaaa aatcaaactg aaccaaacct caggtgtgga cttagtaacc
159240agtttataaa cataccgtgg caagtggagg aggcaggcgg cagaacggca tgaaggtagg
159300actggggttt tccctctaaa agggcaatgg ggggacaaag ggactctagc caagacctga
159360tgcagaacca ggctcagttc ccctgttatc tcaaggctat catcactagg aggcctaagg
159420caatggacca caaggacctt gtccttgtag gacagtcact tcctgcagtg aagtgctctt
159480ctggaagcat actaatagga tgtaggctca ggacagctgg tctctgtcct ttagtatttt
159540tccacatgcc agggatgtta accttccaag cctccatctc ttctaatggg gggggggtgt
159600tgaggggctc agccactctg catccatgtc tttgaaagcc agtggtatta ctccaggacc
159660ctgagcaagc tgtcctagtc agccctggcc acttctggac tccttgcctg agtcagtagg
159720tgccaatcct aggattgtca ccagcaggtt tcttcctagg gaggcaagca ctgtatcacc
159780atggcgcctt ctatgccccc tctatgaggc ccttgggagc cccgccccac tgattgcctg
159840attaatgtac caacaatgag gatggagcct ttgccatgca ttttaacatt gcaaattagc
159900aggaatccaa gtctctgtgg aggggccctg cacctcttct gccagactca tcaagcgcct
159960cttgggcagg gctgccttct acttgagggg gcggaaggga gaagacccag ttccactctc
160020cttcccctcc aggaggtgcc cttcatcgtg ttctgcttcg ttactctcaa gcctccggcc
160080tcccacgcac gtgagctccc aaggggctct acagcctccg tcattccttc ttccattcat
160140acttgccccc tagtctagga gagccatgga agacagtgtg ggaagggctt gacaatgagc
160200atcatgcccc atttgcatat gcggtggcaa taccctggtg ggtaccagga gagtataggg
160260gaaattaaga gaggggccta aggaaagcct ctgctatccc tgggctacca gtcagcattg
160320cttggtcact gatcccctct gtaacaccag cccttctgca acctgccaga gtttttgacc
160380tttgaactag ggctgagaag ggtctgctct gttcagctgc cttggctggg aggggaatct
160440gctcagacct cagcacacac tcaacagaag gcatgcaagc aagggagcta gcagtggcct
160500tgggtcagct ggcaagcccc aaactcttcc tgccaagctg agcatgaaaa gccacctcac
160560catggtccca tgggaccaga cctggtagga taggtggcaa ggctaaggca gcggaatagc
160620atgtgcaaag gcactggggt gggaaagggc ctgtgcttct caccccctct aatggtgcag
160680agcctccaag gaatactgta acctcagctc agctgggctc gggtggccag agagcttggc
160740accagaacca gcatcaacag ggcctgtctg ctaaacccag acctcacaag ccagtttagt
160800aggggccctg tagcaccctg gccaccagaa ctaacgagga agatctgacg ctgggaatat
160860gtctttaatg aaaagccctt ccggaagcca catttgcaca gaagaaaatg aggtgcccag
160920agcatcagtg ggctggttgc agctggagaa cacagcaggg ggacaggtcc taccaagcta
160980ccctgccttc aggctggggc tctagccagc tccctgatgc ctggagtagg taaagcagcc
161040tcgaaatggg ctgggtcagc tttttcaggc tccaaagggt caggacagct gctgcagctt
161100agcacccaag ggggctgccc ctctacccct aagtagaggc atccccatgg cccctgggca
161160ggtcagtggg tctctctgaa gctttgtagg ctgcttcttg gccatgtagc caatctctct
161220ggccttcagt cctccctccc tgcccccagc ctggccagct gctcttctct gagcaatcga
161280tgttaaccga atgctctctt gctgtgggga tggcggcctc aggccaggcc agctgcactc
161340ctggggctgc tggcgcctca ggccacttgg cacttgtgcc acttgtgttc taaacacagg
161400ctccttcctg gctcggccct gaagacaaga aagctggcca gggaacagct gggctcccat
161460ctcagcctcc actgctgtgc agagcggccg gcagcctcct atccatggct gtgagtagaa
161520cagggctgtg gagccagagg cctaagttga atcctggctg ctccttttaa tgcttgcagg
161580agcctcgttt tcctcacctg caaaatgggg cagtcattgg aagctcagcc agtcctccag
161640cccacagata ctggtaccca cctgcccttc ccacatctcc atctgtctaa ctgaaaccat
161700ccaaaccaag ctcttctctt cctctccggc ctcctccgtt cacaacttct ccatcttggg
161760taaagatggt ccctttacat cagctgcctg ggaggaacac ctcagaatca ccgtggctca
161820ctctggggaa attctgctgg ctagatttta gaatgtatcc agtatctatc cacttatcat
161880actcgctatt gctaccattc acccagtagc ctcctggatg ccctcccccc ccccccgcaa
161940gccctgcctc ctcaccccta cacctccttc aacaggaact agggtagtcc agggaaagtg
162000agtcaggaag ggctgctcct tagtctgcat cctccagagt gcccatctaa ctagaagctg
162060ccccaggtct ttctcccagc ccagaggccc tgcctctccc tggctacaca gaccttgctg
162120ccgtccctcc cacatgccag ccctcagcct ccctcctgcc tttgtccatg ctgttccatc
162180tacctggacc ggttcccagt gtgtctgcag ggctgcttcc cagagaagcc actcttgagc
162240agcgatttgc aaggagcttc tttcctggga cattttactc tacagcacgc gacctcttgg
162300acagcacgac ataagtcact tgtttccttt atgtccgctg ccactttgtt ctgatgagtg
162360tgctccctgc acacagtagg tgctcattaa cagttctggg ggagggaatt accttctcaa
162420gtctctggag aattgaatga tgacactcag gaagccctag gctcaagcct ggggcctgga
162480tcatagtagg tgctcgataa atgttggttg taattagtcc tgggagactc agagccttca
162540ggagaacaga cacctgaact tggctcacat caagactcct taggcccatc aaggaagtga
162600ccgtttgttg gatgagcact ctgagaagga cccaatacca gtcctttcct gggcaagggg
162660aaatagactg ggactgggga gtttcccaag gtatgggatt ttcagcacta aactggaata
162720atgctaaaga aaaaaaaaag ttagtcactc taaaaggggc caggaccaaa acctttcaaa
162780cagaaatgtc tgggtttatg aagagaggaa gccagatatg gtggggcaca tctttaatcc
162840aggtgcttgg gaggcaaaga cagatggagc tctgtgagtt tgaggccagc ctattctaca
162900aagtgagttc taggacagcc aaggctacat agagaaaccc acttgacttg ccacccaaat
162960taaaaatctt agtgggggag cagtcaagga ggaaacacac acacacacac acacacacac
163020acacacacac acgttagtat aatatcatac tatggctctg tgcctgcagt ccaggaatga
163080gggctgaact cagagtgtta gtgtgtgcta gtggatattt gagctctgta tttatgtgca
163140tgtctgtgta gatgtgtacc tgaggtgttt atgtgtacac aggtgttggc ctgttgcata
163200tggatggaga catggttgtg tttgctaggc atttgcatgt gtctgagttc atgcacataa
163260actcacatct acctctggag actgagagtg acaacccagg gcccttttat cctgcagcac
163320cccaggccca gcaccccgac ccagcatccc aggcccagca ctccagaccc agcaccccag
163380gcccagcatc ccacgcccag catcccaggc ccagcacccc agacccagca tcccaggccc
163440agcaccccag acccagcacc ccaggcccag catcccaggc ccagcacccc gacccagcat
163500cccaggccca gcaccccagg tccaagcact caatgcccag caccccgacc cagcatccca
163560ggcccagcac cccaggccca gcatccaagg cccagcatcc cagggacagc accccaggcc
163620cagcatccca ggcccagcat cccagggaca gcaccccagg cccagcatcc caggtccagc
163680atcccaggga cagcacccca ggcccagtat cccagggaca gcaccccagg cccagtatcc
163740cagggacagc accccaggcc cagtatccca gggacagcac cccaggcaca gtatcccaca
163800tggaggcagc acatactgaa gatagggaat gtctctgagg cctcttatct tggtccttac
163860cctcattgct ttcagcacct gctctcctca cactcggaat caaacaccct gtgcaggttc
163920tcccagtacc aggattcccc tcagctgagg aatgggtagc taccattttg gcttttgtct
163980gtctggggtt ggcagcccca tgctaattgg actgacagtt tctcctgaga gcaatttggg
164040cagcacatcc tgcccattag gcctaacctt gcctgcaggg gtgtgctgta ggggcaggga
164100tggagcctac cctgtatagc tctgtattga ggcactcccc caagctatga cccatgccag
164160tgggagtcat ttcacctagg caactccaga tgggcacaaa aatctctcca ataagggtag
164220gtatgggaat aggtaaggag agcatagtga gcctggctgg gcacctgaga cctgagcagc
164280ctgcacggga gattgtgtca ctgtggttcc agactgccaa gacatcttgg ctttcacccc
164340aactcaggat ggtccagaat ccagagctct taagagagca gatgctgaga ggcacttaac
164400ccagggctaa gacccttcct tggacggttt cttggctttc tactctgtcc tctgtcccag
164460tctgtcatcc ccatctgtgc ctaacagctc tctgtggaaa acatgaggcg tatgagctct
164520ctacttctcc cagcatccca tgcccgcacc ccagctcact gtgtgccctc atgttactca
164580aatcttctgc taggtttgag ggccccaggt tgaggctgtg ggtttccctc catctgtccc
164640tcccttttac caccaccact aatcctcttc ctcctcttcc tcctcttcct cctcctcctc
164700tttctncncn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
164760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna caaaggacgc
164820aaattgaact tacagaggaa acacaaatga gctgtaatga cagaaaagga atactatttc
164880ccttgtgatc agaaagctat agattaaacc ccagcccttt ttgcctttgt ttttcatatt
164940tagtggttct ggggactgaa cccaaggaag gacagctgct tggaatgttg gagacctctc
165000aggcattgct tatgctaagc tgacaaaggc tttctgaatg catatggtag tatctattca
165060tcttaagatg cctataacca tgggtggagc aattctctaa gagtctgtct gaacttctta
165120agcatgtgct caaaaacaca cataaagatt gtttactgaa gcagtatttg caacagtgaa
165180acaaaaccaa acaacaacaa aaagtactta atagctcatc agtaggaaaa agaacaacaa
165240attgggacac atttatacta ttctcgttct atcacacgac tattaagaag attaaggtgt
165300gaggatgaaa agcccctgca gaacaacgca cacaatgaaa ttgaatagag aataaagaaa
165360aaaatatata gtgtatttat gtaaaacata cataaacaca taaaaagaat agaaaactct
165420ttgactgtat atctttgaaa agaaccagca gagtgctatg gtttataaat gaaaagttcc
165480cccaaaattc ctgagttgga ggttcaatgc agagttcata ctcttaggaa gtaactaaat
165540caggaatgct ctgatctaat caatggattg accaactgat agattaataa tctgaaggca
165600acacttccag gaggcagaaa caaggtgagg cctaactgga ggaagtttgt caccagaggc
165660atgtccttgg agaggctacc tcatccttga tccttccctt tctccgcttc ctggctgtaa
165720aagtatttat ttgactgatg taactgtcat cttggaggct tactggctcc atcagctaac
165780ctaggcctag ccctggaagc ttctagcttc catacaatct aatccaagcc tagaatgttc
165840cagcctttag gacttgctgc tgagatcacc gtttcctgtt ctttctgaac tctagctggc
165900tgattcagtc cccctgttcc gggctcaaac tcctctcccc gatgatttta ttcacaatct
165960gtcttttctc ttggcctctg aattgctctg cttggtctca aactaactct agcaatcttt
166020tctaatctct tgtctccttc acactctctt gcttgttctg tctttactgt gtctagtttg
166080ttctctcttc catccttctc tgtaaagctc tcccggtaaa cctgcctcct cctccccctc
166140tgtgccgctc tactctccct ctcagctcta ctgcactgct ctccacagct ctcctgtatc
166200ctgtgctgca ctctcttctc cggtaccacc tgtgtctccc ttacgtagct tccctttcct
166260ctctcttctc ctgagggttg ggcagatcct atcctgtcaa acctttctct gattcttcac
166320tttgtctgcc actcaattag acatcacttt caagcaggag tgcctcctct acaaaccaac
166380tttaccttca ttgtttcaaa ttaaaggtga gtactaaggg tgtgtctctt tttcagccag
166440tgagagtaaa gatgtgtgct aataaggctg agccaactct agctagaaat agtttctttt
166500tctccataaa taacagaatc ttagggttca caatacgatc aaatatcctg agacagctgg
166560ctgccatgtg gtgaagagaa tgcagagagt taaggatgtg ctcttgaggt ttcagatggg
166620aataagcatt ttcttgggag ttggattaga gtcattcctg tgacactgtg acaaaggact
166680cgactacatt ttgccatgcc ttgagactgt ggagggctga gcgatggatt aatacactgg
166740agtgaatttt aaggcagcca aggctgtggt tggactgtta ctagctacgt ttagctggat
166800ttatattaag aattgggaac aaaaaagcag agtagaaagg acagttttgg cagaaggagg
166860tttcttcata tacaacttag tttttatagt tagcttttta tgttgctatg accaaaatac
166920cttatggaaa ctgaagaata aaaattttta ctgaactctt cttcacccca gaacccgacc
166980cctcccatct agagattgtt cccggaacac tcctgaactc ttcaccccag aatgctttcc
167040tgaactcctc accctagagt tcgaaccctc ccaactaaaa actgttccaa gaacattttt
167100gagataaggg cctcctaaaa caacctcaaa atgaaccggg tacattgcca aataatagga
167160catgacccct tagttacgta gattcccttg gcagaacccc ttgtcccttg acagaacccc
167220ctagtgatgt aaacttgtac tttccctgcc cagctctccc cccttgagtt ttactatata
167280agcctatgaa aaatttggct ggtcgtcgat tctcctctac accactaggt gcatgagttt
167340cgaccccaga gctctggtct atgttccatg tgctttcttg ctgttgttct attaaatctt
167400gccttctaca ttttgagtac ggtctcagtg tcttcttggg tccgcggctg tcccggggct
167460tgagtgcttg agtgagggtc tcccttcggg ggtctttcat tttggtgcat tggccgggaa
167520acagcgcgac cacccagagg tcctagaccc acttagaggt aaggttcttt gttctgtttt
167580ggtctgatgt ttgtgttctg tttctaagtt tggtgcgatc gcagtttcgg ttttgcggat
167640gctcagtgag accgcgctcc gagagggaac gcggggtgga taaggataga cgtgtccagg
167700tgtccaccgt ccgttcaccc tgggagacgt cccaggaaaa acaggggagg accagggacg
167760cctggtggac ccctttggag gccaagagac catttggggt tgcgagatcg tgggtttgag
167820tcccacctcg tgcccagttg cgagatcgtg ggttcgagtc ccacctcgcg ttttgttgcg
167880agaccgtggg ttcaagtccc acctcgcgtt tggtcacgag atcgtgggtt cgagtcccac
167940ctcgtgcaga gggtctcaat cggccggcct tagaaaggcc atctgattct ttgagttgct
168000tgtggtcgac gcagagtcgc cgccgtttct ggtttctttt ttgtcttagt ctcgtgtccg
168060ctcttgttgt gtctactgtt tttctagaaa tgggacaatc tgtgtccact cccctttctc
168120tgactctgga gcattggaag gaggtgcggg tcagagccca caaccagtcg gtggaagtca
168180gaaagggtcc gtggcagacc ttttgcgcct ccgagtggcc aacgtttaga gtaggctggc
168240cacctgaggg tgcttttgac ttgtcactaa tcgctgccgt caggcgaatt gtttttcagg
168300aggaaggggg tcaccctgat cagatcccct acattgtgac ctggcagaat ctcgtccaat
168360tcccacctcc gtgggtcaag ccttggaccc caaactcttc gaaactgacg gtcgcggttg
168420cccagtctga tgcagccgga aagtctggcc catcagcacc ccccaagatc tatccagaga
168480ttgacgacct cctctggata gactcccaac ctccccctta ccccctgccc caacagccac
168540ctgcagctgc cccaccacag ggaccaatag cgagaggggc tcagggaccg gcgggggaga
168600ctcggagtcg ccgaggccga agccccgggg aggaaggggg gccagactca acagttgcct
168660tgccactcag agcacatgtg agagggccag caccaggacc taatgatctc attcctttac
168720agtactggcc tttttcctct tctgatttat ataattgaaa aactaaccac cctcccttct
168780cagagaaccc ctctggactt actgggctcc ttgagtcact tatgttctcc catcaaccca
168840cttgggatga ttgtcagcag cttttgcagg ttctttttac aacagaagaa agaaaaagaa
168900tcctcataga ggcgagaaaa aatgttctgg gagaggacgg cacacccact gccctcccta
168960acctcgtgga cgaggctttc cccttgaacc gccccaactg ggactacaac accgcggaag
169020gtaggggacg cctccttgtc tatcgccgga ctctagtggc aggtctcaga ggagccgcta
169080gacggcccac caatttggct aaggtaagag aggtcttgca ggggcagact gaaccaccct
169140cagtcttcct tgagcgtcta atggaggcat ataggagata cacccctttt gaccccttgt
169200cagaggggca gagagccgct gtagccatgg ccttcattgg tcagtccgct cccgacatta
169260agaaaaagct gcaaaggctg gaggggctcc aagatcatac gctccaagat ttagtaaaag
169320aagcagaaaa agtctatcat aagagggaaa cagaagaaga gaggcaggag agagagaaga
169380aagaaataga ggagagggaa aatagacggg atcgccgtca ggagagaaat ctgagtaaaa
169440ttttggccgc agttgtgaat gatagacagt caggaaaagg taaaataggg ctcctgggca
169500acagggcagt gaaaccgcaa ggtggcaaaa agataccact ggaaaaagac caatgcgcct
169560attgcaaaga gaaaggacac tgggctagag attgccctaa aaagcgggag cgatccaagg
169620tcctaaccct agaagatgat tagggaagtc ggggctcaga ccccctccct gagcctaggg
169680taactttgtc cgtggagggg actcccgtca acttcctgat agacaccgga gcagaacatt
169740cagtactcac taacccccta ggcaagctag gctccaaaaa gaccatggta attggagcca
169800ctggtagtaa attttacccc tggacgacca aacgagctct tcagatagac aaaaatatag
169860tgacccactc ctttctggtg atacctgagt gccctgctcc cctcttgggg cgcgatctgc
169920taaccaaact aaaggctcaa gtccaattta cttcagaagg cccacaagta agctggggaa
169980aggcccctgt tgcctgcctt gtcctcaaca cagaaaaaga gtaccggttg catgaagaac
170040aacccaaaaa tgcagtctct tcaggttggc taactgcgtt ccccaatgtc tgggcagaac
170100aagcaggaat ggggttggct aaacaagtgc ctccggttgt ggtagaactt aaagctgatg
170160ccacccccat ttcggtaaaa caatacccca tgagcaagga agctagaaaa ggcatccggc
170220ctcatatcca gaggttgctg ggccaaggag ttttagtggc ctgtcagtcc ccctggaata
170280caccacttct gccggttcaa aaaccaggga ccaatgacta tcgcccggta caagacctcc
170340gggaggttaa caaaagggtc ctggacattc accccacagt cccgaacccg tacaatttat
170400taagctctct cccacctgag agaacatggt atacagtcct agacttaaaa gatgccttct
170460tttgcctgcg tttgcaccct aagagtcagc tcctgtttgc ttttaaatgg agggacccag
170520agggcggaca gactggtcaa ctaacttgga ctaggctacc acaggggttc aaaaattccc
170580ccaccctgtt tgacgaggcc ctccatcggg atcttgcgcc ttttcgcgct cgaaaccctc
170640agcttaccct actacagtat gtagatgatc tcttggtcgc ggcggcctcg aaggagctgt
170700gtcaccaggg aactgagagg ctcctcacag aactgagtga cttggggtat cgagtttcgg
170760ctaaaaaggc acaaatctgt caaactgagg taaccttcct ggggtatacc ctccgagggg
170820gcaaaagatg gctcacagag gcccggaaaa agactgttat gatgatccca tcgccaacta
170880ccccacggca ggtacgtgag tttctgggga ctgctggctt ttgtagactc tggattccag
170940gctttgcaac cctagcagca cctctatatc ctttgactaa ggaaggggtt cctttcaagt
171000ggaaagaaga acaccaaaga gcttttgagg ctatcaagtc gtctctaatg actgccccca
171060cgctagcatt accagacttg actaagcctt tcgtcctata tgtggacgag agagcgggtg
171120tagccagggg agtattgaca caagcactgg gaccctgaaa aagacctgta gcctatttgt
171180caaaaaaatt agatcctgtt gctagtggat ggcccacatg tctgaaagct attgcagcag
171240tagccctgct gatcaaagat gctgacaaac tgacaatggg acagcaggtg accgttgtag
171300cccctcatgc cttagaaagt atcgtgcgac agccacctga cagataagat gacaaatgcc
171360cgaatgacac actatcagag cctgctgcta aatgagcgtg taacctttgc gccccctgcc
171420atcctcaacc cagctaccct tctccctcta acaaatgatt ccgtcccagt acatcaatgt
171480atggacatcc tcgctgaaga aactgggacc agaagtgacc tgactgacca accctggcct
171540agagctccca gttggtacac ggacggcagc agtttcctga tagaggggaa gcaaaaggct
171600ggagctgcgg tggtagacgg gaaaaaggta atttgggcaa gcgctttgcc tgaaggaaca
171660tcggcacaaa aggctgaact tatagcgctt atacaagccc tccgagaggc taaaggtaag
171720atcgttaata tctacactga cagccgatat gcttttgcta ccgcacacat ccatggggcc
171780atctacaggc agcgagggct attgacctcg gctggtaaag acattaaaaa caaagaaaaa
171840attctggccc tgttagaagc catacatgca cctaaaaagg tagccatcat ccactgcccc
171900ggccacccaa aaaggagaaa acttggtggc caagggcaac cgaatggcag acttagtggc
171960aaaacaagtt gctcaagggg ccatgatctt aactgaaaaa ggtgatccgc ccaaaagccc
172020tgaggatggg aggtataaca taaaagagct atggtagacc agtgatcccc tcccatactt
172080tttttgaaag aaaaatagaa ttaactcccg aagaaggaat aaaatttgta aaaggactac
172140accaattcac ccacctggga gttgaaaaaa tgatgagact aattaaaaat tcccgatacc
172200aagtccccaa cctgaagtca gtggctcaaa agattataga ctcctgcaaa ccatgtgcat
172260tcactaatgc aactaaagcc tacagagaac ctggaaagag acaacgggga gaccatcctg
172320gagtgtattg ggaggtagac tttactgaag ttaaacctga aatgtatggt aacaagtatc
172380tgttagtatt tgtagacacc ttttcaggat gggttgaggc atttcccact aaaacagaga
172440ctgcccagat tgtggccaag aagatccttg aagaaatcct gccaagattt gaaatcccta
172500aggtaatcgg gtccgacaat ggaccagcct ttgttgccca ggtaagtcag ggcttggcca
172560ctcagttggg catcgattgg aaattacact gtgcttaccg ccctcaaagc tcaggacagg
172620tagagaagat aaataggacc ttaaaagaga ccttgactaa attagccatt gagaccggca
172680gaaaagactg ggtggctctc cttcctcttg cgctcaaaca cccctggtcg tttcgggctc
172740actccttttg aagttctgta tggaggacct ccccccttaa tggaagctgg tggaacatta
172800gtttccgact ctgaccctgt cttaccctcc tctttgctta ttcatttaaa ggccctaaaa
172860gtgattagga cccagatttg ggaccaactg aaagcagcct ataccccagg gaccaccgca
172920gtaccccacg ggttccgagt tggagacaaa gtcttggtca gacggcatcg aaccggtagc
172980cttgagccac ggtggaaggg accctatttg gtgttactga caacccctac tgcggtaaaa
173040gttgacggaa tcgcctcctg gatccacgcc tcccacgtca agagggccgc cagtcaagat
173100gaagaaaacc acgacgacaa ttggacagtg gcagtcactg acaatcctct taagcttcgt
173160ctgcgccgca ggcgccactc tagacctagg gaaccttaac cctcatgctc caattcaaca
173220gtcctgggag gtgcttaatg aaaaggaaaa cattgtatgg gcaaccactg cagtccatcc
173280cctctggatt tggtggcctg atctcacgcc tgacatctgt aagttagcgg caggatcccc
173340caattgggac ctctcagatc atactgatct tagcaaccca ccccctgagg agcggtgtgt
173400cccaaatggg atagggagca catatgggtg ttcggggcag ttctaccgag ctaatcttag
173460agctgcacat ttttatgttt gccctggtca gggtcagagc aaaaggcttc aacaaaaatg
173520cgggggggca tcagattact tttgtggtaa atggacatgt gaaacgacag gagatgctta
173580ctggaagccc tcctctaaat gggacctaat cacggtaaaa cgaggtagtg gctatgataa
173640gtcaaacgaa ggagaaagaa acccctataa atatcaagag agtgggtgcg cttttaaaaa
173700cagagcaccc tcaggaccat gcaaagataa atactgtaac cccctacgta taaggttcac
173760cgagaacgga aaacaacacc gtctaagttg gcttaaagga aataggtggg gttggcgagt
173820atacattcca ctaagagatc ctgggttcat tttcacgatc agattgacag tgagagaccc
173880ggcagtgaca ctcgtagggc ccaacaaggt ccttataaaa caggggcccc ccagtcgtac
173940tggctccccc aaaggtcccg actgtaccag ctccaccaac tccacagccc aacacagtgg
174000taccctccct aggaactaat actctcctca taaagcctac cttggcttcc ccaccgcccc
174060taggaacaga ggaccgtctg gtcagtctag tccaaggagc ttttttagtt ctaaatagaa
174120ctaaccctaa tatgactcaa tcatgctggt tatgctatgc ctctagcccc ccttattata
174180aaggaatagc tcagatcagg acttataata ctacttcaga tcattctcaa tgcctttggg
174240gaaaaaacag aaagttgact ctagcagcag tttcaggaag agggctttgt ctgggccggg
174300tacctcagga taaagggcac ctctgtaatc agacccagaa catccagtct agcaaaagcg
174360gtcagtatct ggtgcctccc ctagacacag tgtgggcttg caataccggt ctcactcctt
174420gtgtgtctat gtctgttttt aatagttcca aagatttctg cattttggtt cagcttattc
174480ccagactctt gtatcatgat aatagttctt ttttagataa atttgaacat cgggtccgct
174540gaaaaagaga acccgttacc ttaactttgg cagttctatt aggattggga gtagcagctg
174600gagtaggtac aggaaccgct gccttaatta agaccccccc aatactatga agaactacgt
174660gcagttatgg atattgatct tagaactata gaacagtcta taaccaaatt agaagaatct
174720ttaacttccc tgtccgaagt ggtgctgcaa aatagaaggg aattagactt attattcctt
174780aaaaaaagag gactctgtgc tgccttaaaa gaagaatgtt gtttttatgt tgaccattca
174840ggagtaatca aagattctat ggctaaactt agagaacgcc tagatatacg taaaagagaa
174900agaaaaagcc aacaaagatg gtttgaaagc tggtttaata agtccccttg gctcaccact
174960ctcctctcca ctatagcagg acctttaatt acacttatgc ttttgcttac ttttgggccc
175020tgcatcctta ataagttagt agcttttatt agaaaaagga taaacgcagt ccaggttatg
175080gtactaaggc aacaatatcg ggtccttcag gaggttgaaa actcgctcta agattagagc
175140tatctcctaa aagaagtggg gaatgaagaa taaaaatttt tactgaactc ttcttcaccc
175200cagaacccga cccctcccat ctagagattg ttcccggaac actcctgaac tcttcacccc
175260agaatgcatt cctgaactcc tcaccctaga gttcgaaccc tcccaactaa aaactgttcc
175320tagaacattt ttgagataag ggcctcctaa aacaaccgca aaatgaaccg ggtacattgc
175380caaataatag gacatgaccc cttagttacg tagattccct tggcagaacc ccttgtcccc
175440tgacagaacc ccctagtgat gtaaacttgt actttccctg cccagctctc cccccttgag
175500ttttactata taagcctgta aaaaatttgg ctggtcgtcg attctcctct acaccactag
175560gtgcatgagt ttcgacccca gagctctggt ctatgttcca tgtgctttct tgctgttgtt
175620ctattaaatc ttgccttcta cattttgagt acggtctcag tgtcttcttg ggtccgcggc
175680tgtcccgggg cttgagtgct tgagtgaggg tctcccttcg ggggtctttc aaaactactt
175740cagaggaaaa atgtattctg cctcatgggt tcagggggtt tccctcagca aattcaggga
175800agacaagatg gaacagctca acctgctggc aggagggtgt gggaaaggac aagtgttcat
175860tgtgtggtgg acaggaaaca gagagctgcc tacagtctta caggcctacc accactgacc
175920tacctctgtc cgtcaggccc tacatcttaa aggatctaca gtttattaaa agaacactac
175980cagataggaa ccaagtatca aaccaccagt ttgtagggga taaaaataca aggaacacat
176040ctcaatagga gtgtgttcca ggatgtggac aaggagaaca cagttgttta aaagcttaac
176100gctggccagg agagctgcac acctttaatt ccatcactcg taagagggaa gcaggttcat
176160ctctgtgagt tcaaggcaag cctgggctat acaattctag attagccaga gctacatcgt
176220aggagcctgt ttcaaaacaa acaaaaccaa accataaaaa agcatttctg aggctttggg
176280tttaatcccc atgacctcaa atagccaaac agctctcctc agtccaaacc aaactgcaaa
176340attggagcta gtgagatggc tcaacatatg aaagtccttc ccaaaaatat tgacaactgt
176400agcttatctc tggggacaca cataatggga gaggaccaat ttctacaagt taccctctga
176460cctccacaca tatgcctccc acaaataaga aaatatatat aataaaaaga aagaagtcta
176520cagctgcaca tggtcatgca tgcctataat ccagcactcc agaggctgag gcaggaggat
176580tattagtttg agatcgcata gcaagcagta ggctagacag ggctacatag tgtaaacctg
176640ccttaaaaca caaaaatcaa ttaagcaaca ataacagtaa caaccacaac aaaaacccaa
176700aagagtactt tgtagtaagg acaataccaa aaatgttcct ttaaggacag ttctggaatc
176760agcaatagcc ttccgagtgc tcagggatgt ataaatactt agaaaacttc ccctggagaa
176820atgagcacca gggtacactg ctctcagagc tgcccagaaa gttgtttatc ctggattcat
176880ttcagccttc ctaactgctc aggcattcag aggtcacttc tgtagtagcc aatgtctaaa
176940aaggctaaac tactgctcag catggctgtg gtacttggca ttatcatttt gtgactggtt
177000ttgtagttat gcagaattca agagttatag catcatgaaa gtttccacca agttcctgat
177060ccagtcacct cttaaaggtt ggatgcacca agtgcctttg gggtgataaa ttatattcaa
177120ataatggtat tccaccctaa tccccaaaga cttctggcca tctcataatg taaaatgctg
177180agccatcgca ccagcccatg gccttgaact cttgatggtc ctgtctcagc ctgtgtttgg
177240attataaatc tgttggtgag gtattccttt gctgataata caagcaaatt cttcaagctt
177300ccatcctaga ctgaagacca gcagctctcc aggagtcctc aatgcagact ggcccagctg
177360ggacattgag cctcatggac tcagccgcta ctagattcgc aacctattca gacaagccac
177420tgttggacta cccagacaat actatgtaag ccaatcccat tttaatacac atattcatct
177480gggtgtgtgg cacacacctc tactcccagc acgcaagagg cagaggcagg cagatctctg
177540atttcgaggc ctggtctata gagtgaattc caggccagcc agggctacac agagaaaacc
177600tgtttcaaca aaaccaaaac cgtaaattca ttctatcagc tctatttcct tagagaattc
177660taatacatgt gggtaccagg ggttgaactc aaagtcttca tgtttacgta gcaagtttcc
177720ttctgctagc ctagtgaagc tgaggcaggt acagccggtt ctttactgct ccttgcaaat
177780ggtcctcctg agctttcctt tgagagccta caaagaactc tttttttctt taggtctcca
177840ggttttggtc ttaagaggtt ctggacttgg atctgtagct gtcatatcac agacattcaa
177900catctggcaa atgtcttgac aaaggagatc acttgtgttt gctgcagtgt cccttctggc
177960tgtgagattt tgctcctcac cactgcagga ctgcagatct attctgcctt tttagttgac
178020ttttcattcc tgagaactgg ggaaaactga ctttgtattt gggctttgaa tttgtccatt
178080tgtcaatcca tcacaccaga cctaaccaac tgccaagagt tctgctgact tttgttttct
178140ctagggtggt cactttgctg ggctcatcct catccttggc ctgcagttta tccccaggaa
178200agaaaatggc taacgactgc taagaagcag tctttccttc cagaaatttt agtctatcta
178260gaccttgctg cagtctgaag tctttaaaat gtgtttgtta tggtagaata ttttgagttg
178320ccttaggagt attgcttgct gtcacctatc atattctatc aggaagcaga cgtcccattt
178380accaaatgtg aagaaatatg gcatcaatac ccactgcaaa aagtgtaaat aaataataaa
178440aaaatagatt tattacagag tgcaagggaa aagaaaaaaa tcagccagtt tcagaattgt
178500aactggacaa atgttggtac agttcatgaa gaggttctac aaaatggctg ggggtgggaa
178560cataatgagt tagtttgctt ttttttttct ctttccttcc ctttcctttc cttacaaggt
178620ctcatgtagt ctatggtctc aaactcacca ctgtaaatca ccttgaactt ctgatccttc
178680tgcacgctgg caatgtaagc atgtgccacc aggcctggct cacacatttg gtttttcaat
178740acagaatagc tctgtgatga ttaacttcaa tcatcaactt gacataacca agaatcgtct
178800gaggaagagt ctcagtgact gggtgggcta agggcatgct cataagggat tatcctgatt
178860gttaattgac atggaaagat caagtccatt gtgagcagca acacgccctg aacagaagtc
178920ttctgaagta taagaggaga aagcttgatg agagcaagca ggcaagcaag ccaggatcca
178980cgtgtttatt ctctgtctgt tcttgaccgt agatgtgatg gctgtcttgg cttcctggga
179040aacatgaact gcaccctgga attgcaaggc aaacaaacct tttcctcttc caagttgctt
179100tatgctaaga tattttatcg cagcaataga aatgaaactt agaacaggcc cataactgcc
179160agctttggaa ctgaacctaa ggctgttata attcactagg atagggacca ctggaagtga
179220atctgatttt gatggtaaaa tcatgtgttt gtttctggat atgatagatt tatcaatttg
179280agactcagaa aagaagttag gacttgaatt ccgttttaga gacattccag agaaaactga
179340tgtcattgtt ctgaatgtaa gtgcctcagc tgaaaataca aagagtacag ggaagaaagc
179400ccaggctaga atctgaagga actcctctat tttttgtttg cttgtttgtt tggttggttt
179460tttgagacag ggtttctctg tgtagccctg actgtcctgg aactcacttt gtagaccagg
179520ctggcctcga actaagaaat ctgcctgcct ctgcttccca agtgctggga ttaaaggcgt
179580gtgccaccac accaggctag gaactcgtct attacacatt aacacccctc tttaattaac
179640tgttcctgcc aatgtaccaa atagtcaatt gattcctgtt tatttaccac atgtttctgt
179700tagtaaacca gaataactta tctagccaaa gtctgcctat tagccatatt ttcatcagtt
179760cccaaccatt tttggaattc tgtgagggga atccacagat gctgtagacc gctttagaca
179820tttttcagct tttttcaagt tgcaggtcat gattcagtgg gtcatgaaat taatttagtg
179880ggttctgatt agcatttcaa aatgaggcaa gcagagggca tattgtcaca gcacagcaca
179940tgcggtaagc agccacacac tcttgcttgg aggcttagtc agtttctggc tctaaacgcc
180000ccaggtttgt ttctctatcc taggcctctc tcttaaattc caaacatagt tagacattac
180060cattggggca cgtgcaactc aaacacggag tgtgactcct ttccccatct gcggttccca
180120gatttggcaa tgtcaccctc ctcccttctc cctagggtca gttttacctc tcacactcca
180180caacacaaca cctctcatct caagaattgc cattagggct ggtgagatgg ctcagaggtt
180240aagagcaccg actgctcttc tgaaggttct gagttcaaat cccagcaacc acatggtggc
180300tcacaaccat ctgtaatggg atctgattac ctcttctggt gtgtctgaag acagctacag
180360tgtactcaca tatattaaat aaataaatct aaaaaaaaaa aaaaaaagaa ttgccattaa
180420atgtacctca gagtccaaat gcttcttcct cccctgacta cactcacgct ggcctgagtc
180480cattttctta ttgaggttac tgcttctctg cttctaccct ggctccttct gctgcctatc
180540cttgacacag cagacaagca gttctttaaa gcagggctca ggaccagtga gactgatcgg
180600ctctggtggc acttcctgcc atgactgatg atctaaggtt aagcctagaa cccacgaggt
180660agaagcaaag gacctactct ccaaagccgt cctctgacca ccatgtgtaa actgcacatg
180720tacatgcatg cacatggtac acacacatac acagaagtaa aaagagattt aaattgaaaa
180780tcattaaaaa gaaaaatcag ggctcagcaa actttccgtg tagaaaacta gagtacttag
180840gctttgaaag ccaagaagtg gatattaatt atagttattc attatagcag agatttctaa
180900aaccttttga caaaactaaa aaatataaca gagtgtattt tttttgtaat gtaagtttac
180960taatggcagc agtgggatta gtttcttttt tagattattg ttattatttt tattaattat
181020tagtgttttt gtgtttattc atattccaca gcatgtgtgt ggaattggat ttctgcttcc
181080acctttgtgt gggtcctaga gattgaactc aagtcatcaa gcttgcacag taggtggtca
181140ggcttacaca gtaggtggtc aggcttgtat ctttggaagg caagcatttt acttcctgtg
181200ccagctcact ggccttcttt gtttaaaaaa agaaaaaaaa agtccttttt tgtttaatta
181260gggttcatgg ccagtgctct ttatcttaaa atcaactgca aacttttatc tggtaaaaag
181320ccatccttag ctgtggtcct aggagaaaaa catacagttg gatggcttta tcctgcaggc
181380ttagtttgat catctctctt tgaagatata atcagctcac atcacactca agcctctgcc
181440aacgagtttt ctacttctgt tcaacaaact acccaagctg agcagctcca aacaacagcc
181500agttatgatc ctcacagtcc ggtgggtcag aagcctaagc gggcgtggct acctcgctgc
181560tattgcctga ccctgctcgg tgcatccaca ttcacatcct ttcctggtga gtgtggttct
181620ttgactggtt ttgttccaat ttttagtata tgtgctgctg aaacaatctt tttgcctctg
181680cctccagact gcagggatta atgttcttga ctgccacaga gcactaatat ttactgaaca
181740tgtgatcatg tggtgctcag cactcttgca cccaaggctc ggggaacatg gaggaagagg
181800gggtggaaag attccaagaa ccagaggaag aagaaagtca gaggtgagac tgcatctcct
181860agaaatgtca gggacatttc tagacctctg aagtctcaag aacaaggcct gaaagtctta
181920tttatatagg ttaacctgaa aggggaaaaa attcttacag gggtccaacg ttagacaaag
181980aactctaagc aactaaggaa tgttgggggg ggggtagtct tccccaggga acactcctct
182040acccttcaag ccccacccaa gctggttatc caaaacaaac tggtcagtcc tgaagccata
182100tacgcacaag taacatcata tggatgggca gattgcattt aggaatacac acatacacac
182160acacaactta aaaagagagg ccatgaattt aagagagagc aaagcaaagt gggaaggggt
182220acatgggaag gttggaggca gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
182280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
182340nagagagaga gcctattatg tcgttggttg cttctaatca ttagaaaacc actctcttag
182400gctgagtcag aactagccta gctgagacac tgtccaccca ctgtcccaga gcaaggccat
182460cgctgtccca gatttgcctt tgggggccct tgaaatgaaa gtcaccagca ggctccggaa
182520gctgcctcat gctaatcagt tgaggttgtg aaaatagccc tgcagtggtt cctgggcctg
182580cagctgggcc agagccacta aggggagtct ggtcctttgg agcagagtta acagtcatca
182640gtgctttttt ttttttttta aatgttccct gctttaggct cagtgctgtg cgctacttct
182700aatccttgca ataggctgca agacaggcaa gaatatcatc cctgttttgc cctcaggcaa
182760attctgaagt ctggcaaatg aaagatgtgg gatttgaaca cagacttgtt tggccaaaga
182820attctcactc tacttctgcc tgtgccacct tcctctcatg cacggggagg ggaggggagc
182880ccacctccca tgctcagggg ctaggaagtg gggagaagat ggatgtcctc aaagcagggt
182940gagaatgaag tagaagccag cttcaaatct aaactaagca atgttttatt tccatttccc
183000tgaacataaa gttcagttac atttggttta aaaaaaaaaa tccctacaca actggttctt
183060gagaaatgtc aagtgctaca attcagtgga tgtggatgaa acaatcaaaa tgttgaacac
183120ccccaaacag atacaaacct tcatcaaagt ctcttccaaa ggctgggtct gaaaagagcg
183180actcatgttc cagcccagtt ggctccttct catgtgagct ccgacttcca aagactgctt
183240gcaccaggag gaaatataat agatgtcctt tttaaggggg gtggggtctg tctgacaacc
183300tcccacagtg actgtggata cagcccagtt agtagagttc tcgcctagca agcgtgcggc
183360cctgggcttg agatctagca ccctaaggca tagtggtaca tgcccatgac cacagcactt
183420gggacgtaga ggcagaagga tcagttcaag gtcagatgag gggtggggag gcattccttt
183480aatgccagca tttgagaggc agaaacagat gaatttgtga gttcaaggcc agcctggtct
183540acagactgag ttccaagaca gccaaggcta cacagagaaa ccctgtcttg tcaggaaaaa
183600agatagtggg agagaattca aggttatctt ggactgcata agactttgat tccaaaataa
183660acaaaaatgg agcatgaatg cttgcaactg tggacaatat tgggttcata catattctgt
183720tttgtcacct acataccaat tatacaaatc agattcagct gggcccactg gtgcatgttt
183780gttcccagca tctgggaggt agagatgggc agagctctat gactttaagg ctagcctggt
183840ctacaaagta agttctagga cagccaaccc tacagagaga tacactactt ctaaatcaat
183900caatcaatcc atcagtcaat catgggctgg agagatagct cagtgatcaa tagtgccatt
183960cttccagagg acctgggttt gattcccagc acccacatgg cagctcacag atgtctgtaa
184020ctccaatccc aagggacatg acaacttcta ctggtctctt tggtcaacag gcatgcacgc
184080agcatacaat atatatatgg gtaaaatgct atatatataa aaatcagatt cacaaatcaa
184140gtacagaaag agatcaacta taatgaaaca accataacac atactgtttt aaaagctacc
184200tttcctgtct gacatctgac ttcttgcgtg acccagtgcc tccacaatga gacaagcaga
184260tggtgtagtc cctgtgaccc aggattaggc aaccactgcc cctgggaagc cattcctagt
184320aacaggatca aatcccacag tgctccactg taccccgcca gaggacgtga agcctccctt
184380ccccggctgt ctgtgcagca ccgcccagat gcttggtgtc actgagtgac catcggagcc
184440caactgagga gtgcctcagt gctcctcagg gcagtgtgca attgaaactt gcatgtgatt
184500tctggaattt gtcatttgat atttccagac tccagctgac cttggataac agcacagagg
184560gcagaactgc agaggaaaag gggcactact gtgactgtta tcccctgcat gattatagaa
184620gggtctgtgc tttctcttac aaatcattgc ggcctctctt cacttccctc ctttgaagtc
184680gaaaaaaata gatgcatcct cacagtacag gatggcaggt acgaggcggg ttccgggact
184740gaggcaggac tcatcatgca gcctctttcc ctcaactaca ccgccgcccc tgcagagttc
184800cctctgatca aatcagtttc aggcctggaa agaaacggcc actcaggctg gggatgtggt
184860tccattagag gcctgcatgc acgaagctct ggatttgatc ttcagcacgg gcataagccc
184920agtatggtag tatctgttta gcatggtgtg gaggtatacc tatctctgca cagggagacc
184980agaagttcag agttatcctt gcacttacag taagttcaaa gctagcttgg gcaacatgaa
185040gttttgtctt aacaaaacga aacaagaggg gccggggaga tgcttcgctg tgctgagtca
185100tttgctgcca agtttgatga cctgagtttg gtcccttgag cttatggtgg aaggagagaa
185160atgacgcttg aactccacgc acatgccaca gcccacgcat gaatgtgtat acacacacac
185220actaagtgga taaatgttaa aaataaataa atcaaaggaa tggccactca aaatctacca
185280tcgttgggaa gggaggggaa aaggcaggcg agggagatag ataaccctga tatgaacacg
185340gaaagagcca gtgtgccacc aaagctgccc agtgtgccac caaagctgcc cagtgtgcca
185400ccaaagctgc ccagtgtgcc accaaagctg cccagacttg attacagatt tggccaggga
185460cacaggaggc cagcaggagc agccaggttc cacctcagag gtggagccac aaacctggaa
185520atgaaacgtc tttccctttc ttcagaccac agcagtgaca gctgtcctgc agagtctgga
185580gggctggcag ggctcatcca ctctagtgtg cctgtggcca gaacaggcct cagtcacagg
185640tgcttttcca aggtcttagt gtctaattaa ggttagcagc caaattggag agagaagggt
185700gctggacttt actctgctgt aaggactttg ggcattgttc cattccgtga tcaaatacca
185760ctggctctgc caaccaccat gtcagtgggt cttcagaggt agaagaactc atcctttttt
185820gagaggtttg gtctggtcct tgtctaatgc aaaatgcctg gggcaccagg ttaatgtcaa
185880ctcaaaggca agtgctggtc cagcatgtgt ggaatcctaa gttcaatacc catcagagcc
185940ccaagcccta gaggacagac atgctttaaa aaaaagtcat gctttaaaaa aattctgttg
186000agggggctgg agaaatagct cagcagttaa gagcactagc tgctctttca gaggacccag
186060attccgttcc taacattttc atggtggctc aaagctgtct ataattcaag tcctagaggg
186120gaatctgttg ccctctctgg ttttctcagg caccaagaac acatgtggtg caaacataca
186180cgcaggcaaa acactcatac atatgaaaca ttttttataa acctgctcgg atgtggtggc
186240tcatgccagc gatgctctca gcactcagat ggcagaggca gggggatttt gtgttgagcc
186300cagcctgagc tatagaatga gatgctgtct caaaaagaaa aaaaaaacaa aaaaaaacaa
186360aaaacaaaaa caaatctgtg ggcttaatca ttcctagcca gaaggagctg gctccagcaa
186420caatggatcc ctagagctct gctttgcccc ctggtctgga gagcttgctc tagaaaggaa
186480ttctccatag accacatttc tattttgggg accactctgg gcctgcacat ggaaaaatgg
186540agagttgagg tttacatggt catttttttt gttgttacag taatgagagc tttgaaatga
186600tcacaaaagg aaaataggaa aatatgcctc ctaaaaagag ccgaggcaaa ttattatagc
186660aaccgaatcc taaaaggaat gttcaagtaa aaaaaaaaaa atatggctca tgcgaagttg
186720ttatggcaac tgacatttaa aagtaacagg atttgggcaa gtttctttct ccaccctctc
186780tgcaaagtct tgcaggaact tcatgctaaa attatgcttt aattttaatt agccaaatag
186840gtcataaata tagcttattc ccaaatcctt agaatttcta ccctgcaagg agctgaacta
186900ctaatgagga aatatttcca caaaaaccca cttaccatta agaaccccca tccgatattt
186960ttctaatata atttatcaat ttaaacacat tgcataatgt gccactctgt agcattccat
187020taaaatgata aatagcaata gtggtgaggg gtggggggca aaaaccagga gattaaacat
187080atccaaagca gtgtagctat atttaaatac ctcaatccat tgtagaggaa aacacactgt
187140tctcatccgc agatacagtc tagactcaga gcagcatatc cttgactgta agggtattaa
187200taggacagac gaaggggggc aataagaaat gacaggaaac ttcagaagaa ataaaatttc
187260tattaggctt tgttataaga ttacatcaaa gcagttcata tgttttaatc tggggaggaa
187320aaaaagcaac tacttggggt ttgcgcctgg gggctgcctc tgtgtactga accagacagt
187380ttgcataatg aacaattttc attcaatcag gatctcagca gagatagctc ctactcaaag
187440gaacccggca caggctcata gtttttatct cccagctcca cctgctggag aaaccttgta
187500ttgcagggag agaaagcagt cgggaggcat tgtcctagtg gctgtgtacc taaagttaca
187560gacctgactt taaacagttt ctctctggag gttgaaaggg gctctgtaag ataccagagt
187620ggattgctct caaagactct cggactcctg ttacaggcaa gtaaggtcct agcagatggt
187680agcatggatc tccggccctt ctcactgctt tcttgaatca gggatttaga aattgctatt
187740tgcataccag gaggactgaa gtttggctcc cggtgaccag aggacaaggt cattgtttaa
187800aaccacccaa actcatttcc gacttggttg gtcaaatttt caagtttccc agcagtctaa
187860ggattcataa aataaggcag aggcagagaa acggagggtg tgtgtgtgtg tgtgtgtgtg
187920tacccaaaat ggaactgcat tttcatgcac aatagaaaac ttaaagactg aaccaatcat
187980tttggaaaac tggcacagct gacattggct agaggaagga acggccaggg cgagccagct
188040gcaccaagac ccagggctga ggcctaatcc gcctttatcc gagggtttag tgaggctccc
188100gccgctcacc aatcccggct ggagccgcag aagagctctc ttcacttggc tcagtcccag
188160cacagtcgca ctatgctctc ccgtggggag gccgctccgg gagggggagc gacatcaagc
188220tttgtgaaac tgttttcgaa aacctgggat gatcatttaa atgtttaaaa tatgcacatg
188280gtaattcaaa actaattacc ctgagcacat ttgaaacatt tatgccatca tcttggatcc
188340tgcctactga ttgtgcgctg cagctcactc tggtgtttct ataaactgct tcagcgattt
188400taacttccag gctaaatcag gcagccacag gcgctgcctc cagccctggg ttggtggaga
188460gacccccatc cctgacttcc aggcgaggag gcggcccgtt tctccagaga gccgtttgtc
188520agggtcttgt agttctggct gccgaattat tgctcttatc cgtgttcata attctcatct
188580gcattattta atttaggcta gaatgacctc tttccctccc gagtcttcct ccctcattcc
188640catttcctct tcttcaattc gtggccccca ttttctgatt ggtccaaata tatagacaaa
188700tatccttgat cgtcccaccc cacttggcta catcttcatc tgggagccaa tgtggtgagt
188760tttctgggtt tgcaaggtgg tcaggtccac cagtcatcct aaggtgtgtg agagaggtag
188820accaacatga gcggcgcaca gccgccatca ctgagagagc acgtgccctg cagctcaggc
188880acaggcatgc acacaccggc agacatgtgc acatgcgctt tccccagcaa accctgcttg
188940cagagtaatt aggcctaggc agttcctgaa gcaaattcat ttcccccttt tccagaataa
189000aatgagttct cttcctttgg gggtgctaaa ccagcatgcc agtggctaga agcctgagat
189060gggtgatgtg gctgaaacca tttctgcagc caagcctgtg ggcagaagct aaccttgggc
189120tggggagctg cagtcggaag aggcacaatt ctgggatcaa gaaatgagca ctggtttata
189180ggtacactcc cagaaataga cagatgaggg ctgcctcctt attagcgctt tgaagatgcc
189240catggcgggt ttttagacat ttaggaatat aaaagtaggt tggattccca cagtcagctg
189300aagtttgaca gagtgatatt accgggttta actagagcca ttaagagact cttcattatc
189360ccacaccacc gccacccaag ttatcacatg agccataatg caagagaatt ttcattccat
189420caacaagaga gggagccggt ctatctttgt ccaaaggaaa tgagcagccc agcgtgaagc
189480ttgtgaggaa ttgagtgtac aacactccaa taacatcccc tgcaggattg cctctgcgat
189540ttagtcggtg aagcaggggt aactgcgctc gagcagtctg cctgtgtacc tggcttgcaa
189600gaacaccagc tcgaggaaca ccaaaaaggc cgattaatga caaaggacac tcatagaggc
189660ccgaattcca cagggcttaa gtattaagcc ccaaagaaat caaggtctag gccattctcc
189720tggcgctcag caatctcatt tattatttct ctacaaagat ccaacactca atttcccagg
189780tatcccctgt atctgactca cattctcctg ctcagtaagc catcctggtt tgaaacgggc
189840ctcccctcct cctgcctatg catgctttgc gtcttcacaa cgacagctgg taatttgcaa
189900gaccccctcc actggactct ctcaccccac atacttggaa ctactccttg gaactacttg
189960tttatcaagt gttctgttgg tgagccttct cttgcattaa agctgtgaga aggaaccaca
190020gtttctattt cctttacatt tcttgtagcg tctcacatgg gagacaccca ggttagatat
190080actgagggtc ctggtagttt tagagttgga gttagatgac ccagcaacat gccttccccc
190140accacgcacc aagcaaaaat tgcacccacc cttccctcag atgttcctgg catcttataa
190200ctcgcccaaa gccagattta ttgctcctgc tgtaaagtgt atcttctcta agcctcactt
190260aaaagctacc acttggcaga agatcaagtc agaagtgcag gctagcaggt gacggtgagg
190320acagggcggg atggggcggg tagggtggag cgaataattg aagctccaag agttaccagc
190380tcaatattta acctaactgg taatttgctg tgacaattac gccatgaagg gaacgctgcg
190440actatgcaag aatgttgctc tctaattaag agggctctgc atttcctagt cacccgcact
190500ttaataacac acagaatgag ccttggctcc gggagctaaa ggttccatta ggagcacggg
190560cagcatatgg ctgtgcacat aggccgtgag tgatgcagcc cagttaagcc cgctaacacc
190620ttcaattcgt cctcagatag agcccagaga gcgcggctca ggccctcacg ccacgagccc
190680catttgactg acaggcatct tcccggaaag cctgcgcgtg cctacactgc aaatggacct
190740gcttcccaca gcccggcttt caaccaggaa ggcttggcgt gggtctgatc cttcaagagt
190800aactttaata aggattttct cacagaaaga aaagtccatg ggaacaaatc ctcctcttaa
190860gagcgtgaga caggaatggg gacacaagcc aacaccccaa ttgctaggct aactctgata
190920tgagacaaaa gaatattaat atcttggcta tgaaggagga tggtgccatc ttctgaattg
190980atgggagttt tgaggcatgg ctaagctggg caaaccattt tctttttttt ctcttcttaa
191040ttagtggttc atttatggag ggcttgctgc ccggagagcc catcagaaga gagctcgctt
191100tatggagatg tagcttataa aactactcag attttaaaca aacagtgcag gaggccagag
191160gtagaagtgg tgggggtggg gtggggcaag agaacaattg catctgcaga aggctagccc
191220tgcaccccaa gcctatgttt agggttgatc agcttcccga ggcaagccca gaagcctcta
191280aaattttagg ccaatagaaa tgacctctgc accacggctg actgaagcta taaataagcc
191340tcgagttgag cagtggtgtc aacggagaga gcagaggaaa gtccaatcag agcttcattt
191400ttttttttta aagtccactt gcttgggact cacctgaagg cagggcattg agtagagcct
191460tggctccctg cagcgagagg ctccagtttt cccaggcacc agcccatcgg ttggttacct
191520aaccaccgaa agggaactgc acagcacaca agttaaatat aggctgggtt atctgcattt
191580tacaagctct gagcaagcta tctgaagaag ctgtcatttt taatgacggc acaaacttcc
191640aattaccgac tgggtaatcc actagggagc aggtagtttt ggaagaacag ttcaccatta
191700ttaaaagttt acacaatcac ttttgagttg actataagta tttcacacga ggcaggtggg
191760attagggact ttttgggtgg tttactcgag gctgcaacca acaatgagtg ttttctcaag
191820aattatacat tgagatttgt caactgctgg ggagtagtgg agggtcctgg taatgcagaa
191880aggttatgaa atggccaggt aaggttgggt gcttccaagt ctcaaatata ctcctaaggc
191940cagctccaag tcataagctc aaacaagtct tcaaggggcc tggagagtta agacaaataa
192000ggatcactta ggctacccac ggacaagcac ttctcataca aggaccggct acctccaaca
192060ccatcttccc aacatggctt ctatgttgct tcaacaacca gggcagggtg aattaggggt
192120gggtctctcc aatgtggact caaatcatga ctacagcntg gggttttttt tttttttttt
192180tttttttttt tntttggttt ttcgagacag ggtttctcca tatagccctg gctgtcctgg
192240aactcacttt gtagaccagg ctggcctcgg actcagaaac ccgcctgcct ctgcctctgc
192300ctcctgagtg ctggaattaa aggtgtatgc taccacgccc ggccgagtcc gtcttgataa
192360tgaagttccc agtgacctgg atgtcaactg aagttggatt ttactgtgat gactactgag
192420tccggctcag aattttgggg ggacaaggta ccttgattta actgggcact acacgactgt
192480aacccccaca ttgggagagg cagaggcaga ggcagaggca gaaagttggt tggaggctag
192540gcaaggctac acagcaagaa gctgtctcaa aaccaaagac atctttcttg atccaaatcc
192600tgtcggaggg tgtgaggcct tgggggccag aacaaggtgg tcaaggaaga ccactgactc
192660tgtcctttgc tccattactt aatcagaatc gccatcacag atatagctag gagattttaa
192720gccttggtgg ctgcaatctg catttaagag ctaagtggga taaactcagg ggtgggccca
192780atgcctccct ccccaccctc cctgcatccc tccatttacc tgtttccagg gatctgctta
192840atttacctgc cagcctttgg tgggacacag gcttagtggc ttagcgctgc tcggggcacc
192900agagaccctc acagaagcac ctgaatgtac tttcagcgct gcagagcacg cacggctcag
192960gcccatcaga agaacccagg cttatgctaa ggagccagaa agtagaagca gctggcaaga
193020gtgattcagc cccataaatt tacacatccg tacagccaaa cccacttgaa gtgatccaga
193080gccactttta ttgaaataga aaagatgcct attctggagt gctaagtggt acaggagggt
193140gggtatataa gagataatcc catgttgtct ttgatgtggt gctagggaga taacccagga
193200cctcacgcct gcctgcaagg tagccaccaa gccacaccca caacctctat ttatacacac
193260actaagtgtg gaggtatgga taaaaaaaaa tgtcccaaga cctcacgaat ctgcaaacat
193320ggtgcctggt tggtggcacc gtttggggag gcagtggacc atttggtctt gcaggaggaa
193380gttatgtcac tgggtatggg ctttgagagt ttgtagcttt gctccccttc cagttaactc
193440tgctctcgta aggttcctgc caccatgttt cctctgccat tatggacacc tggtcctcta
193500gaactgtaag ccacttactc tcaggtctct ttcagtcctg gagtcttatc atagcaatga
193560aaagtaactt gtgtggcagc cagctaagca agggctgtgg ccgactgctt gggattatgg
193620ttgtgtctgt ctgtctgtct gtcattccat ttatatagtc ctgagaattg aaccacttta
193680ccactgacat gtctcagtcc tcttggtatc atatattcac ttaagacaag atctcattaa
193740gtcattcaga ctggttttga gcttgcaatc ctcctgcctc tgcctcaagg cgataggatc
193800cctagggtac tcgaccagac tgggagtagc aggttctgtt ctcttagctt tctacagtga
193860ttgtggatta tttgtgtata aagatctgat ggcccgaccg actcccttcc ctttaagtga
193920acatcaacag tatttagcat caacttaata aactcatttg gtaaagccat ctccccacct
193980cttgaacaaa tgaaaatcaa acagcagtac ctgttctcct agagcagcgg ctctcagcct
194040tccggccttt taatacagtt cctcgtgttg cggtgacccc cccccccccc agccgtagaa
194100ttatttcatt gcttaaccag agttaactgg aagggttaat aataaaacca gtctgggaga
194160ctaaggttac ccaaccacgc taggaaggag aggaaagggc cactcgcaca aacctgtctt
194220tgagatgaag aacaatcaac ataacaggga cagagcagtc cttgtaacaa gtgcaaagga
194280gagagagagg ctgagtttct acttctataa ataaaccctt ggcaggcgga tcactaaagg
194340aacacaagtc aatataaacc tttagacatg gggctgccaa acttcacttt tcgacagtat
194400attaattatg tagtcaatag ccatgggttt cattagcgta ttaaatacca cgatcaatat
194460tatttatact tttcgaagac aagccactca gggaaaaaat ggtgggggga ggaggaggaa
194520caatttgacc ctgtagttca aaaaaagtca gaacagcaca ctagagatta gcaagggttt
194580aatggaaggc ataaaacact ggaaatatgg acagaaatca gatccctgcc ttcatttttc
194640tgccttttac aaagagactg gagggaattc agaaactatt taaaataaag gcaaaatgat
194700tagagcccct ccctcccctc agctgcttaa cactggggtt gtggtggacg caaaataagc
194760attgagctct aagtgataga tgagaatcag aacaggaaca gtgtttttga ggcaaaatat
194820gtccaagaga attcaaagaa ctgtgggcca gaatctactt aggcagtcct ctgggacccg
194880aatccctcac aggcgttaac agtggaacca atttccaagg cagccctgct ggtgatctga
194940tttttgagta gggaaatctg ttaaacatcg tcccacgagg gagcccagct ctttcactcc
195000ccacgggttt ctacatgcag ctgtgctaga tctgctgaag tggccggtga ggaggtgtgg
195060ggattggttc agcgacctca gaggacattc ttgttcacta gccctcgtgc actggggcga
195120tgaccgaatg ctgtgagcag gagatatcaa aggccggcta ctggactgaa aactagatca
195180ccatctctaa cctgcaattt gtcaatctca gacagcaatg aagactgtga ttttctagtc
195240aacgctttgt aagcaaggtc agatagaggc tccataaaaa ttgttcaggg ttcaggcaga
195300gaatcaagtg taactcaatc cctatctcct gagattaggg aagggaagga aggctgtgtc
195360tactaaacca gtgagcctca agcaaagcct gtctgttctc agcaaggtga gccacccacc
195420aaagatgcca acagctaagg gccagggatg tagtgcaggg tgctgtgata tcaacagctg
195480ggagacagaa acaggaggat caggacttca aggtagtttg ggctataaaa tataagcttg
195540aagctaccca cttgaagact gtccccaaca aaacaaacaa gctgggtatg gtggtcgatg
195600cttgtatttc tagtgtatga gacgaaggaa gaaagctcaa gcccgagacc tgcttgggtt
195660acatagggaa gatggtgcct caaaaacagg acagccgagg agcagacaga cagggcagac
195720agggtgcatc gatctaaatc cacatacctg gatttaaagt aatatctggg agactggtct
195780gtgagggccg ttccagagat ttaataaaga cccaccctga ctgagtatgg gcaacaccca
195840tgggtggccc aggggtccag actgaataaa gggaaaatgg gaggaagttc agctggtagt
195900gtttccaagt attaggacca cagcctggcc cctggcatgt gctggccagc tagtctagct
195960ccagtatcaa gcttcaggcc agcggcaggg cactggacag ttcccacaca cgacacacac
196020acacacagag cactagcatt cacctcctgg tctcttcttg acaacagata aaatgtaact
196080ggctgccaca gtgagagtcc cctaccttcc tcaccgttaa ggatggtaca aactgtgagc
196140cagcagcagc catttctcca gtaacttgct ttccacagat actgttatag cactaagaaa
196200agcaactgaa acatggggtg ctgtgacccc ttggcaccac aaagccatgg caagctgaag
196260tgcacacatc acaggccagg cctgaagatg ctgggggact gcaatgctgc ctggattctg
196320gcagagatgt gcagcagatg ccaagaggtg ggctgcagca accagagata attaatatga
196380ttaggaacac actgagcagg catgctcttg ccgaatgaaa agcctcgcag tgtaatgact
196440gttttcttcc tcgatcacgg tctccacgtt tcagagttgg cttggtgtta ggctgccgcg
196500taaacatcaa tccaaccccg aggggccaga tcatcggtgt tcctgggctc aatcgccttt
196560ccttttgtgt tttcattcat ttaaagatgc attccagggt tgcaaacatt agtgagaatc
196620atctccaggc ctcagtctaa tctctgagtc tgtaatgagt taacatcttt ccctagtgaa
196680tatttattat gaaggctaat taattgcttt ccagttacaa gaatccttta cagtcaaaga
196740aagtaggatc cacaaagata tactgtttat tcaaacaaag caaaggaaac aaagcttctt
196800tcttaaattc tatttaacat agctttaata aaggtacaca ggtccgcctg gcaaccgaac
196860ggtaactgat gcaaactgaa gccatgctct gtagcagcct ggatgtccca gtgccacctc
196920tgtctgcagg ctttgtcgga tttactaaga ttctgttatc ttcaaacagg gattgtgtct
196980caagtaactg accccactat gtggataatg aagtaaatta tgcaatttgg gggtttgctt
197040ttccccaagg ggacagcaag ccagtgctta tcagccgtcc tcagaggaga caattctgat
197100taatatcaga gtcatctgac tcagtctatt aaacctatca aaccctgaag gaaggatatt
197160cagatattaa cgataggcct ttgattaata attctacctt gttgccattc taagcattaa
197220caaccatgca gtaactctgc aaaacagacc ctttgattcc aggcagacgc accctctgaa
197280cacctgggtt ctcccctact cttctccccc caggaggaac tcaagacaaa aaggtgccac
197340cactggaaaa gcacactcca ggttacataa tttgcctcat tatccagagt ggggttaatg
197400acttgtgaca taatttctgt ttgaagataa caaaatttca tgaaatccga caaagccgga
197460aggcaggagg aggggactgc tgccacacta ccggtggctg agaactggag cggaaggttc
197520acacacagcc ctctgagctc actgtctttg cttatcagtg agtcccaaga ggggcccaga
197580tgggttgcca gcctccccta gaggatcttc attgtggagc tgtcccatgg ggcgggaagg
197640aagccattct atttctgttc ttctctcttc cgttctggcc accagtggta cttgctccca
197700tcacatgttc ttcctgatgt tcgcgatcag ccgtctgcca tagtctctga agtccacggg
197760cttcacgtcc atcacagtgg ccttaattcg agattcatcc tttagaaaag agagaagctg
197820tttgtgagtg gcagagcctg gcgtgcagcg gaagagagaa ctttctttgc ttcagtggct
197880tcaatgagtc cagcaggaag aaggaaagtt tacaagtctc agagagaaag tgctgtgact
197940tcctggagtt gggccagatc ctcttccaca gaccctttcc ccatcctagg tgccctgtgc
198000tcagacctag catcctcccg gagaagcctc tgtctttcta tgggtgcagt gggggcccag
198060agcagacagg taactcaccc taaagcatca ctttcatcta gaggagctct gtggtagtag
198120ggactgaggc ttctgctcca gctctgggca aggttacttc tctgctcttc accattcctg
198180tccatcccag gaagacagaa aatccctaca ctctcccttg atctacccga ctttctgaca
198240ccagcctacc tatgttcatt taatacaaca actaaaatat ctattcacag gcactaagct
198300ggtgataacg cagaatgcac aaactctgcg gctgcagggg agacggcaga gttcctcctc
198360cacttgtctc cttgaactaa acagtgtctt tgaggcagaa cagggtgaca cctagggaca
198420cacaagtcta gctgggggcc ttcatgcttc catgtgctta gtaattaatt actacatgca
198480ccgctgttta caagtatggt taggagcccg actgcctggg ttggcctctc gcctctgcca
198540ctccatggct ttaggttcag agtcattctc tgcatgcctc tgcctgtctc tccgttggta
198600aagcttgcaa caacagctcc aacacagaaa gtgctgtgag ggtcgacagt ggatagatgg
198660ctagatagat ggggcaggac ggactgtcca gtaagcaggg ttcatcatgg ctatgcagct
198720ctggacatca ggattagttt aaacacttgt caggtggggc acttttacca gcacgtgcta
198780tttgtttaat attctgagtt ttagaaccta aactgtggga aacaagagtc cacacataac
198840annnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
198900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngccttgaac ttgcttctgc
198960ctcctgctgg ggttacaggc ttgagccacc atgacagctt tagcaatagc tttgtaaatc
199020cacagtgtca agctggatat gatggcacat gcttgcaata ctaacctcca aagattccct
199080gaactcgagt tggtgaaata gtccaccagg taaaagagct tgctgcccaa gcctgaatct
199140gattccctgg tcacatgctt taaggaaaga acttgccgaa gatgtcctct gagtgccacg
199200tgtaccgatg catgcttgca gccacccaca cacccacaca agtgcactgt ctcacacagt
199260gagaacagca agtgaacaaa caaacaagcc gggggggggg ggattgtgac cagaataatt
199320gagggggggt gtaaagctct tggcaggtgg ctggctcctg gtaacactcc ataagtgggg
199380aagttccaca tgtaaggtca tgtgatcgag tacatctggg cctccaacag tccttgnnga
199440agaaacagat gcagtctgtc atatctaaac cattgttgtc gtatctctgg gtagtctttc
199500ttttctcctt cctttctttt ttctctccct ttctctttta aaaaattatt tatttattat
199560tatatctaag tacactgtag ctgtcttcaa acacaccaga agagggtgtc tgtcagatct
199620cattatggat ggttgtgagc catcatgtgg ttgctgggat ttgaactcag gaccttcaga
199680agaacagtca gtgctcttaa ccgctgagcc atctctccat cccccaaccc ctttctcttt
199740tgagttaggt tttgtgtagc cctgggtggc gttaccttaa ctacactggc tttgaacttg
199800caatgatact ctgcctgatc tgtcttaatc attttgagat agggactcac tacatagcct
199860ttgctggtct ggaactaaca gagatctgcc tgtttctgcc ttgcaaatgc tgggaataaa
199920gttatgtacc accacacctg gagtttaagg gttttttgtt tgtttgtttt tcgagacagg
199980gtttctctgg gtagtcctgg ctgtcctgga actcgctctg tagaccaggc tggccttgaa
200040ctcagaaatc cgcctgcctc tgcctcccaa gtgctgggat taaaggcgtg tgccaccacg
200100cccagtttaa gggttttttt ttgtttgttt tttcctgaga cagggtttct ctgtgtagct
200160ctggatgttc tggaactcac tctgtagagc aggttagcct tgaactcaga aatctgactg
200220cctctgcctc ccaagtgctg ggattaaagg cgtgtgccat cactgcccag tgattttttt
200280ttttttaatg tgtgtatttg tatgggtgtg tgggtgcttg tggaagccag gtgtcagatc
200340cccagagcgg aagtgttctt aaccgctgaa ccatctctct tccctcttcc ctaactctga
200400ttttaaaggc accaaactct taggtaggag actatacaca cacacacaca cacacacaca
200460cacacccgta cacacccgta cacaccacat gaccatgcct gagcacacaa gtggttttat
200520tgctggtctg gcctgtgtat gagctggaac caaaaccttt gtcgggagat ccgcagtctg
200580cagtttgagc acaggctctc tggtttctgt tctctgtcct gtgtcgcatc ttgactagag
200640gcagagaagc atctgcaagg ctgtgaccac gctggctggt gctctgccat ctacattttc
200700aacaggaaat ctcaggagag tatttccttt taagaacgcc agacttttgt gcctgggcca
200760cttctctact tcccagaaca ttgtgtgcca agtggcaagt tattaaccaa gtgctttgga
200820aaattaaact ccttggtttg cagagtagca tgggagcatt gagagggtgt atgcctaaag
200880gcctggttct gctgctggca gagctgacac ttggctaaag ggctggcatt tctgagatga
200940gcctcactag atccgcgtct cagagtctgc aggagaaatc agagagggga gaaggtccag
201000tggcctgttc aggatgatct tcctctgcat ttaagggcgg ctggtttgcc cacgtagccc
201060cagaaccaaa cgagcctcgg acgaagcccc ctaaaggcag taggagagac tgagccttgg
201120ctcttcagca ggggtgggga caagagcaag aggcgggatc tcgcccggcc ctttagagac
201180acgtgcggtt gtttccgtgt ctgggagatc acatgacccg catcagctga cccgtcacgg
201240tggagctcag cgctggtgct tcgcgctccc cgccctgctg cgccccggag cgcaggaccc
201300tgcggagggg taagaaaacc cccaggcttt ctttcctttg tcgctggttc gcgcagtcac
201360ctgcacccta ccccccgctc ctcgttcatc ccagtcttcc cggcctggca ccccggaagc
201420cactgcgagg agggccgtgg ccaggctcag ccttgcgctg cccccaggcg gccaggacca
201480aatggcccag gggagcagaa ggcggaaagt ggttcttaca gcagggtccg agggctggtc
201540cccttcctca ggacctgaca tggaggagct gctccggagc gtggagagag atctgaacat
201600tgatgcccgg cagctggccc tggcgccggg gggcactcat gtagtggccc tagtgtccac
201660gcgttggctg gctagtctcc gggagcgccg actgggaccc tgtccccggg ctgagggcct
201720gggtgaagca gaagtcagga ctttactgca acgttcggta cagaggctgc ccccaggctg
201780gactcgagtg gaggtgcatg ggctgcggaa acggagactg tcctacccgc tgggtggagg
201840cgtgcccttt gaggaggggt cctgtagccc tgaaactctc actcggttca tgcaggaggt
201900ggctgcccag aattaccgga acctgtggcg ccatgcatac cacacttatg gacagcctta
201960cagccacagc actgccccct cagctctacc tgccctagac tctatacgac aagctctcca
202020gagggtgtat ggatgcacct tcttgccagt gggtgaatcc atcccatgtc tatcaaatgt
202080cagggatggg ccctgcccct ctcggggcag ccctgcctgc cccagccttt tgcgagctga
202140ggctttgctg gagtcgcccg agatgctcta tgtggtacac ccttatgtgc aattctccct
202200gcatgatgta gttaccttca gccctgccaa gctgaccaac agccaagcca aggtgctctt
202260tcttctcttc cgtgttctga gggccatgga tgcctgtcac cgccaggggc tggcctgtgg
202320ggctctgtct ttgcaccaca ttgctgtaga cgagaagcta tgcagtgagc tccggctgga
202380cctgagcgct tacgagatgc cttccgagga tgaaaaccag gagggctctg aagagaaaaa
202440tgggacaggc attaagtctg aaaaagaggg ggaagggaga actgagtgtc ccacctgcca
202500gaaagaactt cggggccttg tgctagactg ggtccatggc cgaatcagca acttccacta
202560cctcatgcag ctgaatcggt tggcaggtcg acggcagggg gatcccaact atcacccagt
202620gctgccctgg gtggtggact ttaccacacc ttatgggcgc ttccgagacc ttcgtaaatc
202680caagttccga ctcaacaagg gagataagca attggacttc acctatgaga tgacccggca
202740ggcatttgtt gcaggtggtg caggaagtgg ggagccaccc catgttcctc accacatctc
202800tgacgtgctc tctgacatca cgtactatgt atacaaggcc cgtcgcacac cgcgctcggt
202860gctctgtgga catgtccgag cgcagtggga accccacgag tatcctgcca ccatggagcg
202920gatgcagacc tggacaccgg atgagtgcat acccgagttc tacacggacc cctctatctt
202980ttgctctatc caccctgaca tgcccgacct ggatgtgccg gcctggtgca gttctaacca
203040ggaatttgtg gctgcccatc gagccctcct ggagagctgg gaggtgtccc aagacctgca
203100tcactggatt gatcttacct ttggctacaa actccagggc aaagaagctg tgaaggagaa
203160gaatgtgtgt ctgcacctgg tggacgctca cacccatctg accagctatg gcgtggtaca
203220gctatttgat cagccacacc cccaacgcct ggctggatct cctgccctgg cccctgaacc
203280tccactcatc ccccggctgt tggtccagcc tattcgggag gccacaggcc aggaggacat
203340ttcaggacaa cttataaatg gtgcgggcag gcttgtcgta gaggccactc catgtgagac
203400tggctggact agagataggc ctgggacagg agaagatgat ttagaacagg ctacagaagc
203460tctggattcc atctccctcc ccgggaaagc aggtgaccag ccaggctctt cctccagtca
203520agcatcacct ggcctgttgt ctttttctgc accctcgggg tctcgaccag gccgtaggag
203580caaagctgcc gggttggacc ctggggaggg tgaagagggc aagattgtcc ttccagaggg
203640cttcagtccc atacaggcct tggaagagct ggagaaagtg ggtaacttcc tggccaaagg
203700cctagggagc cagttggagg agcctgaaaa gcctcacgcc cagccacctg tgcacctgca
203760gagcctcttc catcgagaca tgcaggtcct gggtgtcctg ttggctgaga tggtgtttgc
203820caccagggtc cggatactgc agcctgatgc acctttgtgg gtacgctttg aggctgttcg
203880gggtctctgc atacgccact ccaaggacat ccccgtgtct ctgcagcctg tgctagacac
203940actcctacag ctgagcggac ccaaaagtcc catggtgtcg aagaagggca agctagaccc
204000actgtttgag tataggccgg tttcccaggg attaccccca cccagcccag cccagctcct
204060cagccccttc agctccgtgg tccccttccc tccatacttc ccagcactgc acaagttcat
204120tcttttatat caggcccggc gtgtggagga tgaggtccag ggtcgggagc tggcgtttgc
204180tctgtggcag cagctgggtg cggtgttaaa tgacatcact cccgagggct tagagatcct
204240cctgcctttc gtgctgtcgc tcatgtctga ggagcacacg gctgtgtaca cagcctggta
204300cctatttgaa cccgttgcca aggccctggg ccccaaaaat gccaacaagt acctcctgaa
204360gcctctcatc ggtgcctatg agagcccctg ccgcctgcat ggccgcttct acctgtacac
204420cgactgtttt gtggcccagt tggtggtgcg gctgggcttg caggccttcc tcacccacct
204480gctgccccat gtcctccagg tactggctgg ggtggaggct tcccaggagg agggcaaagg
204540cctggtcggg accactgagg atgaggaaag tgagctcccg gtgtccgggc ctggctcctg
204600tgcctttggg gaagagattc agatggatgg gcagccggct gcttcctcag gactggggct
204660cccagactac aggtcgggcg tcagcttcca tgaccaggcc gacctgccgg acacggagga
204720cttccaagct ggactctacg tggctgaatc tccacagccc caggaggctg aggccgtgag
204780cctgggccag ctgagtgata agagcagtac cagcgaagcc tcccagggcg aggagagggg
204840tggggatgat ggcggtgccc ctgcggacaa gaacagcgtc aagtcagggg acagcagcca
204900ggacttgaag cagagcgaag gctctgagga agaggaggag gaggaaggct gtgtggtgtt
204960ggaggaggac caggaggatg aagtcacggg aacatccgag ctcactctgt ctgacacgat
205020gctgtccatg gagacggtgg tggctcctgg tgatgggaga gacagagaag aggaagagga
205080gccgctgaca gagcagacag aaggcaaaga acaaaagatc ctccttggtg agcccgtggg
205140ctgagggggc atgggtcagg tgcttttcct tcaggctctc atatgctggg tgtgggtcca
205200accagatcca ctgtagcacg cacagccaca gtcagacaca gtgcatggaa tgtggaagtg
205260ctgtgtgtga gtggaaagtg gggcttagat ttagctttca ggagacagaa agctccttta
205320aaagccatac cttgggctga ggctgggagt ggagttgagt ggtagagcac ttgtctggta
205380tactcgaggg ccctgggtgt cttatctcta gccccagaag aagtattaag aaataaaagc
205440aagtggtggt tgagatgtga atggagccag aactggccgg aacagtcggg tggaagtggg
205500aagagtgttc cagacaggga acagtgtgtg tgtacctctg aggctctcat ggttccatca
205560gagaggcagg gaaaggctaa aatggttttc ttaagagagt ccagaagggc tgggcttggt
205620ggtgcaggcc tttaatccca gcactcagga ggcagaggca ggcggatttt tgagttcgag
205680gccagcctgg tctacaaagt gagttccagg acagccaggg ctatacagag aaaccctgtc
205740tcgaaaaaaa aaaaaaaaaa aaagagtgta gaagggtgga agccagggac aagtctgtac
205800aagaaggaac ttgggagcat tgccgaaagg atgacctctc tgcaggtcct gcccgaggag
205860ccagtttctg gggaccttga ccatggctag gtgaatggac ccaggatggg atggtcaggc
205920ttgctagcag agccacagcc gagttggctg ggtggggtgg ggtggggtgg gaagggtgag
205980ttatctgatg agctcaggac cttttcctgc cctgcagata cagcctgcaa gatggtccgc
206040tggctgtctg ccaagcttgg ccccacagta gcctctcgcc atgtggcccg gaacctgctg
206100cgcctgctga catcttgtta tgttggtaag gtctgtggtt agtgctggag accaggttcc
206160ccagccaggc ttctgcccat ccttagccct ctctaggcga ctccttccct aacttcccag
206220cactccctga gcagggcctg ggtctcaccc attaagctgg gttttcttgg gtaagtgggg
206280aagagcccag tattgaatga atagaagcca ccccacagtc tcagaaggcc ggcttccctc
206340ctgccctcca ctggcttctc aacgctgctg cccttccttg gtagggccca ctcgacagca
206400gttcaccgtc agcagtgatg acacccctcc actgaatgcc ggcaacatct accagaagag
206460gccagtccta ggtgacatcg tgtcggggcc tgtgctcagc tgcctcctcc acattgccta
206520cctgtatgga gaacccgttc tcacctacca gtacctgccc tacatcagct acctggtcag
206580tccctggttc gtcaaacccc ggcttggggg tgggggcaag gatccaagga ccagccccag
206640gtcttggggg ttccaggagg tctgtggggt gacctgtccc tccctcatct attctgtggt
206700tctaggtagc cccagggagc aactcaaacc ccagccgact gaacagccgc aaggaggccg
206760ggctgctggc agcggtgaca ctgacgcaga aaatcatcgt atacctctct gacacgaccc
206820tcatggacat tctgccccgc attagccacg aggtcttgct gcctgtgctt ggcttcctca
206880cctccttcgt cacagggtag gcccctgctg cttgggagag ccacctggct gagggggccc
206940ccaggaaggg ctaggaagct cagggagaag cagataccgg cctgagtcat ggttctgatg
207000ttgggggtag tggcacaggt ctttcattcc agcacccaga ggagggcaag tttctgtgag
207060tctgagacta gcctggtcta cagagagagc tccaggctat ctaaggctcc atagtaagac
207120tctgacttaa gaaaagagtc gtggttcatt ctgggttgtg ggtgtggctt ggtgatggga
207180cactttccca gcatgcagga ggagctatgc ttgagttcca gcccttcaga aaaacaaaaa
207240tgggggctgg aaagaatagc tcagggttta agagcactgg ttgctcttcc agaggatcca
207300ggttagattc ccagctgcca catggtagct cataaccatc cggcagttct atggaacctg
207360ccaccctcct tcggtctctg tgggcactgc aaacatgtgc acagacatac atgcaggcag
207420aaaaaacacc catacacata aaattagacc aaaaaagttc atgttctctc ctacctgtag
207480ctctgactaa gctacactgc ttccctgtgc ctcagtttcc tcccctggtc tggactgatc
207540agccttacat gcagctcctg ttatttgaag ttcctggtaa attggtcaag tccttcaggg
207600aagggctggg aactcttgca ctttgattct aggttcccca gtggggccca ggcccggact
207660gtcctatgcg tgaaaaccat cagtctcatc gccctcatct gcttgcgcat cgggcaggag
207720atggtccagc agcacctgag tgagccagtg gccaccttct tccaagtctt ctctcatctg
207780catgagcttc ggcagcaggt aggcaggcag cttctgggct gggtgggcca ggccaggcca
207840ggccaggcca gggcagtgga cccactgaat ctgtggtctt cctacccgca ggatctgcca
207900ctggatccta agggctgtac tgagggccag ctgccagagg cgaccttctc tgatgggcag
207960cgacgaccag tggaccccac cctgctggaa gagctgcaga aggtgttcac cctggaaatg
208020gcgtacacaa tctacgtacc tttctcctgc ctgttgggta ttgcccatca cgttcctttg
208080cacagagttg gtgactacat ctcttccctg gggtgggccc cgatgctttc acctccagag
208140tcagcaatgg aatcttttta tttttatttt gacatggggt ctcatttagc ccaggctgac
208200ctttaactcc agctccttcc agcttccacc gtctcctgtt ggcattgtag tcatgtggca
208260ttgctcaggc ttcttncatg ttcttatttt taaatgacct gtgtgtgtgt gtgatatctg
208320tgtgagtgtg gaggtgacag aataacagtt ggggggtcag cagatgcctt gcctgatgag
208380catctctcta gatccagttt ttggttttgt gggcttttat gtgtgtgttt gtttgtctgt
208440ttttgtagac agggtctctc tgtgtagcct ggccatcctg gaactcattc agtagaccaa
208500gctggccttg agctcacaga gattcacctg cctctgcctc ccagtgctgg gattaaaggc
208560gtgtaccact cctgcctggc tttgtttttg ttaaccacca tcctcctgcc tcagcatctg
208620cctcccctgt gctgggatta caggtgtgtg ctatcacacc cagctaacag tggatttaaa
208680cgtaggaatt ttaggatcag agtgaccaga tttggtccta gggcccaatt tccacagtga
208740ttatctatct tagttaggat ctctgttgaa aatcatggtg gaacatcatt accaaatgca
208800acttggggag gaaaaggttt attttgtctg acaactctca ggtcaccaag ggaagtcagg
208860gcaggaactc gaggcagaag ctgaagcaaa agccatggaa gaactctggc ttgttcctca
208920tggcttgctc agtctggtgt accccctccc cacccacctc cccacaatgg tttctctgct
208980tatgcctggc tgtcctagaa ctcactctgt agactaggct ggcctcaaac tcaagagatc
209040cccctgcctc tgcatctcaa gtgctaagat taaaggcggg tgccatcacc cctgccccag
209100gggtggcact acccactgta aattggtccc ttcccatatc agttgttaaa taagaaaact
209160cctccatagg ccaatctggt gggggaattt tctcagttga gggtttctct tctcaaatga
209220ctgtagctga tgccaaattg ataaaacaaa tctcaaacca ccaccaccaa caacaataaa
209280accaaacaaa ccaaacaact aaccaagaca gtgacttata aagagaatct gaacattttc
209340cagcaggaaa ggctcaggag ctggccattc aagtctgggg aacagaatgt aggggaatat
209400gatggtctcc agaagctacc tgcaaaggaa tgaacagctt gctgggtttt gtggcttccc
209460ttatgggatg ggcgctgtac tgggcttctc tctgagtagg atgggccacc ctgtagttgg
209520gaatattttg ctcctacaga attgtaagtt cccagaggca ggacacatct gtcttattct
209580tcattgtgtg tctgatgcta gaatggtgcc tggcatacac gtgtgtgtct ctatagagac
209640agcactcatg tctacgtatc gataaaggaa gctgttttgg ggggaggaaa caggcttaca
209700gacgagaact taataaccca gagtagccca gtcagtacct tgccttggct tctgttgttt
209760ctaagctctg ggtagatagc taccttgcca tcttccctga tcttagaact ttccccactc
209820ccctgtaggt gacatcatcc ggaaaatcat ccccaaccat gagttggtcg gggagctggc
209880agggctctat ctggaaagca tgagcccgag ctctcgaaac ccagccagca tggaacccac
209940catggctagt gccggccctg aatgggaccc tcagagtggg agctgtctcc aggacgatgg
210000ccactcaggg acctttggga gtgtcctggt tggaaatcgc atccagatcc ctgactctca
210060gccccagagt cctgggccac tgggctccct ctctggagtg ggtagtagcg gaggcctcag
210120caacaggaat gaagacaacg ccctgaagcg ggagctgcct cggagtgccc atgggctgag
210180cgggaactgg ctggcgtact ggcagtacga gatcggtgtg agccagcagg atgcccactt
210240ccacttccac cagatccgcc tgcagagctt cccagggcac acgggggccg tcaaatgcgt
210300ggccgccctg agcagtgaag acttctttct gagtggcagc aaggaccgga ctgtgcgcct
210360ctggccgctg tacaactatg gggacgggac caatgagacg gcttcccgcc tcatctatgc
210420ccagcaccgc aaaagcgtct tctacgtggg ccagcttgag gccccgcagt atgtggtgag
210480ctgtgatggg gcagtgcacg tctgggaccc cttcacaggt gagcgggccc aggtgaggcc
210540tgttcgacgg ctgctttact gtgccttagc caggcctctg ggaacgggac ctagtgcgaa
210600acgtacaatg gcgtattttg acggggaaga ttcagtgagg caggaagaga agaagagtca
210660ggacttagaa tctgtgggac ccaagtttga atccactccc ccaacttacc agcaatcggc
210720tcagttgctg caggcgtctg ccttctacct gtaagaacca aaaatttaga agattccacg
210780agtatggctt tggcttcttg tacgacgtca cctgtcgtcg ttgtaaagag aagtatcgag
210840tggaggaggg tcagggcaga cggaggtcgc agctagttag agcatgctat gtgaagagag
210900cagactgttc tggggctgga cccttgactt cactgtggaa gcagcaagat gagaaagccc
210960tgagattgtg ttttctgagg gtcactgggg aatgggatgc aggtgtgggg tgagttggag
211020tttgaagtag ccagggctct ttgatagcca ctaagtcccc agatgtgtcc tttttcagga
211080aagacccttc gcacagtgga tccttcagac agccgggtgc ccctgacggc tgtggctgtc
211140atgcctgccc cacacaccag catcaccatg gccagctccg actccactct gcgctttgtg
211200gactgcagga agccaggctt gcaggtcagg aggggtgcag ttcctgggct actgggggtc
211260tctaggtacc agtcaggaaa gacactcagg ggactccacc aggaacgctg cagtgacagg
211320cagccctgtg tgggtggggc gctggcacgg atggggcttt tctcttccgg ggatggagtg
211380ggagggtcag gcctactggt ttcgtgggcc tgaatggggt gagctgcagt agggtgggtg
211440gcagtgatgg atggcgacgg gcacttgaac acaatctcct cctatagcat gagttccgac
211500tgggtggagg gctgaaccct gggcttgttc gctcgttggc cgtcagcccc agtggccgga
211560gtgttgtggc tggcttctcc tcgggcttca tggtgctcct agatacccgc acgggcctgg
211620ttctacgagg ctggccagcc catgaagggg acattctaca gatcaaggtg actgactgcc
211680tgaggtccta tcctttcatt tctacttagg gcctggtctg ggagaggaca ggtttatgct
211740ggtgtccctt ataactactc ggggacattc agtggggtgg gaaaatggcc ctcgtaggcc
211800agctcaggaa ccagctgcac aggaggcagg ctaggggcag gaatcagggc tagaactgac
211860cctgatgctc cacagcgatg ttctaatgag taacccttgt ccatatttgt cttgcttgga
211920ggatcagggg tcacgccctg tccgtgaccc agttcaggtt aaataaagcc aggaggctgt
211980ttactgcctg gagaccactg agcagagtcc atgccccctg ctgggctgtc ctgatggggg
212040gcaggaacag gcgcaggcct gcgcatcgtg ttcctgcctc ctatattcaa tcatagacct
212100cagagctcag caggttctgg gaggggagaa atagggctgc ttgtgggagg atttctccct
212160gcagtgggaa ctctcctccc ccgccgtcca gatggaggtg aaagacaggc actgttgctt
212220acagggaagg caggctgccc ccagctctat ccaggacccc aggggaccct gggtctcagt
212280gtctctaaat cccaacattc taagaaagtg tcaggatggc tctggggtca tcctgggtgt
212340tagtcccagc tctcggagtc ttctctgagc accagttctt ttcctatggg aagtaaggac
212400atgccaggtg ttctttgaga ggacctgagt ttggttctca gcactgtcta gctctggctc
212460cagggggttc aacacccttt atggcttctg tggacacata ttcttatgtg gcctgcacgc
212520acacacacga acacaaataa aaataaatgt taaaagaaga cagcggcacc ttgtacctca
212580catgttagta cgattggatg tggcagtgcc tcacagaatc tgtgggactt tatttattta
212640tttatttttt ggtcttaaaa ttttaaaaga ttggtttagg agtggtggta cacaccatta
212700atcgcaacac tcaggagcag aggcaggtgg atctctatgg gtttgaggcc agcctggtct
212760acagagcaag tttcagggca gccaaggtta cacagagaaa ctctttctca aaaaataaaa
212820acaaaatatt taaaagattt acttacttct tatttgagct aggatctccc tattagccct
212880ggctgtcctg gaactcactg tatagaccag gctggcacct taaactcacg aagatcctcc
212940tgcttctgcc tcctaagtgc tgagattaaa gtagtgttat accatgcccc actattttct
213000ttatagattt ggtgttttgc ctgcttgtgt atatatgcac taccttcatg cagtgctaat
213060aggggtcaga ggcgagtatc agctcttcct ggaactagag ttatggaagg ttgggagtca
213120ccatgctggg actgggtcat ttgcaagagt tacaagtact tctgagccat ctccagcccc
213180ctagagtttt tttcccccct ggctgtcctg aagtagaatc tgttcttgtt ttgttttgtt
213240tttcgagaca gggtttctct acataggcct ggctgtcctg gaactcactc tgtagaccag
213300gctggcctcg aactcagaaa tccgcctgcc tctggctctc agaatactgg aattaaaggt
213360gtgcgccacc acgcctggct cagaatctgt ttttaaatga gagtaatagt tacaggtttt
213420ttgttttgtt tttttctttt ctttttttgt ttttgttttt tggctcattt gttttatttg
213480ttttgagaca ggtctcactc tgaaccccta ggtggcctgg agcttgctat gtagaacaca
213540ctgactttaa acttgttttc tgagtgctgg atttatgggc ttgtgctatt ttgcccagcc
213600tctgatggtt gttaataaca atattattta gcttttcttt tggagatagg ctctcactgt
213660atatcaccca gacagtggct ggtctggaaa tcactgtgta ggccaggctg accttgaatt
213720cacagagatc tgcctcccga gttcagaaat taaaagcact ctgggatggt tttggagttt
213780ggtgagtacc caagcctcca ttgatgctat ctgtccctcc cgctctctgc aggctgtaga
213840gggcagcgtg ctcatcagct cctcttccga ccattccttg actgtttgga aggagctgga
213900acagaagccc acgcaccact acaagtcagc gtccgaccca atccacacct ttgacctgta
213960cggcagcgag gtggtcaccg gcactgtagc caacaagatt ggtgtctgtt ccctgcttga
214020gccaccctct caggccacca caaagctcag ttccgagaac ttccgtggca cgctcactag
214080tctggctttg ctgcccacga aacgccacct cctgctgggc tcggacaatg gcatcatccg
214140cctcctggca tagggccagc caggagttgg ctgagggcag ggcgagatga catctctcag
214200ggcccgctcc tcattcttga tctcgaagcc gattcttcta ggcaagcccc aggctctggc
214260tacccacatg gcctgctgtc tgggattgca cagctcctga atctccaaag ccttgaagtg
214320gcttcatgaa actcgggaga tactgttcct aaccagcaag aattggggca aggaaagcac
214380tgtgatcccc attgctcccc agttctgcct tctggattca catggggaca gggcagctcc
214440aggaaatgaa aggagttggg cctttgctca gccagcttcc tctagccacg ctctccttag
214500ctctgtttct cccttgggta ggaaactgct cctgtctagg gttctgatgg tactgggact
214560ccaggctcag gagggctggc caggacctac gactttcagg gcttggtctg gggttttagc
214620attcattcag ccaggtcttc agtatgggac cagaaaaaag gggatgtgag aacagggcta
214680gggaaggggt tatatgggcc cagctggtcc aggaatgaat ccatgccttg ccttggtacc
214740cctaaccaca gcgtttgtgc cttcagccgg ggaggcagcc cttgggacca gcatccctag
214800ggacaggagg cagcgggaat catctctgta tctcgggttc tgcccagggg atgggcagac
214860tctgccatct cttgagtgtt cgtttggaga agcctgagat gtggcccctg ctgccttctc
214920actagttgca gtctatgtaa ataaggtcaa taaattcttt ggaagagcca cggagctgag
214980tgaggctgtg ttgtgttttg ctttgcctag gctgggctca ggcagctctg cctcagcctc
215040ccaaggagct ggggaactgg tatatgtcac tgtatatgtc actgtgcctg gcttatggct
215100tggcttggct ttttttcaga tggtctcaag tgcctcaggt tggccttgat cttgggatga
215160ccttcctgct tgaaacagag tagtgggctt ataggcatga cccaccaggt ccaattttta
215220ttttttaaag gcattgattt ttatacgtgt atggttgttt tgcccacttg tacatatgca
215280caccatactt gtgtctggtc cctgcggagg tcagaagagg gcatcgggat cacctggaac
215340cgaagttaat gaatggttat gagccacatc tcgatgctga agattgaacc tggatccttt
215400gcaagagcag ccagtgttct tacccactga gccatctcta agccccacac ccagcttctt
215460ttgatacaag gtctggtagc tcaaacttga tatgcagccg aggaggttga cctggtattc
215520cctacctacc ctcttctctc taccttccaa gtgctgatat tatacatagg catggatagt
215580catgcccacc agtttgcctt gatggcacca gagtcaggaa agtccaaacc tggtagttgc
215640aaacacagca agagggtaga ggcagccatt gtcctctggc tgccttggat acagagcttc
215700tgggttgggt ggccttgggt cagttttccg aatggttcac ccttggggaa agggaacact
215760gctgaagagg tgggaccctg ggagggccgg cctccagctg ggtctctcca gccctcgcct
215820tggaacctag gctggaggga gccaaccagg atcctggact tgctacagtt aggtgaacag
215880gctcctgcag cctccccttc ccttgggtag ctgtggtggt ggtggtggtg gtggtggtgg
215940tggtggtggt ggtggtggtg gtgggggggg gggngnngnt
21598017473PRTMus sp. 17Met Lys Arg Ala Ser Ser Gly Gly Ser Arg Leu Leu
Ala Trp Val Leu1 5 10
15Trp Leu Gln Ala Trp Arg Val Ala Thr Pro Cys Pro Gly Ala Cys Val
20 25 30Cys Tyr Asn Glu Pro Lys Val
Thr Thr Ser Cys Pro Gln Gln Gly Leu 35 40
45Gln Ala Val Pro Thr Gly Ile Pro Ala Ser Ser Gln Arg Ile Phe
Leu 50 55 60His Gly Asn Arg Ile Ser
His Val Pro Ala Ala Ser Phe Gln Ser Cys65 70
75 80Arg Asn Leu Thr Ile Leu Trp Leu His Ser Asn
Ala Leu Ala Arg Ile 85 90
95Asp Ala Ala Ala Phe Thr Gly Leu Thr Leu Leu Glu Gln Leu Asp Leu
100 105 110Ser Asp Asn Ala Gln Leu
His Val Val Asp Pro Thr Thr Phe His Gly 115 120
125Leu Gly His Leu His Thr Leu His Leu Asp Arg Cys Gly Leu
Arg Glu 130 135 140Leu Gly Pro Gly Leu
Phe Arg Gly Leu Ala Ala Leu Gln Tyr Leu Tyr145 150
155 160Leu Gln Asp Asn Asn Leu Gln Ala Leu Pro
Asp Asn Thr Phe Arg Asp 165 170
175Leu Gly Asn Leu Thr His Leu Phe Leu His Gly Asn Arg Ile Pro Ser
180 185 190Val Pro Glu His Ala
Phe Arg Gly Leu His Ser Leu Asp Arg Leu Leu 195
200 205Leu His Gln Asn His Val Ala Arg Val His Pro His
Ala Phe Arg Asp 210 215 220Leu Gly Arg
Leu Met Thr Leu Tyr Leu Phe Ala Asn Asn Leu Ser Met225
230 235 240Leu Pro Ala Glu Val Leu Met
Pro Leu Arg Ser Leu Gln Tyr Leu Arg 245
250 255Leu Asn Asp Asn Pro Trp Val Cys Asp Cys Arg Ala
Arg Pro Leu Trp 260 265 270Ala
Trp Leu Gln Lys Phe Arg Gly Ser Ser Ser Glu Val Pro Cys Asn 275
280 285Leu Pro Gln Arg Leu Ala Asp Arg Asp
Leu Lys Arg Leu Ala Ala Ser 290 295
300Asp Leu Glu Gly Cys Ala Val Ala Ser Gly Pro Phe Arg Pro Ile Gln305
310 315 320Thr Ser Gln Leu
Thr Asp Glu Glu Leu Leu Ser Leu Pro Lys Cys Cys 325
330 335Gln Pro Asp Ala Ala Asp Lys Ala Ser Val
Leu Glu Pro Gly Arg Pro 340 345
350Ala Ser Ala Gly Asn Ala Leu Lys Gly Arg Val Pro Pro Gly Asp Thr
355 360 365Pro Pro Gly Asn Gly Ser Gly
Pro Arg His Ile Asn Asp Ser Pro Phe 370 375
380Gly Thr Leu Pro Ser Ser Ala Glu Pro Pro Leu Thr Ala Leu Arg
Pro385 390 395 400Gly Gly
Ser Glu Pro Pro Gly Leu Pro Thr Thr Gly Pro Arg Arg Arg
405 410 415Pro Gly Cys Ser Arg Lys Asn
Arg Thr Arg Ser His Cys Arg Leu Gly 420 425
430Gln Ala Gly Ser Gly Ala Ser Gly Thr Gly Asp Ala Glu Gly
Ser Gly 435 440 445Ala Leu Pro Ala
Leu Ala Cys Ser Leu Ala Pro Leu Gly Leu Ala Leu 450
455 460Val Leu Trp Thr Val Leu Gly Pro Cys465
47018283PRTArtificial SequenceConsensus sequence 18Cys Pro Xaa Xaa
Cys Xaa Cys Tyr Xaa Xaa Pro Xaa Xaa Thr Xaa Ser1 5
10 15Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Xaa Xaa Xaa Pro Xaa Xaa 20 25
30Xaa Xaa Arg Xaa Phe Leu Xaa Xaa Asn Xaa Ile Xaa Xaa Xaa Xaa Xaa
35 40 45Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Leu Trp Xaa Xaa Ser 50 55
60Asn Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa65
70 75 80Leu Glu Xaa Leu Asp
Leu Xaa Asp Asn Xaa Xaa Leu Xaa Xaa Xaa Xaa 85
90 95Pro Xaa Thr Phe Xaa Gly Leu Xaa Xaa Leu Xaa
Xaa Leu Xaa Leu Xaa 100 105
110Xaa Cys Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Phe Xaa Gly Leu Xaa
115 120 125Xaa Leu Gln Tyr Leu Tyr Leu
Gln Xaa Asn Xaa Xaa Xaa Xaa Leu Xaa 130 135
140Asp Xaa Xaa Phe Xaa Asp Leu Xaa Asn Leu Xaa His Leu Phe Leu
His145 150 155 160Gly Asn
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Arg Gly Leu Xaa
165 170 175Xaa Leu Asp Arg Leu Leu Leu
His Xaa Asn Xaa Xaa Xaa Xaa Val His 180 185
190Xaa Xaa Ala Phe Xaa Xaa Leu Xaa Arg Leu Xaa Xaa Leu Xaa
Leu Phe 195 200 205Xaa Asn Xaa Leu
Xaa Xaa Leu Xaa Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa 210
215 220Xaa Leu Xaa Xaa Leu Arg Leu Asn Xaa Asn Xaa Trp
Xaa Cys Xaa Cys225 230 235
240Arg Xaa Arg Xaa Leu Trp Xaa Trp Xaa Xaa Xaa Xaa Arg Xaa Ser Ser
245 250 255Ser Xaa Val Xaa Cys
Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Asp Leu 260
265 270Xaa Xaa Leu Xaa Xaa Xaa Asp Xaa Xaa Xaa Cys
275 2801950PRTArtificial SequenceConsensus sequence
19Asn Xaa Trp Xaa Cys Xaa Cys Arg Ala Arg Xaa Leu Trp Xaa Trp Xaa1
5 10 15Xaa Xaa Xaa Arg Xaa Ser
Ser Ser Xaa Val Xaa Cys Xaa Xaa Pro Xaa 20 25
30Xaa Xaa Xaa Xaa Xaa Asp Leu Xaa Xaa Leu Xaa Xaa Xaa
Asp Xaa Xaa 35 40 45Xaa Cys
50
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