Patent application title: DELTA-5 DESATURASES AND THEIR USE IN MAKING POLYUNSATURATED FATTY ACIDS
Inventors:
Howard G. Damude (Hockessin, DE, US)
Quinn Qun Zhu (West Chester, PA, US)
Assignees:
E. I. DU PONT DE NEMOURS AND COMPANY
IPC8 Class: AC12N902FI
USPC Class:
Class name:
Publication date: 2015-06-25
Patent application number: 20150175977
Abstract:
Isolated nucleic acid fragments and recombinant constructs comprising
such fragments encoding delta-5 desaturases along with a method of making
long-chain polyunsaturated fatty acids (PUFAs) using these delta-5
desaturases in plants are disclosed.Claims:
1. (canceled)
2. An isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:13.
3. The isolated polypeptide of claim 2, wherein the amino acid sequence has at least 90% sequence identity, based on the Clustal V method of alignment when compared to SEQ ID NO:13.
4. The isolated polypeptide of claim 3, wherein the amino acid sequence has at least 95% sequence identity, based on the Clustal V method of alignment when compared to SEQ ID NO:13.
5-38. (canceled)
Description:
[0001] This application claims the benefit of U.S. Provisional Application
No. 60/915,733, filed May 3, 2007, the entire content of which is herein
incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of biotechnology, in particular, this pertains to polynucleotide sequences encoding delta-5 desaturases and the use of these desaturases in making long-chain polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
[0003] The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: "essential" fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further elongation and desaturation of linoleic acid (LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg et al., Amer. J. Clin. Nutr. 28:958-966 (1975); Dyerberg et al., Lancet. 2(8081):117-119 (1978); Shimokawa, H., World Rev. Nutr. Diet 88:100-108 (2001); von Schacky et al., World Rev. Nutr. Diet 88:90-99 (2001)). Numerous other studies document wide-ranging health benefits conferred by administration of omega-3 and/or omega-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
[0004] Today, a variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production via numerous divergent efforts. Although the natural PUFA-producing abilities of the host organisms are sometimes essential to a given methodology, genetic engineering has also proven that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs. Whether this effect is the result of natural abilities or recombinant technology, production of arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) all require expression of either the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid (EDA; 20:2 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3)) or the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of γ-linoleic acid (GLA; 18:3 ω-6) and/or stearidonic acid (STA; 18:4 ω-3) (FIG. 1). A delta-6 elongase is also known as a C18/20 elongase.
[0005] The delta-8 desaturase enzymes identified thus far have the ability to convert both EDA to dihomo-γ-linolenic acid (DGLA; 20:3) and ETrA to eicosatetraenoic acid (ETA; 20:4) (wherein ARA are EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a delta-5 desaturase, while DHA synthesis requires subsequent expression of an additional C20/22 elongase and a delta-4 desaturase).
[0006] Based on the role delta-8 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has been effort to identify and characterize these enzymes. Initial efforts on the isolation and characterization of delta-8 desaturases from Euglena gracilis; and, several sequence variations within the Euglena gracilis delta-8 desaturase have been reported (see, e.g., Wallis et al., Arch. Biochem. and Biophys. 365(2):307-316 (1999); PCT Publication No. WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO 2004/057001). Also, Applicants' Assignee's co-pending applications having U.S. application Ser. Nos. 11/166,003 and 11/166,993 filed Jun. 24, 2005 (Attorney Docket Nos. BB-1547 and CL-3150, respectively (PCT Publication Nos. WO 2006/012325 and WO 2006/012326; both published Feb. 2, 2006)) discloses amino acid and nucleic acid sequences for a Euglena gracilis delta-8 desaturase.
[0007] More recently, PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885). Sayanova et al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 delta-8 desaturase. Also, Applicants' Assignee's co-pending application having U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007; Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S. patent application Ser. No. 11/876,115 (filed Oct. 22, 2007; Attorney Docket No. BB-1574) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf--gymnastica CCMP1594.
[0008] Based on the utility of expressing delta-8 desaturases in conjunction with delta-9 elongases, there has also been effort to identify and characterize delta-9 elongases from various sources. Most delta-9 elongase enzymes identified so far have the ability to convert both LA to EDA and ALA to ETrA (wherein DGLA and ETA are subsequently synthesized from EDA and ETrA, respectively, following reaction with a Δ8 desaturase; ARA and EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a Δ5 desaturase; and, DHA synthesis requires subsequent expression of an additional C20/22 elongase and a Δ4 desaturase). A delta-9 elongase from Isochrysis galbana has been publicly available (described in GenBank Accession No. AAL37626, as well as PCT Publication No. WO 02/077213). Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562), discloses a delta-9 elongase from Euglena gracilis. Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/601,564 filed Nov. 16, 2006 (Attorney Docket No. CL-3600), discloses a delta-9 elongase from Eutreptiella sp. CCMP389.
[0009] Most delta-5 desaturase enzymes identified so far have the primary ability to convert dihomo-gamma-linolenic acid [20:3, DGLA] to ARA, with secondary activity in converting eicosatetraenoic acid [20:4, ETA] to EPA (where DHA is subsequently synthesized from EPA following reaction with an additional C20/22 elongase and a delta-4 desaturase). The delta-5 desaturase has a role in both the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of gamma-linoleic acid ["GLA"; 18:3 ω-6] and/or stearidonic acid ["STA"; 18:4 ω-3]) and the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid ["EDA"; 20:2 ω-6] and/or eicosatrienoic acid ["ETrA"; 20:3 ω-3]) (FIG. 1).
[0010] Furthermore, based on the role delta-5 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has also been an effort to identify and characterize these enzymes from various sources. As such, delta-5 desaturases have been disclosed in both the open literature (e.g., GenBank Accession Nos. AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654 and AB022097) and the patent literature (e.g., U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183).
[0011] Applicants' Assignee has a number of patent applications concerning the production of PUFAs in oleaginous yeasts (i.e., Yarrowia lipolytica), including: PCT Publication Nos. WO 2004/101757 and WO 2004/101753 (both published Nov. 25, 2004); U.S. application Ser. No. 11/265,761 (filed Nov. 2, 2005); U.S. application Ser. No. 11/264,784 (filed Nov. 1, 2005); and U.S. application Ser. No. 11/264,737 (filed Nov. 1, 2005).
[0012] Relatedly, PCT Publication No. WO 2004/071467 (published Aug. 26, 2004; Attorney Docket No. BB-1538) concerns the production of PUFAs in plants, while PCT Publication No. WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants; both are Applicants' Assignee's copending applications.
SUMMARY OF THE INVENTION
[0013] The present invention concerns an isolated polynucleotide comprising:
[0014] (a) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:13;
[0015] (b) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:12;
[0016] (c) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:12; or
[0017] (d) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
[0018] In a second embodiment, the invention concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the invention operably linked to at least one regulatory sequence.
[0019] In a third embodiment, the invention concerns a plant cell comprising in its genome the recombinant DNA construct of the invention.
[0020] In a fourth embodiment, the invention concerns a method for transforming a plant cell, comprising transforming a plant cell with a recombinant construct of the invention or an isolated polynucleotide of the invention and selecting those plant cells transformed with the recombinant construct or the isolated polynucleotide.
[0021] In a fifth embodiment, the invention concerns transgenic seed comprising in its genome the recombinant construct of the invention or a transgenic seed obtained from a plant made by a method of the invention. Also of interest is oil or by-products obtained from such transgenic seeds.
[0022] In a sixth embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising:
[0023] (a) transforming a plant cell with the recombinant construct of the invention; and
[0024] (b) selecting those transformed plant cells that make long-chain polyunsaturated fatty acids.
[0025] In a seventh embodiment, the invention concerns a method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising:
[0026] (a) transforming an oilseed plant cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase;
[0027] (b) regenerating an oilseed plant from the transformed cell of step (a); and
[0028] (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.
[0029] In an eighth embodiment, the invention concerns an oilseed plant comprising in its genome the recombinant construct of the invention. Suitable oilseed plants include, but are not limited to, soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
[0030] In a ninth embodiment, the invention concerns an oilseed plant comprising:
[0031] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and
[0032] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase.
[0033] Also of interest are transgenic seeds obtained from such oilseed plants as well as oil or by-products obtained from these transgenic seeds. A preferred by-product is lecithin.
[0034] In a tenth embodiment, the invention concerns food or feed incorporating an oil or seed of the invention or food or feed comprising an ingredient derived from the processing of the seeds.
[0035] In an eleventh embodiment, the invention concerns progeny plants obtained from obtained from a plant made by the method of the invention or an oilseed plant of the invention.
Biological Deposits
[0036] The following plasmid has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bears the following designation, Accession Number and date of deposit (Table 1).
TABLE-US-00001 TABLE 1 ATCC Deposit Plasmid Accession Number Date of Deposit pKR72 PTA-6019 May 28, 2004
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0037] The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.
[0038] FIG. 1 is a representative omega-3 and omega-6 fatty acid pathway providing for the conversion of myristic acid through various intermediates to DHA.
[0039] FIG. 2A is a schematic of EgD5.
[0040] FIG. 2B is a map of plasmid pZUF17 (SEQ ID NO:7).
[0041] FIG. 2C is a map of plasmid pDMW267 (SEQ ID NO:8).
[0042] FIG. 3 is a map of plasmid pY115 (SEQ ID NO:19).
[0043] FIG. 4 is a map of plasmid pY159 (SEQ ID NO:23).
[0044] FIG. 5 is a map of plasmid pY169 (SEQ ID NO:24).
[0045] FIG. 6 are the fatty acid profiles for Yarrowia lipolytica expressing pY169.
[0046] FIG. 7 is a map of pKR1153 (SEQ ID NO:44).
[0047] FIGS. 8A, 8B and 8C shows a comparison of the nucleotide sequences of EaD5 (same as EaD5Des1) (SEQ ID NO:12) and EaD5S (SEQ ID NO:45).
[0048] FIG. 9 is a map of plasmid pEaD5S (SEQ ID NO:46).
[0049] FIG. 10 shows a chromatogram of the lipid profile of an Euglena anabaena cell extract as described in the Examples.
[0050] FIG. 11 shows ten events having the highest average correct delta-5 desaturase activities (average of the 5 somatic soybean embryos analyzed) from approximately 30 events transformed with pKR1153 (Experiment MSE2140). Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA and EPA. Fatty acid compositions for an individual embryo were expressed as the weight percent (wt. %) of total fatty acids and the average fatty acid composition is an average of six individual embryos for each event. The activity of the delta-5 desaturase is expressed as percent delta-5 desaturation ("% delta-5 desat"), calculated according to the following formula: ([product]/[substrate+product])*100. More specifically, the percent delta-5 desaturation was determined as: ([ARA+EPA]/[DGLA+ETA+ARA+EPA])*100.
[0051] The sequence descriptions summarize the Sequences Listing attached hereto. The Sequence Listing contains one letter codes for nucleotide sequence characters and the single and three letter codes for amino acids as defined in the IUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219(2):345-373 (1984).
[0052] SEQ ID NO:1 is the cDNA sequence of the Euglena anabaena delta-5 desaturase 1 (EaD5Des1).
[0053] SEQ ID NO:2 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase coding sequence (EgD5).
[0054] SEQ ID NO:3 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL794.
[0055] SEQ ID NO:4 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL797.
[0056] SEQ ID NO:5 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL796.
[0057] SEQ ID NO:6 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL795.
[0058] SEQ ID NO:7 is the nucleotide sequence of plasmid pZUF17.
[0059] SEQ ID NO:8 is the nucleotide sequence of plasmid pDMW367.
[0060] SEQ ID NO:9 is the nucleotide sequence of the M13F universal primer.
[0061] SEQ ID NO:10 is the nucleotide sequence of M13-28Rev.
[0062] SEQ ID NO:11 is the nucleotide sequence of plasmid pLF119.
[0063] SEQ ID NO:12 is the nucleotide sequence of the Euglena anabaena delta-5 desaturase 1 coding sequence (EaD5Des1).
[0064] SEQ ID NO:13 is the amino acid sequence of the Euglena anabaena delta-5 desaturase 1 (EaD5Des1).
[0065] SEQ ID NO:14 is the amino acid sequence of the Thalassiosira pseudonana delta-8 fatty acid desaturase.
[0066] SEQ ID NO:15 is the amino acid sequence of the Phaeodactylum tricornutum delta-5 fatty acid desaturase.
[0067] SEQ ID NO:16 is the amino acid sequence of the Euglena gracilis delta-5 desaturase (EgD5).
[0068] SEQ ID NO:17 is the nucleotide sequence of plasmid pDMW263.
[0069] SEQ ID NO:18 is the nucleotide sequence of plasmid pDMW237.
[0070] SEQ ID NO:19 is the nucleotide sequence of plasmid pY115.
[0071] SEQ ID NO:20 is the nucleotide sequence of oligonucleotide oYFBA1.
[0072] SEQ ID NO:21 is the nucleotide sequence of oligonucleotide oYFBA1-6.
[0073] SEQ ID NO:22 is the nucleotide sequence of plasmid pY158.
[0074] SEQ ID NO:23 is the nucleotide sequence of plasmid pY159.
[0075] SEQ ID NO:24 is the nucleotide sequence of plasmid pY169.
[0076] SEQ ID NO:25 is the nucleotide sequence of the Euglena gracilis delta-9 elongase (EgD9e).
[0077] SEQ ID NO:26 is the nucleotide sequence of the Euglena gracilis delta-8 desaturase (EgD8).
[0078] SEQ ID NO:27 is the nucleotide sequence of the Euglena gracilis elongase sense oligonucleotide oEugEL1-1.
[0079] SEQ ID NO:28 is the nucleotide sequence of the Euglena gracilis elongase anti-sense oligonucleotide oEugEL1-2.
[0080] SEQ ID NO:29 is the nucleotide sequence of plasmid pKR906.
[0081] SEQ ID NO:30 is the nucleotide sequence of plasmid pKR72.
[0082] SEQ ID NO:31 is the nucleotide sequence of plasmid KS102.
[0083] SEQ ID NO:32 is the nucleotide sequence of plasmid pKR197.
[0084] SEQ ID NO:33 is the nucleotide sequence of plasmid pKR911.
[0085] SEQ ID NO:34 is the nucleotide sequence of plasmid pKR680.
[0086] SEQ ID NO:35 is the nucleotide sequence of plasmid pKR913.
[0087] SEQ ID NO:36 is the nucleotide sequence of oligonucleotide oEAd5-1-1.
[0088] SEQ ID NO:37 is the nucleotide sequence of oligonucleotide oEAd5-1-2.
[0089] SEQ ID NO:38 is the nucleotide sequence of plasmid pKR1136.
[0090] SEQ ID NO:39 is the nucleotide sequence of plasmid pKR767.
[0091] SEQ ID NO:40 is the nucleotide sequence of the Mortierella alpine delta-5 desaturase coding sequence (MaD5).
[0092] SEQ ID NO:41 is the nucleotide sequence of plasmid pKR974.
[0093] SEQ ID NO:42 is the nucleotide sequence of the Saprolegnia diclina delta-5 desaturase coding sequence (SdD5).
[0094] SEQ ID NO:43 is the nucleotide sequence of plasmid pKR1139.
[0095] SEQ ID NO:44 is the nucleotide sequence of plasmid pKR1153.
[0096] SEQ ID NO:45 is the nucleotide sequence of the codon-optimized Euglena anabaena delta-5 desaturase gene (EaD5S).
[0097] SEQ ID NO:46 is the nucleotide sequence of plasmid pEaD5S.
DETAILED DESCRIPTION OF THE INVENTION
[0098] The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.
[0099] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes a plurality of such plants, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.
[0100] The present invention relates to delta-5 desaturase enzymes and nucleic acid for encoding the same isolated from Euglena anabaena. These are useful for, inter alia, for the manipulation of biochemical pathways for the production of PUFAs. Thus, the subject invention finds many applications.
[0101] PUFAs, or derivatives thereof, made by the methodology disclosed herein can be used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use (human or veterinary). In this case, the PUFAs are generally administered orally but can be administered by any route by which they may be successfully absorbed, e.g., parenterally (e.g., subcutaneously, intramuscularly or intravenously), rectally, vaginally or topically (e.g., as a skin ointment or lotion).
[0102] Supplementation of humans or animals with PUFAs produced by recombinant means can result in increased levels of the added PUFAs, as well as their metabolic progeny. For example, treatment with EPA can result not only in increased levels of EPA, but also downstream products of EPA such as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes). Complex regulatory mechanisms can make it desirable to combine various PUFAs, or add different conjugates of PUFAs, in order to prevent, control or overcome such mechanisms to achieve the desired levels of specific PUFAs in an individual.
[0103] In the context of this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
[0104] "Open reading frame" is abbreviated ORF.
[0105] "Polymerase chain reaction" is abbreviated PCR.
[0106] "American Type Culture Collection" is abbreviated ATCC.
[0107] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).
[0108] "Triacylglycerols" are abbreviated TAGs.
[0109] The term "fatty acids" refers to long-chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C12 to C22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C16 and C22. Additional details concerning the differentiation between "saturated fatty acids" versus "unsaturated fatty acids", "monounsaturated fatty acids" versus "polyunsaturated fatty acids" (or "PUFAs"), and "omega-6 fatty acids" (ω-6 or n-6) versus "omega-3 fatty acids" (ω-3 or n-3) are provided in PCT Publication No. WO 2004/101757.
[0110] Fatty acids are described herein by a simple notation system of "X:Y", wherein X is number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds. The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the "c" affix for the cis-configuration of the double bond (e.g., palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1, 6c), LA (18:2, 9c, 12c), GLA (18:3, 6c, 9c, 12c) and ALA (18:3, 9c, 12c, 15c)). Unless otherwise specified, 18:1, 18:2 and 18:3 refer to oleic, LA and ALA fatty acids, respectively. If not specifically written as otherwise, double bonds are assumed to be of the cis configuration. For instance, the double bonds in 18:2 (9,12) would be assumed to be in the cis configuration.
[0111] Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 2. In the column titled "Shorthand Notation", the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the table summarizes the common names of omega-3 and omega-6 fatty acids and their precursors, the abbreviations that will be used throughout the remainder of the specification, and each compounds' chemical name.
TABLE-US-00002 TABLE 2 Nomenclature of Polyunsaturated Fatty Acids and Precursors Common Shorthand Name Abbreviation Chemical Name Notation myristic -- tetradecanoic 14:0 palmitic PA hexadecanoic 16:0 palmitoleic -- 9-hexadecenoic 16:1 stearic -- octadecanoic 18:0 oleic -- cis-9-octadecenoic 18:1 linoleic LA cis-9,12-octadecadienoic 18:2 ω-6 gamma- GLA cis-6,9,12- 18:3 ω-6 linolenic octadecatrienoic eicosadienoic EDA cis-11,14-eicosadienoic 20:2 ω-6 dihomo- DGLA cis-8,11,14-eicosatrienoic 20:3 ω-6 gamma- linolenic sciadonic SCI cis-5,11,14-eicosatrienoic 20:3b ω-6 arachidonic ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoic alpha-linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoic stearidonic STA cis-6,9,12,15- 18:4 ω-3 octadecatetraenoic eicosatrienoic ETrA or ERA cis-11,14,17- 20:3 ω-3 eicosatrienoic eicosa- ETA cis-8,11,14,17- 20:4 ω-3 tetraenoic eicosatetraenoic juniperonic JUP cis-5,11,14,17- 20:4b ω-3 eicosatrienoic eicosa- EPA cis-5,8,11,14,17- 20:5 ω-3 pentaenoic eicosapentaenoic docosa- DPA cis-7,10,13,16,19- 22:5 ω-3 pentaenoic docosapentaenoic docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3 hexaenoic docosahexaenoic
[0112] A metabolic pathway, or biosynthetic pathway, in a biochemical sense, can be regarded as a series of chemical reactions occurring within a cell, catalyzed by enzymes, to achieve either the formation of a metabolic product to be used or stored by the cell, or the initiation of another metabolic pathway (then called a flux generating step). Many of these pathways are elaborate, and involve a step by step modification of the initial substance to shape it into a product having the exact chemical structure desired.
[0113] The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see PCT Publication No. WO 2006/052870). Simplistically, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulim membrane. More specifically, "PUFA biosynthetic pathway enzyme" refers to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase.
[0114] The term "omega-3/omega-6 fatty acid biosynthetic pathway" refers to a set of genes which, when expressed under the appropriate conditions encode enzymes that catalyze the production of either or both omega-3 and omega-6 fatty acids. Typically the genes involved in the omega-3/omega-6 fatty acid biosynthetic pathway encode PUFA biosynthetic pathway enzymes. A representative pathway is illustrated in FIG. 1, providing for the conversion of myristic acid through various intermediates to DHA, which demonstrates how both omega-3 and omega-6 fatty acids may be produced from a common source. The pathway is naturally divided into two portions where one portion will generate omega-3 fatty acids and the other portion, omega-6 fatty acids.
[0115] The term "functional" as used herein in context with the omega-3/omega-6 fatty acid biosynthetic pathway means that some (or all of) the genes in the pathway express active enzymes, resulting in in vivo catalysis or substrate conversion. It should be understood that "omega-3/omega-6 fatty acid biosynthetic pathway" or "functional omega-3/omega-6 fatty acid biosynthetic pathway" does not imply that all the PUFA biosynthetic pathway enzyme genes are required, as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.
[0116] The term "delta-9 elongase/delta-8 desaturase pathway" refers to a biosynthetic pathway for production of long-chain PUFAs. This pathway, at a minimum, comprises a delta-9 elongase and a delta-8 desaturase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized. This pathway may be advantageous in some embodiments, as the biosynthesis of GLA and/or STA is excluded.
[0117] The term "intermediate fatty acid" refers to any fatty acid produced in a fatty acid metabolic pathway that can be further converted to an intended product fatty acid in this pathway by the action of other metabolic pathway enzymes. For instance, when EPA is produced using the delta-9 elongase/delta-8 desaturase pathway, EDA, ETrA, DGLA, ETA and ARA can be produced and are considered "intermediate fatty acids" since these fatty acids can be further converted to EPA via action of other metabolic pathway enzymes.
[0118] The term "by-product fatty acid" refers to any fatty acid produced in a fatty acid metabolic pathway that is not the intended fatty acid product of the pathway nor an "intermediate fatty acid" of the pathway. For instance, when EPA is produced using the delta-9 elongase/delta-8 desaturase pathway, sciadonic acid (SCI) and juniperonic acid (JUP) also can be produced by the action of a delta-5 desaturase on either EDA or ETrA, respectively. They are considered to be "by-product fatty acids" since neither can be further converted to EPA by the action of other metabolic pathway enzymes.
[0119] The terms "triacylglycerol", "oil" and "TAGs" refer to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule (and such terms will be used interchangeably throughout the present disclosure herein). Such oils can contain long-chain PUFAs, as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. Thus, "oil biosynthesis" generically refers to the synthesis of TAGs in the cell.
[0120] "Percent (%) PUFAs in the total lipid and oil fractions" refers to the percent of PUFAs relative to the total fatty acids in those fractions. The term "total lipid fraction" or "lipid fraction" both refer to the sum of all lipids (i.e., neutral and polar) within an oleaginous organism, thus including those lipids that are located in the phosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fraction and triacylglycerol (TAG or oil) fraction. However, the terms "lipid" and "oil" will be used interchangeably throughout the specification.
[0121] The terms "conversion efficiency" and "percent substrate conversion" refer to the efficiency by which a particular enzyme (e.g., a desaturase) can convert substrate to product. The conversion efficiency is measured according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it.
[0122] "Desaturase" is a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a fatty acid or precursor of interest. Despite use of the omega-reference system throughout the specification to refer to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the substrate using the delta-system. Of particular interest herein are delta-5 desaturases that will desaturate a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of DGLA to ARA and/or ETA to EPA. Other useful fatty acid desaturases include, for example: (1) delta-8 desaturases that catalyze the conversion of EDA to DGLA and/or ERA to ETA; (2) delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; (3) delta-4 desaturases that catalyze the conversion of DPA to DHA; (4) delta-12 desaturases that catalyze the conversion of oleic acid to LA; (5) delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; (6) delta-17 desaturases that catalyze the conversion of ARA to EPA and/or DGLA to ETA; and (7) delta-9 desaturases that catalyze the conversion of palmitic acid to palmitoleic acid (16:1) and/or stearic acid to oleic acid (18:1). In the art, delta-15 and delta-17 desaturases are also occasionally referred to as "omega-3 desaturases", "w-3 desaturases", and/or "ω-3 desaturases", based on their ability to convert omega-6 fatty acids into their omega-3 counterparts (e.g., conversion of LA into ALA and ARA into EPA, respectively). In some embodiments, it is most desirable to empirically determine the specificity of a particular fatty acid desaturase by transforming a suitable host with the gene for the fatty acid desaturase and determining its effect on the fatty acid profile of the host.
[0123] The term "delta-5 desaturase" refers to an enzyme that desaturates a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule. Preferably, a delta-5 desaturase converts dihomo-gamma-linolenic acid [20:3, DGLA] to arachidonic acid [20:4, ARA] or converts eicosatetraenoic acid [20:4, ETA] to eicosapentaenoic acid [20:5, EPA].
[0124] For the purposes herein, the terms "EaD5Des1" or "EaD5" refers to a delta-5 desaturase enzyme (SEQ ID NO:13) isolated from Euglena anabaena, encoded by SEQ ID NO:12 herein. Likewise, the term "EaD5S" (SEQ ID NO:45) refers to a delta-5 desaturase codon-optimized for expression in Yarrowia lipolytica.
[0125] For the purposes herein, the term "IgD9e" refers to a delta-9 elongase (SEQ ID NO:15) (NCBI Accession No. AAL37626 [GI 17226123], locus AAL37626, CDS AF390174; GenBank Accession No. AF390174) isolated from Isochrysis galbana. In contrast, the term "IgD9eS" refers to a synthetic (codon-optimized) delta-9 elongase derived from the DNA sequence of the Isochrysis galbana delta-9 elongase which can be used for expression in Yarrowia lipolytica.
[0126] Similarly for the purposes herein, the term "EgD9e" refers to a delta-9 elongase isolated from Euglena gracilis. EgD9e is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562).
[0127] Similarly, the term "EgD8" refers to a delta-8 desaturase enzyme isolated from Euglena gracilis. EgD8 is 100% identical and functionally equivalent to "Eg5", as described in PCT Publication Nos. WO 2006/012325 and WO 2006/012326 (SEQ ID NO:2 of U.S. Publication No. 20050287652-A1).
[0128] The term "elongase system" refers to a suite of four enzymes that are responsible for elongation of a fatty acid carbon chain to produce a fatty acid that is two carbons longer than the fatty acid substrate that the elongase system acts upon. More specifically, the process of elongation occurs in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., Plant Cell 8:281-292 (1996)). In the first step, which has been found to be both substrate-specific and also rate-limiting, malonyl-CoA is condensed with a long-chain acyl-CoA to yield carbon dioxide (CO2) and a β-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA, LA to EDA, ALA to ETrA and EPA to DPA.
[0129] For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-CoA and long-chain acyl-CoA to β-ketoacyl-CoA) will be referred to generically as an "elongase". In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree of unsaturation. Accordingly, elongases can have different specificities. For example, a C14/16 elongase will utilize a C14 substrate (e.g., myristic acid), a C16/18 elongase will utilize a C16 substrate (e.g., palmitate), a C18/20 elongase will utilize a C18 substrate (e.g., GLA, STA) and a C20/22 elongase will utilize a C20 substrate (e.g., EPA). Similarly, a "delta-9 elongase" may be able to catalyze the conversion of LA to EDA and/or ALA to ETrA. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions. Thus, for example, a delta-9 elongase may also act as a C16/18 elongase, C18/20 elongase and/or C20/22 elongase and may have alternate, but not preferred, specificities for delta-5 and delta-6 fatty acids such as EPA and/or GLA, respectively.
[0130] The term "conservative amino acid substitution" refers to a substitution of an amino acid residue in a given protein with another amino acid, without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene that result in the production of a chemically equivalent amino acid at a given site (but that do not affect the structural and functional properties of the encoded, folded protein) are common. For the purposes of the present invention, "conservative amino acid substitutions" are defined as exchanges within one of the following five groups:
[0131] 1. small aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [T] (Pro [P], Gly [G]);
[0132] 2. polar, negatively charged residues and their amides: Asp [D], Asn [N], Glu [E], Gln [Q];
[0133] 3. polar, positively charged residues: His [H], Arg [R], Lys [K];
[0134] 4. large aliphatic, nonpolar residues: Met [M], Leu [L], Ile [I], Val [V] (Cys [C]); and,
[0135] 5. large aromatic residues: Phe [F], Tyr [Y], Trp [W]. Conservative amino acid substitutions generally maintain: 1) the structure of the polypeptide backbone in the area of the substitution; 2) the charge or hydrophobicity of the molecule at the target site; or 3) the bulk of the side chain. Additionally, in many cases, alterations of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
[0136] As used herein, "nucleic acid" means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deosycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridlate, "T" for deosythymidylate, "R" for purines (A or G), "Y" for pyrimidiens (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
[0137] The terms "subfragment that is functionally equivalent" and "functionally equivalent subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of chimeric genes to produce the desired phenotype in a transformed plant. Chimeric genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.
[0138] The term "conserved domain" or "motif" means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or "signatures", to determine if a protein with a newly determined sequence belongs to a previously identified protein family.
[0139] The terms "homology", "homologous", "substantially similar" and "corresponding substantially" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
[0140] Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
[0141] The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
[0142] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
[0143] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.
[0144] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120, or 240 minutes.
[0145] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
[0146] Thus, "percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. These identities can be determined using any of the programs described herein.
[0147] Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized.
[0148] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191) and found in the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.
[0149] "BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters.
[0150] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. Indeed, any integer amino acid identity from 50% to 100% may be useful in describing the present invention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, of interest is any full-length or partial complement of this isolated nucleotide fragment.
[0151] "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.
[0152] The term "genome" as it applies to a plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.
[0153] A "codon-optimized gene" is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
[0154] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ that plant is heterozygous at that locus.
[0155] "Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to: promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
[0156] "Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B. Biochemistry of Plants 15:1-82 (1989).
[0157] "Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D., Mol. Biotechnol. 3:225-236 (1995)).
[0158] "3' non-coding sequences", "transcription terminator" or "termination sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell 1:671-680 (1989).
[0159] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. A RNA transcript is referred to as the mature RNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a DNA that is complementary to, and synthesized from, a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.
[0160] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.
[0161] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods are well known to those skilled in the art and are described infra.
[0162] "PCR" or "polymerase chain reaction" is a technique for the synthesis of large quantities of specific DNA segments and consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double-stranded DNA is heat denatured, the two primers complementary to the 3' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a "cycle".
[0163] The term "recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
[0164] The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host (i.e., to a discrete nucleic acid fragment into which a nucleic acid sequence or fragment can be moved.)
[0165] The terms "recombinant construct", "expression construct", "chimeric construct", "construct", and "recombinant DNA construct" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.
[0166] The term "expression", as used herein, refers to the production of a functional end-product (e.g., a mRNA or a protein [either precursor or mature]).
[0167] The term "introduced" means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
[0168] "Mature" protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed). "Precursor" protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.
[0169] "Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.
[0170] As used herein, "transgenic" refers to a plant or a cell which comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression construct. Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
[0171] "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020). Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808 (2000)). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. More recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication No. WO 99/53050, published Oct. 21, 1999; PCT Publication No. WO 02/00904, published Jan. 3, 2002). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or "silencing", of proximal mRNA encoding sequences (PCT Publication No. WO 98/36083, published Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although genetic evidence has begun to unravel this complex situation (Elmayan et al., Plant Cell 10:1747-1757 (1998)).
[0172] The term "oleaginous" refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). A class of plants identified as oleaginous are commonly referred to as "oilseed" plants. Examples of oilseed plants include, but are not limited to: soybean (Glycine and Soja sp.), flax (Linum sp.), rapeseed (Brassica sp.), maize, cotton, safflower (Carthamus sp.) and sunflower (Helianthus sp.).
[0173] Within oleaginous microorganisms the cellular oil or TAG content generally follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)). The term "oleaginous yeast" refers to those microorganisms classified as yeasts that make oil. It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
[0174] The term "Euglenophyceae" refers to a group of unicellular colorless or photosynthetic flagellates ("euglenoids") found living in freshwater, marine, soil, and parasitic environments. The class is characterized by solitary unicells, wherein most are free-swimming and have two flagella (one of which may be nonemergent) arising from an anterior invagination known as a reservoir. Photosynthetic euglenoids contain one to many grass-green chloroplasts, which vary from minute disks to expanded plates or ribbons. Colorless euglenoids depend on osmotrophy or phagotrophy for nutrient assimilation. About 1000 species have been described and classified into about 40 genera and 6 orders. Examples of Euglenophyceae include, but are no means limited to, the following genera: Euglena, Eutreptiella and Tetruetreptia.
[0175] The term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. "Progeny" comprises any subsequent generation of a plant.
An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols
[0176] In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium. This process, leading to the de novo synthesis of free palmitate (16:0) in oleaginous microorganisms, is described in detail in PCT Publication No. WO 2004/101757. Palmitate is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases (FIG. 1).
[0177] TAGs (the primary storage unit for fatty acids) are formed by a series of reactions that involve: (1) the esterification of one molecule of acyl-CoA to glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; (2) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); (3) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and (4) the addition of a third fatty acid by the action of an acyltransferase to form TAG. A wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids.
Biosynthesis of Omega Fatty Acids
[0178] The metabolic process wherein oleic acid is converted to long chain omega-3/omega-6 fatty acids involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds. This requires a series of special desaturation and elongation enzymes present in the endoplasmic reticulim membrane. However, as seen in FIG. 1 and as described below, there are often multiple alternate pathways for production of a specific long chain omega-3/omega-6 fatty acid.
[0179] Specifically, all pathways require the initial conversion of oleic acid to LA, the first of the omega-6 fatty acids, by a delta-12 desaturase. Then, using the "delta-9 elongase/delta-8 desaturase pathway", long chain omega-6 fatty acids are formed as follows: (1) LA is converted to EDA by a delta-9 elongase; (2) EDA is converted to DGLA by a delta-8 desaturase; and (3) DGLA is converted to ARA by a delta-5 desaturase. Alternatively, the "delta-9 elongase/delta-8 desaturase pathway" can be utilized for formation of long chain omega-3 fatty acids as follows: (1) LA is converted to ALA, the first of the omega-3 fatty acids, by a delta-15 desaturase; (2) ALA is converted to ETrA by a delta-9 elongase; (3) ETrA is converted to ETA by a delta-8 desaturase; (4) ETA is converted to EPA by a delta-5 desaturase; (5) EPA is converted to DPA by a C20/22 elongase; and (6) DPA is converted to DHA by a delta-4 desaturase. Optionally, omega-6 fatty acids may be converted to omega-3 fatty acids; for example, ETA and EPA are produced from DGLA and ARA, respectively, by delta-17 desaturase activity.
[0180] Alternate pathways for the biosynthesis of omega-3/omega-6 fatty acids utilize a delta-6 desaturase and C18/20 elongase (also known as delta-6 elongase, the terms can be used interchangeably) (i.e., the "delta-6 desaturase/delta-6 elongase pathway"). More specifically, LA and ALA may be converted to GLA and STA, respectively, by a delta-6 desaturase; then, a C18/20 elongase converts GLA to DGLA and/or STA to ETA.
[0181] It is contemplated that the particular functionalities required to be introduced into a specific host organism for production of omega-3/omega-6 fatty acids will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate, and the desired end product(s). For example, expression of the delta-9 elongase/delta-8 desaturase pathway may be preferred in some embodiments, as opposed to expression of the delta-6 desaturase/delta-6 elongase pathway, since PUFAs produced via the former pathway are devoid of GLA.
[0182] One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for omega-3/omega-6 fatty acid biosynthesis. Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo. Although the particular source of the desaturase and elongase genes introduced into the host is not critical, considerations for choosing a specific polypeptide having desaturase or elongase activity include: (1) the substrate specificity of the polypeptide; (2) whether the polypeptide or a component thereof is a rate-limiting enzyme; (3) whether the desaturase or elongase is essential for synthesis of a desired PUFA; and/or (4) cofactors required by the polypeptide. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell (see PCT Publication No. WO 2004/101757 for additional details).
[0183] In additional embodiments, it will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of unpurified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired omega-3/omega-6 fatty acid, as well as various upstream intermediary PUFAs. Thus, consideration of each enzyme's conversion efficiency is also a variable when optimizing biosynthesis of a desired fatty acid that must be considered in light of the final desired lipid profile of the product.
[0184] With each of the considerations above in mind, candidate genes having the appropriate desaturase and elongase activities (e.g., delta-6 desaturases, C18/20 elongases, delta-5 desaturases, delta-17 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C14/16 elongases, C16/18 elongases, delta-9 elongases, delta-8 desaturases, delta-4 desaturases and C20/22 elongases) can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of organisms having the ability to produce PUFAs. These genes will be suitable for introduction into a specific host organism, to enable or enhance the organism's synthesis of PUFAs.
Sequence Identification of Novel Delta-5 Desaturases
[0185] In the present invention, nucleotide sequences encoding delta-5 desaturases have been isolated from Euglena anabaena (designated herein as "EaD5Des1").
[0186] Thus, the present invention concerns an isolated polynucleotide comprising:
[0187] (a) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:13 [EaD5Des1];
[0188] (b) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:12 [EaD5Des1]; or,
[0189] (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
[0190] In still another aspect, this invention concerns an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 90% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:12.
[0191] The instant EaD5Des1 sequences can be codon-optimized for expression in a particular host organism (see SEQ ID NO:45). As is well known in the art, this can be a useful means to further optimize the expression of the enzyme in the alternate host, since use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide. In general, host-preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for a polypeptide of interest having e.g., desaturase activity can be synthesized in whole or in part using the codons preferred in the host species.
[0192] EaD5Des1 could be codon-optimized for expression in Yarrowia lipolytica, as taught in PCT Publication No. WO 04/101757 and U.S. Pat. No. 7,125,672. In one embodiment, it may be desirable to modify a portion of the codons encoding EaD5Des1 (as set forth in SEQ ID NO:12) to enhance expression of the gene in a host organism including, but not limited to, a plant or plant part.
[0193] One skilled in the art would be able to use the teachings herein to create various other codon-optimized delta-5 desaturase proteins suitable for optimal expression in alternate hosts, based on the wildtype EaD5Des1 sequence. Accordingly, the instant invention relates to any codon-optimized delta-5 desaturase protein that is derived from the wildtype EaD5Des1 (i.e., encoded by SEQ ID NO:12).
Identification and Isolation of Homologs
[0194] Any of the instant desaturase sequences (i.e., EaD5Des1) or portions thereof may be used to search for delta-5 desaturase homologs in the same or other bacterial, algal, fungal, euglenoid or plant species using sequence analysis software. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
[0195] Alternatively, any of the instant desaturase sequences or portions thereof may also be employed as hybridization reagents for the identification of delta-5 desaturase homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest and a specific hybridization method. Probes of the present invention are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable" to the nucleic acid sequence to be detected. Although the probe length can vary from 5 bases to tens of thousands of bases, typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
[0196] Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added (e.g., guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, cesium trifluoroacetate). If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
[0197] Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
[0198] Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
[0199] In additional embodiments, any of the delta-5 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used to isolate genes encoding homologous proteins from the same or other bacterial, algal, fungal, euglenoid or plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: (1) methods of nucleic acid hybridization; (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and (3) methods of library construction and screening by complementation.
[0200] For example, genes encoding similar proteins or polypeptides to the delta-5 desaturases described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from e.g., any desired yeast or fungus using methodology well known to those skilled in the art (wherein those organisms producing ARA and/or EPA would be preferred). Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.
[0201] Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, "The use of oligonucleotide as specific hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
[0202] Generally two short segments of the instant sequences may be used in PCR protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. PCR may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding eukaryotic genes.
[0203] Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (Gibco/BRL, Gaithersburg, Md.), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
[0204] In other embodiments, any of the delta-5 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used for creation of new and improved fatty acid desaturases. As is well known in the art, in vitro mutagenesis and selection, chemical mutagenesis, "gene shuffling" methods or other means can be employed to obtain mutations of naturally occurring desaturase genes. Alternatively, improved fatty acids may be synthesized by domain swapping, wherein a functional domain from any of the delta-5 desaturase nucleic acid fragments described herein are exchanged with a functional domain in an alternate desaturase gene to thereby result in a novel protein. As used herein, "domain" or "functional domain" refer to nucleic acid sequence(s) that are capable of eliciting a biological response in plants.
Methods for Production of Various Omega-3 and/or Omega-6 Fatty Acids
[0205] It is expected that introduction of chimeric genes encoding the delta-5 desaturases described herein (i.e., EaD5Des1 or other mutant enzymes, codon-optimized enzymes or homologs thereof), under the control of the appropriate promoters will result in increased production of ARA and/or EPA in the transformed host organism, respectively. As such, the present invention encompasses a method for the direct production of PUFAs comprising exposing a fatty acid substrate (i.e., DGLA and/or ETA) to the desaturase enzymes described herein (e.g., EaD5Des1), such that the substrate is converted to the desired fatty acid product (i.e., ARA and/or EPA).
[0206] More specifically, it is an object of the present invention to provide a method for the production of ARA in a plant host cell (e.g. soybean), wherein the plant host cell comprises:
[0207] (a) a recombinant construct encoding a delta-5 desaturase polypeptide selected from the group consisting of SEQ ID NO:13; and,
[0208] (b) a source of DGLA; wherein the host plant cell is grown under conditions such that the delta-5 desaturase is expressed and the DGLA is converted to ARA, and wherein the ARA is optionally recovered.
[0209] In alternate embodiments of the present invention, the delta-5 desaturase may be used for the use of the enzyme for the conversion of ETA to EPA. Accordingly the invention provides a method for the production of EPA, wherein the host cell comprises:
[0210] (a) a recombinant construct encoding a delta-5 desaturase polypeptide selected from the group consisting of SEQ ID NO:13; and,
[0211] (b) a source of ETA; wherein the host plant cell is grown under conditions such that the delta-5 desaturase is expressed and the ETA is converted to EPA, and wherein the EPA is optionally recovered.
[0212] Alternatively, each delta-5 desaturase gene and its corresponding enzyme product described herein can be used indirectly for the production of various omega-6 and omega-3 PUFAs, including e.g., DGLA, ETA, ARA, EPA, DPA and/or DHA (see FIG. 1; see also PCT Publication No. WO 2004/101757). Indirect production of omega-3/omega-6 PUFAs occurs wherein the fatty acid substrate is converted indirectly into the desired fatty acid product, via means of an intermediate step(s) or pathway intermediate(s). Thus, it is contemplated that the delta-5 desaturases described herein (i.e., EaD5Des1, or other mutant enzymes, codon-optimized enzymes or homologs thereof) may be expressed in conjunction with additional genes encoding enzymes of the PUFA biosynthetic pathway (e.g., delta-6 desaturases, C18/20 elongases, delta-17 desaturases, delta-8 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C14/16 elongases, C16/18 elongases, delta-9 elongases, delta-5 desaturases, delta-4 desaturases, C20/22 elongases) to result in higher levels of production of longer-chain omega-3/omega-6 fatty acids (e.g., ARA, EPA, DPA and DHA).
[0213] In preferred embodiments, the delta-5 desaturases of the present invention will minimally be expressed in conjunction with a delta-9 elongase and a delta-8 desaturases (e.g., a delta-8 desaturase or a codon-optimized delta-8 desaturase). The delta-5 desaturase could also be minimally expressed in conjunction with a delta-6 desaturase and C18/20 elongases. However, the particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s).
[0214] When the desired end product is SCI and/or JUP, then the delta-5 desaturase will be minimally expressed in conjunction with a delta-9 elongase in an organism which makes EDA and/or ERA, respectively.
[0215] The term "delta-6 desaturase/delta-6 elongase pathway" also refers to a biosynthetic pathway for production of long-chain PUFAs. This pathway, at a minimum, comprises a delta-6 desaturase and a delta-6 elongase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized. Occasionally, a delta-6 elongase may elongate fatty acids other than the intended fatty acid. For instance, delta-6 elongases generally convert GLA to DGLA but some delta-6 elongases may also convert unintended substrates such as LA or ALA to EDA or ETrA, respectively. In a delta-6 desaturase/delta-6 elongase pathway, EDA and ETrA would be considered "by-product fatty acids" as defined herein. Addition of a delta-8 desaturase to a delta-6 desaturase/delta-6 elongase pathway would provided a means to convert the "by-product fatty acids" EDA and ETrA back into the "intermediate fatty acids" (as defined previously) DGLA and ETA, respectively.
Plant Expression Systems, Cassettes and Vectors, and Transformation
[0216] In one embodiment, this invention concerns a recombinant construct comprising any one of the delta-5 desaturase polynucleotides of the invention operably linked to at least one regulatory sequence suitable for expression in a plant. A promoter is a DNA sequence that directs cellular machinery of a plant to produce RNA from the contiguous coding sequence downstream (3') of the promoter. The promoter region influences the rate, developmental stage, and cell type in which the RNA transcript of the gene is made. The RNA transcript is processed to produce mRNA which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' non-translated leader sequence is a region of the mRNA upstream of the protein coding region that may play a role in initiation and translation of the mRNA. The 3' transcription termination/polyadenylation signal is a non-translated region downstream of the protein coding region that functions in the plant cell to cause termination of the RNA transcript and the addition of polyadenylate nucleotides to the 3' end of the RNA.
[0217] The origin of the promoter chosen to drive expression of the delta-5 desaturase coding sequence is not important as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the desired nucleic acid fragments in the desired host tissue at the right time. Either heterologous or non-heterologous (i.e., endogenous) promoters can be used to practice the invention. For example, suitable promoters include, but are not limited to: the alpha prime subunit of beta conglycinin promoter, the Kunitz trypsin inhibitor 3 promoter, the annexin promoter, the glycinin Gy1 promoter, the beta subunit of beta conglycinin promoter, the P34/Gly Bd m 30K promoter, the albumin promoter, the Leg A1 promoter and the Leg A2 promoter.
[0218] The annexin, or P34, promoter is described in PCT Publication No. WO 2004/071178 (published Aug. 26, 2004). The level of activity of the annexin promoter is comparable to that of many known strong promoters, such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension protein promoters (Callis et al., J Biol. Chem. 265(21):12486-93 (1990)); (4) a tomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76 (1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol Gen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone gene promoter (Atanassova et al., Plant Mol Biol. 37(2):275-85 (1989)).
[0219] Another useful feature of the annexin promoter is its expression profile in developing seeds. The annexin promoter is most active in developing seeds at early stages (before 10 days after pollination) and is largely quiescent in later stages. The expression profile of the annexin promoter is different from that of many seed-specific promoters, e.g., seed storage protein promoters, which often provide highest activity in later stages of development (Chen et al., Dev. Genet. 10:112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al., Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)). The annexin promoter has a more conventional expression profile but remains distinct from other known seed specific promoters. Thus, the annexin promoter will be a very attractive candidate when overexpression, or suppression, of a gene in embryos is desired at an early developing stage. For example, it may be desirable to overexpress a gene regulating early embryo development or a gene involved in the metabolism prior to seed maturation.
[0220] Following identification of an appropriate promoter suitable for expression of a specific delta-5 desaturase coding sequence, the promoter is then operably linked in a sense orientation using conventional means well known to those skilled in the art.
[0221] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J. et al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989 (hereinafter "Sambrook et al., 1989") or Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; In Current Protocols in Molecular Biology; John Wiley and Sons: New York, 1990 (hereinafter "Ausubel et al., 1990").
[0222] Once the recombinant construct has been made, it may then be introduced into a plant cell of choice by methods well known to those of ordinary skill in the art (e.g., transfection, transformation and electroporation). Oilseed plant cells are the preferred plant cells. The transformed plant cell is then cultured and regenerated under suitable conditions permitting expression of the long-chain PUFA which is then optionally recovered and purified.
[0223] The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells.
[0224] Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.
[0225] The desired long-chain PUFAs can be expressed in seed. Also within the scope of this invention are seeds or plant parts obtained from such transformed plants.
[0226] Plant parts include differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.
[0227] The term "plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term "genome" refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.
[0228] Thus, this invention also concerns a method for transforming a cell, comprising transforming a cell with the recombinant construct of the invention and selecting those cells transformed with the recombinant construct of the invention.
[0229] Also of interest is a method for producing a transformed plant comprising transforming a plant cell with the delta-5 desaturase polynucleotides of the instant invention and regenerating a plant from the transformed plant cell.
[0230] Methods for transforming dicots (primarily by use of Agrobacterium tumefaciens) and obtaining transgenic plants have been published, among others, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep. 15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995)); papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grant et al. Plant Cell Rep. 15:254-258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C. A. (Mol. Biotechnol. 16:53-65 (2000)). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT Publication No. WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol. 3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)), microinjection and particle bombardement (McCabe, D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).
[0231] There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic: San Diego, Calif. (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
[0232] In addition to the above discussed procedures, practitioners are familiar with the standard resource materials which describe specific conditions and procedures for: the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.); the generation of recombinant DNA fragments and recombinant expression constructs; and, the screening and isolating of clones. See, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: NY (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis: Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al., Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).
[0233] Examples of oilseed plants include, but are not limited to: soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
[0234] Examples of PUFAs having at least twenty carbon atoms and four or more carbon-carbon double bonds include, but are not limited to, omega-3 fatty acids such as EPA, DPA and DHA and the omega-6 fatty acid ARA. Seeds obtained from such plants are also within the scope of this invention as well as oil obtained from such seeds.
[0235] Thus, in one embodiment this invention concerns an oilseed plant comprising:
[0236] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,
[0237] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-9 elongase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase.
[0238] Additional desaturases are discussed, for example, in U.S. Pat. Nos. 6,075,183, 5,968,809, 6,136,574, 5,972,664, 6,051,754, 6,410,288 and PCT Publication Nos. WO 98/46763, WO 98/46764, WO 00/12720 and WO 00/40705.
[0239] The choice of combination of cassettes used depends in part on the PUFA profile and/or desaturase/elongase profile of the oilseed plant cells to be transformed and the long-chain PUFA which is to be expressed.
[0240] In another aspect, this invention concerns a method for making long-chain PUFAs in a plant cell comprising:
[0241] (a) transforming a plant cell with the recombinant construct of the invention; and,
[0242] (b) selecting those transformed plant cells that make long-chain PUFAs.
[0243] In still another aspect, this invention concerns a method for producing at least one PUFA in a soybean cell comprising:
[0244] (a) transforming a soybean cell with a first recombinant DNA construct comprising:
[0245] (i) an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,
[0246] (ii) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-9 elongase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase;
[0247] (b) regenerating a soybean plant from the transformed cell of step (a); and,
[0248] (c) selecting those seeds obtained from the plants of step (b) having an altered level of PUFAs when compared to the level in seeds obtained from a nontransformed soybean plant.
[0249] In other preferred embodiments, the at least one additional recombinant DNA construct encodes a polypeptide having delta-9 elongase activity, e.g., the delta-9 elongase isolated or derived from Isochrysis galbana (GenBank Accession No. AF390174; IgD9e) or the delta-9 elongase isolated or derived from Euglena gracilis.
[0250] In other preferred embodiments, the at least one additional recombinant DNA construct encodes a polypeptide having delta-8 desaturase activity. For example, PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885). Sayanova et al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 delta-8 desaturase. Also, Applicants' Assignee's co-pending application having U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007; Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S. patent application Ser. No. 11/876,115 (filed Oct. 22, 2007; Attorney Docket No. BB-1574) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf--gymnastica CCMP1594.
Microbial Expression Systems, Cassettes and Vectors, and Transformation
[0251] The delta-5 desaturase genes and gene products described herein (i.e., EaD5Des1, or other mutant enzymes, codon-optimized enzymes or homologs thereof) may also be produced in heterologous microbial host cells, particularly in the cells of oleaginous yeasts (e.g., Yarrowia lipolytica).
[0252] Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of any of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the encoded enzymes.
[0253] Vectors or DNA cassettes useful for the transformation of suitable microbial host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell and the proposed means of separating transformed cells versus nontransformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene that controls transcriptional initiation (e.g., a promoter) and a region 3' of the DNA fragment that controls transcriptional termination (i.e., a terminator). It is most preferred when both control regions are derived from genes from the transformed microbial host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
[0254] Initiation control regions or promoters which are useful to drive expression of the instant delta-5 desaturase ORFs in the desired microbial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of directing expression of these genes in the selected host cell is suitable for the present invention. Expression in a microbial host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species (e.g., see PCT Publication Nos. WO 2004/101757 and WO 2006/052870 for preferred transcriptional initiation regulatory regions for use in Yarrowia lipolytica). Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like.
[0255] Nucleotide sequences surrounding the translational initiation codon `ATG` have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.
[0256] The termination region can be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property. Preferably, when the microbial host is a yeast cell, the termination region is derived from a yeast gene (particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces). The 3'-regions of mammalian genes encoding γ-interferon and α-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. Although not intended to be limiting, termination regions useful in the disclosure herein include: ˜100 bp of the 3' region of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession No. M17741); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No. XP--503244) terminators; the Pex20 (GenBank Accession No. AF054613) terminator; the Pex16 (GenBank Accession No. U75433) terminator; the Lip1 (GenBank Accession No. Z50020) terminator; the Lip2 (GenBank Accession No. AJ012632) terminator; and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator.
[0257] As one of skill in the art is aware, merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the microbial host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: (1) the nature of the relevant transcriptional promoter and terminator sequences; (2) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; (3) the final cellular location of the synthesized foreign protein; (4) the efficiency of translation and correct folding of the protein in the host organism; (5) the intrinsic stability of the mRNA and protein of the cloned gene within the host cell; and (6) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the delta-5 desaturase described herein.
[0258] Once the DNA encoding a polypeptide suitable for expression in an appropriate microbial host cell (e.g., oleaginous yeast) has been obtained (e.g., a chimeric gene comprising a promoter, ORF and terminator), it is placed in a plasmid vector capable of autonomous replication in a host cell, or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
[0259] The preferred method of expressing genes in Yarrowia lipolytica is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired [e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP--503244; or, Aco3: GenBank Accession No. AJ001301), the delta-12 desaturase gene locus (PCT Publication No. WO2004/104167), the Lip1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].
[0260] Advantageously, the Ura3 gene can be used repeatedly in combination with 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; "5-FOA") selection (infra), to readily permit genetic modifications to be integrated into the Yarrowia genome in a facile manner.
[0261] Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
[0262] Constructs comprising the gene of interest may be introduced into a microbial host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl. Microbiol. Biotechnol., 48(2):232-235 (1997)).
[0263] For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as "transformed" or "recombinant" herein. Thus, the term "transformed" and "recombinant" are used interchangeably herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified or is present on an extrachromosomal element having multiple copy numbers.
[0264] The transformed host cell can be identified by various selection techniques, as described in PCT Publication Nos. WO 2004/101757 and WO 2006/052870. Preferred selection methods for use herein are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-FOA is used for selection of yeast Ura- mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5'-monophosphate decarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOA is especially useful for the selection and identification of Ura.sup.- mutant yeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp 109-147, 1997). More specifically, one can first knockout the native Ura3 gene to produce a strain having a Ura- phenotype, wherein selection occurs based on 5-FOA resistance. Then, a cluster of multiple chimeric genes and a new Ura3 gene can be integrated into a different locus of the Yarrowia genome to thereby produce a new strain having a Ura+ phenotype. Subsequent integration produces a new Ura3- strain (again identified using 5-FOA selection), when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in combination with 5-FOA selection) can be used as a selection marker in multiple rounds of transformation.
[0265] Following transformation, substrates suitable for the instant delta-5 desaturase (and, optionally other PUFA enzymes that are co-expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.
[0266] Microbial host cells for expression of the instant genes and nucleic acid fragments may include hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, fatty acids, organic acids, oils and alcohols, and/or hydrocarbons over a wide range of temperature and pH values. Based on the needs of the Applicants' Assignee, the genes described in the instant invention will be expressed in an oleaginous yeast (and in particular Yarrowia lipolytica); however, it is contemplated that because transcription, translation and the protein biosynthetic apparatus is highly conserved, any bacteria, yeast, algae and/or fungus will be a suitable microbial host for expression of the present nucleic acid fragments.
[0267] Preferred microbial hosts, however, are oleaginous yeasts. These organisms are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight, and most preferably greater than about 40% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica).
[0268] Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)).
[0269] Historically, various strains of Y. lipolytica have been used for the manufacture and production of: isocitrate lyase; lipases; polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid; γ-decalactone; γ-dodecalatone; and pyruvic acid. Specific teachings applicable for engineering ARA, EPA and DHA production in Y. lipolytica are provided in U.S. patent application Ser. No. 11/264,784 (WO 2006/055322), U.S. patent application Ser. No. 11/265,761 (WO 2006/052870) and U.S. patent application Ser. No. 11/264,737 (WO 2006/052871), respectively.
[0270] Other preferred microbial hosts include oleaginous bacteria, algae and other fungi; and, within this broad group of microbial hosts, of particular interest are microorganisms that synthesize omega-3/omega-6 fatty acids (or those that can be genetically engineered for this purpose [e.g., other yeast such as Saccharomyces cerevisiae]). Thus, for example, transformation of Mortierella alpina (which is commercially used for production of ARA) with any of the present delta-5 desaturase genes under the control of inducible or regulated promoters could yield a transformant organism capable of synthesizing increased quantities of DGLA. The method of transformation of M. alpina is described by Mackenzie et al. (Appl. Environ. Microbiol., 66:4655 (2000)). Similarly, methods for transformation of Thraustochytriales microorganisms are disclosed in U.S. Pat. No. 7,001,772.
Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesis in Microbes
[0271] Methods for manipulating biochemical pathways are well known to those skilled in the art; and, it is expected that numerous manipulations will be possible to maximize omega-3 and/or omega-6 fatty acid biosynthesis in oleaginous yeasts, and particularly, in Yarrowia lipolytica. This manipulation may require metabolic engineering directly within the PUFA biosynthetic pathway or additional coordinated manipulation of various other metabolic pathways.
[0272] In the case of manipulations within the PUFA biosynthetic pathway, it may be desirable to increase the production of LA to enable increased production of omega-6 and/or omega-3 fatty acids. Introducing and/or amplifying genes encoding delta-9 and/or delta-12 desaturases may accomplish this. To maximize production of omega-6 unsaturated fatty acids, it is well known to one skilled in the art that production is favored in a host microorganism that is substantially free of ALA; thus, preferably, the host is selected or obtained by removing or inhibiting delta-15 or omega-3 type desaturase activity that permits conversion of LA to ALA. Alternatively, it may be desirable to maximize production of omega-3 fatty acids (and minimize synthesis of omega-6 fatty acids). In this example, one could utilize a host microorganism wherein the delta-12 desaturase activity that permits conversion of oleic acid to LA is removed or inhibited; subsequently, appropriate expression cassettes would be introduced into the host, along with appropriate substrates (e.g., ALA) for conversion to omega-3 fatty acid derivatives of ALA (e.g., STA, ETrA, ETA, EPA, DPA, DHA).
[0273] In alternate embodiments, biochemical pathways competing with the omega-3 and/or omega-6 fatty acid biosynthetic pathways for energy or carbon, or native PUFA biosynthetic pathway enzymes that interfere with production of a particular PUFA end-product, may be eliminated by gene disruption or down-regulated by other means (e.g., antisense mRNA).
[0274] Detailed discussion of manipulations within the PUFA biosynthetic pathway as a means to increase ARA, EPA or DHA (and associated techniques thereof) are presented in PCT Publication Nos. WO 2006/055322, WO 2006/052870 and WO 2006/052871, respectively, as are desirable manipulations in the TAG biosynthetic pathway and the TAG degradation pathway (and associated techniques thereof).
[0275] Within the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the strategies described above. For example, the present invention provides methods whereby genes encoding key enzymes in the delta-9 elongase/delta-8 desaturase biosynthetic pathway are introduced into plants for the production of omega-3 and/or omega-6 fatty acids. It will be particularly useful to express the present the delta-5 desaturase genes in plants that do not naturally possess omega-3 and/or omega-6 fatty acid biosynthetic pathways and coordinate the expression of these genes, to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism.
Microbial Fermentation Processes for PUFA Production
[0276] The transformed host cell is grown under conditions that optimize expression of chimeric desaturase genes and produce the greatest and the most economical yield of desired PUFAs. In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the amount of different mineral ions, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time and method of cell harvest. Yarrowia lipolytica are generally grown in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
[0277] Fermentation media may contain a suitable carbon source. Suitable carbon sources are taught in PCT Publication No. WO 2004/101757. Although it is contemplated that the source of carbon utilized in may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol, and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.
[0278] Nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) or organic (e.g., urea or glutamate) source. In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins and other components known to those skilled in the art suitable for the growth of the oleaginous host and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions (e.g., Mn+2, Co+2, Zn+2, Mg+2) that promote synthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
[0279] Preferred growth media are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the transformant host cells will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.
[0280] Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be "balanced" between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica). This approach is described in PCT Publication No. WO 2004/101757, as are various suitable fermentation process designs (i.e., batch, fed-batch and continuous) and considerations during growth.
Purification and Processing of PUFA Oils
[0281] PUFAs may be found in the host microorganisms and plants as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cells through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).
[0282] In general, means for the purification of PUFAs may include extraction with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. One is referred to the teachings of PCT Publication No. WO 2004/101757 for additional details. Methods of isolating seed oils are well known in the art: (Young et al., Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al., eds., Chapter 5 pp 253-257; Chapman & Hall: London (1994)). For example, soybean oil is produced using a series of steps involving the extraction and purification of an edible oil product from the oil-bearing seed. Soybean oils and soybean byproducts are produced using the generalized steps shown in Table 3.
TABLE-US-00003 TABLE 3 Generalized Steps for Soybean Oil and Byproduct Production Process Impurities Removed and/or Step Process By-Products Obtained # 1 soybean seed # 2 oil extraction meal # 3 degumming lecithin # 4 alkali or physical refining gums, free fatty acids, pigments # 5 water washing soap # 6 bleaching color, soap, metal # 7 (hydrogenation) # 8 (winterization) stearine # 9 deodorization free fatty acids, tocopherols, sterols, volatiles # 10 oil products
[0283] More specifically, soybean seeds are cleaned, tempered, dehulled and flaked, thereby increasing the efficiency of oil extraction. Oil extraction is usually accomplished by solvent (e.g., hexane) extraction but can also be achieved by a combination of physical pressure and/or solvent extraction. The resulting oil is called crude oil. The crude oil may be degummed by hydrating phospholipids and other polar and neutral lipid complexes that facilitate their separation from the nonhydrating, triglyceride fraction (soybean oil). The resulting lecithin gums may be further processed to make commercially important lecithin products used in a variety of food and industrial products as emulsification and release (i.e., antisticking) agents. Degummed oil may be further refined for the removal of impurities (primarily free fatty acids, pigments and residual gums). Refining is accomplished by the addition of a caustic agent that reacts with free fatty acid to form soap and hydrates phosphatides and proteins in the crude oil. Water is used to wash out traces of soap formed during refining. The soapstock byproduct may be used directly in animal feeds or acidulated to recover the free fatty acids. Color is removed through adsorption with a bleaching earth that removes most of the chlorophyll and carotenoid compounds. The refined oil can be hydrogenated, thereby resulting in fats with various melting properties and textures. Winterization (fractionation) may be used to remove stearine from the hydrogenated oil through crystallization under carefully controlled cooling conditions. Deodorization (principally via steam distillation under vacuum) is the last step and is designed to remove compounds which impart odor or flavor to the oil. Other valuable byproducts such as tocopherols and sterols may be removed during the deodorization process. Deodorized distillate containing these byproducts may be sold for production of natural vitamin E and other high-value pharmaceutical products. Refined, bleached, (hydrogenated, fractionated) and deodorized oils and fats may be packaged and sold directly or further processed into more specialized products. A more detailed reference to soybean seed processing, soybean oil production and byproduct utilization can be found in Erickson, Practical Handbook of Soybean Processing and Utilization, The American Oil Chemists' Society and United Soybean Board (1995). Soybean oil is liquid at room temperature because it is relatively low in saturated fatty acids when compared with oils such as coconut, palm, palm kernel and cocoa butter.
[0284] Plant and microbial oils containing PUFAs that have been refined and/or purified can be hydrogenated, to thereby result in fats with various melting properties and textures. Many processed fats (including spreads, confectionary fats, hard butters, margarines, baking shortenings, etc.) require varying degrees of solidity at room temperature and can only be produced through alteration of the source oil's physical properties. This is most commonly achieved through catalytic hydrogenation.
[0285] Hydrogenation is a chemical reaction in which hydrogen is added to the unsaturated fatty acid double bonds with the aid of a catalyst such as nickel. For example, high oleic soybean oil contains unsaturated oleic, LA and linolenic fatty acids and each of these can be hydrogenated. Hydrogenation has two primary effects. First, the oxidative stability of the oil is increased as a result of the reduction of the unsaturated fatty acid content. Second, the physical properties of the oil are changed because the fatty acid modifications increase the melting point resulting in a semi-liquid or solid fat at room temperature.
[0286] There are many variables which affect the hydrogenation reaction, which in turn alter the composition of the final product. Operating conditions including pressure, temperature, catalyst type and concentration, agitation and reactor design are among the more important parameters that can be controlled. Selective hydrogenation conditions can be used to hydrogenate the more unsaturated fatty acids in preference to the less unsaturated ones. Very light or brush hydrogenation is often employed to increase stability of liquid oils. Further hydrogenation converts a liquid oil to a physically solid fat. The degree of hydrogenation depends on the desired performance and melting characteristics designed for the particular end product. Liquid shortenings (used in the manufacture of baking products, solid fats and shortenings used for commercial frying and roasting operations) and base stocks for margarine manufacture are among the myriad of possible oil and fat products achieved through hydrogenation. A more detailed description of hydrogenation and hydrogenated products can be found in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and Practice. The American Oil Chemists' Society (1994).
[0287] Hydrogenated oils have become somewhat controversial due to the presence of trans-fatty acid isomers that result from the hydrogenation process. Ingestion of large amounts of trans-isomers has been linked with detrimental health effects including increased ratios of low density to high density lipoproteins in the blood plasma and increased risk of coronary heart disease.
PUFA-Containing Oils for Use in Foodstuffs
[0288] The market place currently supports a large variety of food and feed products, incorporating omega-3 and/or omega-6 fatty acids (particularly ARA, EPA and DHA). It is contemplated that the plant/seed oils, altered seeds and microbial oils of the invention comprising PUFAs will function in food and feed products to impart the health benefits of current formulations. Compared to other vegetable oils, the oils of the invention are believed to function similarly to other oils in food applications from a physical standpoint (for example, partially hydrogenated oils such as soybean oil are widely used as ingredients for soft spreads, margarine and shortenings for baking and frying).
[0289] Plant/seed oils, altered seeds and microbial oils containing omega-3 and/or omega-6 fatty acids as described herein will be suitable for use in a variety of food and feed products including, but not limited to: food analogs, meat products, cereal products, baked foods, snack foods and dairy products. Additionally, the present plant/seed oils, altered seeds and microbial oils may be used in formulations to impart health benefit in medical foods including medical nutritionals, dietary supplements, infant formula as well as pharmaceutical products. One of skill in the art of food processing and food formulation will understand how the amount and composition of the plant and microbial oils may be added to the food or feed product. Such an amount will be referred to herein as an "effective" amount and will depend on the food or feed product, the diet that the product is intended to supplement or the medical condition that the medical food or medical nutritional is intended to correct or treat.
[0290] Food analogs can be made using processes well known to those skilled in the art. There can be mentioned meat analogs, cheese analogs, milk analogs and the like. Meat analogs made from soybeans contain soy protein or tofu and other ingredients mixed together to simulate various kinds of meats. These meat alternatives are sold as frozen, canned or dried foods. Usually, they can be used the same way as the foods they replace. Meat alternatives made from soybeans are excellent sources of protein, iron and B vitamins. Examples of meat analogs include, but are not limited to: ham analogs, sausage analogs, bacon analogs, and the like.
[0291] Food analogs can be classified as imitation or substitutes depending on their functional and compositional characteristics. For example, an imitation cheese need only resemble the cheese it is designed to replace. However, a product can generally be called a substitute cheese only if it is nutritionally equivalent to the cheese it is replacing and meets the minimum compositional requirements for that cheese. Thus, substitute cheese will often have higher protein levels than imitation cheeses and be fortified with vitamins and minerals.
[0292] Milk analogs or nondairy food products include, but are not limited to, imitation milks and nondairy frozen desserts (e.g., those made from soybeans and/or soy protein products).
[0293] Meat products encompass a broad variety of products. In the United States "meat" includes "red meats" produced from cattle, hogs and sheep. In addition to the red meats there are poultry items which include chickens, turkeys, geese, guineas, ducks and the fish and shellfish. There is a wide assortment of seasoned and processed meat products: fresh, cured and fried, and cured and cooked. Sausages and hot dogs are examples of processed meat products. Thus, the term "meat products" as used herein includes, but is not limited to, processed meat products.
[0294] A cereal food product is a food product derived from the processing of a cereal grain. A cereal grain includes any plant from the grass family that yields an edible grain (seed). The most popular grains are barley, corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat and wild rice. Examples of a cereal food product include, but are not limited to: whole grain, crushed grain, grits, flour, bran, germ, breakfast cereals, extruded foods, pastas, and the like.
[0295] A baked goods product comprises any of the cereal food products mentioned above and has been baked or processed in a manner comparable to baking (i.e., to dry or harden by subjecting to heat). Examples of a baked good product include, but are not limited to: bread, cakes, doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above, oils of the invention can be used as an ingredient.
[0296] A snack food product comprises any of the above or below described food products.
[0297] A fried food product comprises any of the above or below described food products that has been fried.
[0298] A health food product is any food product that imparts a health benefit. Many oilseed-derived food products may be considered as health foods.
[0299] A beverage can be in a liquid or in a dry powdered form.
[0300] For example, there can be mentioned non-carbonated drinks such as fruit juices, fresh, frozen, canned or concentrate; flavored or plain milk drinks, etc. Adult and infant nutritional formulas are well known in the art and commercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum® from Ross Products Division, Abbott Laboratories).
[0301] Infant formulas are liquids or reconstituted powders fed to infants and young children. "Infant formula" is defined herein as an enteral nutritional product which can be substituted for human breast milk in feeding infants and typically is composed of a desired percentage of fat mixed with desired percentages of carbohydrates and proteins in an aquous solution (e.g., see U.S. Pat. No. 4,670,285). Based on the worldwide composition studies, as well as levels specified by expert groups, average human breast milk typically contains about 0.20% to 0.40% of total fatty acids (assuming about 50% of calories from fat); and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2 (see, e.g., formulations of Enfamil LIPIL® (Mead Johnson & Company) and Similac Advance® (Ross Products Division, Abbott Laboratories)). Infant formulas have a special role to play in the diets of infants because they are often the only source of nutrients for infants; and, although breast-feeding is still the best nourishment for infants, infant formula is a close enough second that babies not only survive but thrive.
[0302] A dairy product is a product derived from milk. A milk analog or nondairy product is derived from a source other than milk, for example, soymilk as was discussed above. These products include, but are not limited to: whole milk, skim milk, fermented milk products such as yogurt or sour milk, cream, butter, condensed milk, dehydrated milk, coffee whitener, coffee creamer, ice cream, cheese, etc.
[0303] Additional food products into which the PUFA-containing oils of the invention could be included are, for example, chewing gums, confections and frostings, gelatins and puddings, hard and soft candies, jams and jellies, white granulated sugar, sugar substitutes, sweet sauces, toppings and syrups, and dry-blended powder mixes.
PUFA-Containing Oils for Use in Health Food Products and Pharmaceuticals
[0304] A health food product is any food product that imparts a health benefit and include functional foods, medical foods, medical nutritionals and dietary supplements. Additionally, the plant/seed oils, altered seeds and microbial oils of the invention may be used in standard pharmaceutical compositions (e.g., the long-chain PUFA containing oils could readily be incorporated into the any of the above mentioned food products, to thereby produce a functional or medical food). More concentrated formulations comprising PUFAs include capsules, powders, tablets, softgels, gelcaps, liquid concentrates and emulsions which can be used as a dietary supplement in humans or animals other than humans.
PUFA-Containing Oils for Use in Animal Feeds
[0305] Animal feeds are generically defined herein as products intended for use as feed or for mixing in feed for animals other than humans. The plant/seed oils, altered seeds and microbial oils of the invention can be used as an ingredient in various animal feeds.
[0306] More specifically, although not limited therein, it is expected that the oils of the invention can be used within pet food products, ruminant and poultry food products and aquacultural food products. Pet food products are those products intended to be fed to a pet (e.g., dog, cat, bird, reptile, rodent). These products can include the cereal and health food products above, as well as meat and meat byproducts, soy protein products, grass and hay products (e.g., alfalfa, timothy, oat or brome grass, vegetables). Ruminant and poultry food products are those wherein the product is intended to be fed to an animal (e.g., turkeys, chickens, cattle, swine). As with the pet foods above, these products can include cereal and health food products, soy protein products, meat and meat byproducts, and grass and hay products as listed above. Aquacultural food products (or "aquafeeds") are those products intended to be used in aquafarming, i.e., which concerns the propagation, cultivation or farming of aquatic organisms and/or animals in fresh or marine waters.
EXAMPLES
[0307] The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
[0308] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "d" means day(s), "μL" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "μM" means micromolar, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "μmole" mean micromole(s), "g" means gram(s), "μg" means microgram(s), "ng" means nanogram(s), "U" means unit(s), "bp" means base pair(s) and "kB" means kilobase(s).
General Methods:
[0309] Transformation and Cultivation of Yarrowia lipolytica:
[0310] Yarrowia lipolytica strains with ATCC Accession Nos. #20362, #76982 and #90812 were purchased from the American Type Culture Collection (Rockville, Md.). Yarrowia lipolytica strains were typically grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).
[0311] Transformation of Yarrowia lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol. Biotechnol. 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C. for approximately 18 h. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer, comprising: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M lithium acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μg sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 1004 of resuspended cells, and maintained at 39° C. for 1 h with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30° C. for 2 to 3 days.
[0312] For selection of transformants, minimal medium ("MM") was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (Difco Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1). Supplements of uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMU" selection media, prepared with 20 g/L agar).
[0313] Alternatively, transformants were selected on 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (Difco Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L agar.
Fatty Acid Analysis of Yarrowia lipolytica:
[0314] For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G. and Nishida I., Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30 m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.
[0315] For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (1004 of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 4004 hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.
Example 1
[0316] Synthesis of a cDNA Library from Euglena anabaena UTEX 373
[0317] The present Example describes the synthesis of a cDNA library from Euglena anabaena UTEX 373. This work included the generation of RNA, synthesis of cDNA, and generation of a cDNA library.
Growth of Euglena anabaena UTEX 373 and Preparation of RNA
[0318] Euglena anabaena UTEX 373 was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). Approximately 2 mL of culture was removed for lipid analysis and centrifuged at 1,800×g for 5 min. The pellet was washed once with water and re-centrifuged. The resulting pellet was dried for 5 min under vacuum, resuspended in 1004 of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this step, 0.5 mL of hexane was added and the vials were incubated for 15 min at room temperature with shaking. Fatty acid methyl esters (5 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Supelco Inc., Cat. No. 24152). The oven temperature was programmed to hold at 170° C. for 1.0 min, increase to 240° C. at 5° C./min and then hold for an additional 1.0 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc. Cat. No. U-99-A) and the resulting chromatogram is shown in FIG. 10. The presence of EDA, ERA, EPA and DHA in the fatty acid profile, with the absence of GLA and STA, suggested that Euglena anabaena uses the delta-9 elongase/delta-8 desaturase pathway for LC-PUFA biosynthesis and would be a good source for LC-PUFA biosynthetic genes such as, but not limited to, delta-5 desaturases.
[0319] The remaining 5 mL of an actively growing culture was transferred into 25 mL of AF-6 Medium (Watanabe & Hiroki, NIES-Collection List of Strains, 5th ed., National Institute for Environmental Studies, Tsukuba, 127 pp (2004)) in a 125 mL glass flask. Euglena anabaena cultures were grown at 22° C. with a 16 h light, 8 h dark cycle for 2 weeks with very gentle agitation.
[0320] After 2 weeks, the culture (25 mL) was transferred to 100 mL of AF-6 medium in a 500 mL glass bottle and the culture was grown for 1 month as described above.
[0321] After this time, two 50 mL aliquots were transferred into two separate 500 mL glass bottles containing 250 mL of AF-6 medium and the cultures were grown for two months as described above (giving a total of ˜600 mL of culture). Next, the cultures were pelleted by centrifugation at 1,800×g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from one of the resulting pellets using the RNA STAT-60® reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water). In this way, 340 μg of total RNA (680 μg/mL) was obtained from the pellet. The remaining pellet was frozen in liquid nitrogen and stored at -80° C. The mRNA was isolated from all 340 μg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 9.0 μg of mRNA was obtained.
Preparation of Euglena anabaena cDNA and Generation of cDNA Library eug1c
[0322] A cDNA library was generated using the Cloneminer® cDNA Library Construction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad, Calif.) and following the manufacturer's protocol provided (Version B, 25-0608). Using the non-radiolabeling method, cDNA was synthesized from 5.12 μg of mRNA (described above) using the Biotin-attB2-Oligo(dT) primer. After synthesis of the first and second strand, the attB1 adapter was added, ligated and the cDNA was size fractionated using column chromatography. DNA from fractions were concentrated, recombined into pDONR®222 and transformed into E. coli ElectroMAX® DH10B® T1 Phage-Resistant cells (Invitrogen Corporation). The Euglena anabaena library was named eug1c.
[0323] The cDNA library eug1c was plated onto LBKan plates (approx. 100,000 colonies), the colonies were scraped off and DNA was isolated using the QIAprep® Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) following the manufacturer's protocol. In this way, a plasmid DNA sub-library from eug1c was obtained.
Example 2
Isolation of the Full-Length Delta-5 Desaturases from Euglena anabaena UTEX 373
[0324] The present Example describes the identification of a cDNA (SEQ ID NO:1) encoding delta-5 desaturase from Euglena anabaena UTEX 373. This work included the generation of a probe derived from the Euglena gracilis delta-5 desaturase (EgD5; SEQ ID NO:2; which is described in U.S. Provisional Application No. 60/801,172 (filed May 17, 2006; Attorney Docket No. CL-3486)) and the hybridization of the probe to the cDNA library eug1c in order to identify delta-5 desaturase homologs from Euglena anabaena UTEX 373.
Generation of Construct pDMW367, Comprising EgD5:
[0325] Based on the cDNA sequence of the Euglena gracilis delta-5 desaturase (EgD5; SEQ ID NO:2) oligonucleotides YL794 and YL797 (SEQ ID NOs:3 and 4, respectively) were used as primers to amplify the first portion of EgD5 (FIG. 2A). Primer YL794 contained a NcoI site and primer YL797 contained a HindIII site. Then, primers YL796 and YL795 (SEQ ID NOs:5 and 6, respectively) were used as primers to amplify the second portion of EgD5. Primer YL796 contained a HindIII site, while primer YL797 contained a NotI site. The PCR reactions, using primer pairs YL794/YL797 or YL796/YL795, with Euglena gracilis cDNA (the generation of which is described in U.S. Provisional Application No. 60/801,172 (filed May 17, 2006; Attorney Docket No. CL-3486)) as template, were individually carried out in a 50 pt total volume comprising: PCR buffer (containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO4, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of Pfu DNA polymerase (Stratagene, San Diego, Calif.). The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by a final extension at 7° C. for 10 min. The individual PCR products were purified using a Qiagen PCR purification kit. The PCR product from the reaction amplified with primers YL794/797 was digested with NcoI and HindIII, while the PCR product from the reaction amplified with primers YL796/YL795 was digested with HindIII and NotI. The NcoI/HindIII and the HindIII NotI digested DNA fragments were purified following gel electrophoresis in 1% (w/v) agarose, and then directionally ligated with NcoI/NotI digested pZUF17 (FIG. 2B; SEQ ID NO:7; comprising a synthetic delta-17 desaturase gene ["D17st"] derived from S. diclina (U.S. Publication No. 2003/0196217 A1), codon-optimized for Yarrowia lipolytica (PCT Publication No. WO 2004/101757)). The product of this ligation was pDMW367 (FIG. 2C; SEQ ID NO:8), which thereby contained the following components:
TABLE-US-00004 TABLE 4 Components of Plasmid pDMW367 (SEQ ID NO: 8) RE Sites and Nucleotides Within Description Of Fragment And SEQ ID NO: 8 Chimeric Gene Components EcoR I/BsiW I FBAIN::EgD5::Pex20, comprising: (7416-1617) FBAIN: Yarrowia lipolytica FBAIN promoter (WO 2005/049805) EgD5: Euglena gracilis delta-5 desaturase (SEQ ID NO: 2 described herein) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 2707-1827 ColE1 plasmid origin of replication 3637-2777 ampicillin-resistance gene (AmpR) for selection in E. coli 4536-5840 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) 7373-5886 Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)
[0326] The term "FBAIN promoter" or "FBAIN promoter region" refers to the 5' upstream untranslated region in front of the `ATG` translation initiation codon of the Yarrowia lipolytica fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and that is necessary for expression, plus a portion of 5' coding region that has an intron of the fba1 gene.
Colony Lifts:
[0327] Approximately 17,000 clones of cDNA library eug1c were plated onto three large square (24 cm×24 cm) petri plates (Corning, Corning, N.Y.) each containing LB+50 μg/mL kanamycin agar media. Cells were grown overnight at 37° C. and plates were then cooled to room temperature.
[0328] Biodyne B 0.45 μm membrane (Cat. No. 60207, Pall Corporation, Pensacola, Fla.) was trimmed to approximately 22 cm×22 cm and the membrane was carefully layed on top of the agar to avoid air bubbles. After incubation for 2 min at room temperature, the membrane was marked for orientation, lifted off with tweezers and placed colony-side up on filter paper soaked with 0.5 M sodium hydroxide and 1.5 M sodium chloride. After denaturation for 4 min, the sodium hydroxide was neutralized by placing the membrane on filter paper soaked with 0.5 M Tris-HCL (pH 7.5) and 1.5 M sodium chloride for 4 min. This step was repeated and the membrane was rinsed briefly in 2×SSC buffer (20×SSC is 3M sodium chloride, 0.3 M sodium citrate; pH 7.0) and air dried on filter paper.
Hybridization:
[0329] Membranes were pre-hybridized at 65° C. in 200 mL hybridization solution for 2 h. Hybridization solution contained 6×SSPE (20×SSPE is 3 M sodium chloride, 0.2 M sodium phosphate, 20 mM EDTA; pH 7.4), 5×Denhardt's reagent (100×Denhardt's reagent is 2% (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone, 2% (w/v) acetylated bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), 100 μg/mL sheared salmon sperm DNA and 5% dextran sulfate.
[0330] A DNA probe was made using an agarose gel purified NcoI/NotI DNA fragment, containing the Euglena gracilis delta-5 desaturase gene, from pDMW367 (SEQ ID NO:8) labeled with P32 dCTP using the RadPrime DNA Labeling System (Cat. No. 18428-011, Invitrogen, Carlsbad, Calif.) following the manufacture's instructions. Unincorporated P32 dCTP was separated using a NICK column (Cat. No. 17-0855-02, Amersham Biosciences, Piscataway, N.J.) following the manufacturer's instructions. The probe was denatured for 5 min at 100° C., placed on ice for 3 min and half was added to the hybridization solution.
[0331] The membrane was hybridized with the probe overnight at 65° C. with gentle shaking and then washed the following day twice with 2×SSC containing 0.5% SDS (5 min each) and twice with 0.2×SSC containing 0.1% SDS (15 min each). After washing, hyperfilm (Cat. No. RPN30K, Amersham Biosciences, Piscataway, N.J.) was exposed to the membrane overnight at -80° C.
[0332] Based on alignment of plates with the exposed hyperfilm, positive colonies were picked using the blunt end of a Pasteur pipette into 1 mL of water and vortexed. Several dilutions were made and plated onto small round Petri dishes (82 mm) containing LB media plus 50 μg/mL kanamycin to obtain around 100 well isolated colonies on a single plate. Lifts were done as described above except NytranN membrane circles (Cat, No. 10416116, Schleicher & Schuell, Keene, N.H.) were used and hybridization was carried out in 100 mL using the remaining radiolabeled probe. In this way, positive clones were confirmed.
[0333] Individual positive clones were grown at 37° C. in LB+50 μg/mL kanamycin liquid media and plasmid was purified using the QIAprep® Spin Miniprep Kit (Qiagen Inc.) following the manufacturer's protocol.
[0334] DNA inserts were end-sequenced in 384-well plates, using vector-primed M13F universal primer (SEQ ID NO:9), M13rev-28 primer (SEQ ID NO:10) and the poly(A) tail-primed WobbleT oligonucleotides, with the ABI BigDye version 3 Prism sequencing kit. For the sequencing reaction, 100-200 ng of template and 6.4 pmol of primer were used, and the following reaction conditions were repeated 25 times: 96° C. for 10 sec, 50° C. for 5 sec and 60° C. for 4 min. After ethanol-based cleanup, cycle sequencing reaction products were resolved and detected on Perkin-Elmer ABI 3700 automated sequencers. The WobbleT primer is an equimolar mix of 21 mer poly(T)A, poly(T)C, and poly(T)G, used to sequence the 3' end of cDNA clones.
[0335] Sequences were aligned and compared using Sequencher® (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.) and in this way, it was determined that all all of the CDS in each cDNA were identical. A representative clone containing a cDNA (pLF119) is shown in SEQ ID NO:11 and the gene contained within the cDNA was called EaD5Des1. The coding sequence for EaD5Des1 is shown in SEQ ID NO:12. The corresponding amino acid sequence for EaD5Des1 is shown in SEQ ID NO:13.
Example 3
Primary Sequence Analysis of the Delta-5 Desaturase Sequence of Euglena anabaena UTEX 373 (EaD5Des1) and Comparison to the Delta-5 Desaturase Sequence of Euglena gracilis (EgD5)
[0336] The amino acid sequence for EaD5Des1 (SEQ ID NO:13) was evaluated by BLASTP (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993)) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL and DDBJ databases) using default parameters with the filter turned off. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins.
[0337] BLASTP analysis with EaD5Des1 yielded a pLog value of 76.52 (P value of 3e-77) versus the Thalassiosira pseudonana delta-8 fatty acid desaturase (TpsD8; SEQ ID NO:14) (NCBI Accession No. AAX14502(GI 60172920), locus AAX14502, CDS AY817152; Tonon et al., FEBS J. 272:3401-3412 (2005)) when compared to the "nr" database. Although identified as a delta-8 fatty acid desaturase in the NCBI database, AY817152 was identified as a delta-5 desaturase in Tonon et al. and the NCBI designation as a delta-8 fatty acid desaturase is likely an error. BLASTP analysis with EaD5Des1 also yielded a pLog value of 75.70 (P value of 2e-76) versus the Phaeodactylum tricornutum delta-5 fatty acid desaturase (SEQ ID NO:15) (NCBI Accession No. AAL92562(GI 19879687), locus AAL92562, CDS AY082392; Domergue et al., Eur. J. Biochem. 269:4105-4113 (2002)) when compared to the "nr" database.
[0338] The amino acid sequence for EaD5Des1 (SEQ ID NO:13) was compared to the Thalassiosira pseudonana delta-8 fatty acid desaturase (SEQ ID NO:14) and the Euglena gracilis delta-5 desaturase amino acid sequence (EgD5; SEQ ID NO:16; which is described in U.S. Provisional Application No. 60/801,172 (filed May 17, 2006; Attorney Docket No. CL-3486)) using BlastP, Clustal V and the Jotun Hein methods of sequence comparison. The % identity against the TpsD8 and EgD5 using each method is shown in Table 5 and Table 6, respectively.
[0339] Sequence percent identity calculations performed by the BlastP method are as described above. Sequence percent identity calculations were performed by the Clustal V method (Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci. 5:151-153 (1989); Higgins et al., Comput. Appl. Biosci. 8:189-191 (1992)) using the MegAlign® v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10).
[0340] Sequence percent identity calculations performed by the Jotun Hein method (Hein, J. J., Meth. Enz. 183:626-645 (1990)) were done using the MegAlign® v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=2).
TABLE-US-00005 TABLE 5 Sequence Comparison of EaD5Des1 (SEQ ID NO: 13) to TpsD8 (SEQ ID NO: 14) % Identity to % Identity to % Identity to TpsD8 TpsD8 by the TpsD8 by the Desaturase by BLASTP Jotun Hein Method Clustal V Method EaD5Des1 37% 40.8% 30.8% (SEQ ID NO: 13)
TABLE-US-00006 TABLE 6 Sequence Comparison of EaD5Des1 (SEQ ID NO: 13) to EqD5 (SEQ ID NO: 16) % Identity to % Identity to % Identity to EgD5 EgD5 by the EgD5 by the Desaturase by BLASTP Jotun Hein Method Clustal V Method EaD5Des1 73% 72.4% 77.1% (SEQ ID NO: 13)
Example 4
Functional Analysis of the Euglena gracilis UTEX 373 Delta-5 Desaturase (EaD5Des1) in Yarrowia lipolytica
[0341] The present Example describes functional analysis of EaD5Des1 (SEQ ID NO:13) in Yarrowia lipolytica. This work included the following steps: (1) Construction of Gateway®-compatible Yarrowia expression vector pY159; (2) transfer of EaD5Des1 (SEQ ID NO:12) into pY159 to produce pY169; and, (3) comparison of lipid profiles within transformant organisms comprising pY169.
Construction of Gateway®-Compatible Yarrowia Expression Vector pY159
[0342] Plasmid pY5-30 (which was previously described in PCT Publication No. WO 2005/003310 (the contents of which are hereby incorporated by reference)), is a shuttle plasmid that can replicate both in E. coli and Yarrowia lipolytica. Plasmid pY5-30 contains the following: a Yarrowia autonomous replication sequence (ARS18); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (AmpR), for selection in E. coli; a Yarrowia LEU2 gene, for selection in Yarrowia; and a chimeric TEF::GUS::XPR gene. Plasmid pDMW263 (SEQ ID NO:17) was created from pY5-30, by replacing the TEF promoter with the Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO 2005/049805) using techniques well known to one skilled in the art. Briefly, this promoter refers to a modified promoter which is located in the 5' upstream untranslated region in front of the `ATG` translation initiation codon of the fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and that is necessary for expression, plus a portion of 5' coding region that has an intron, wherein FBAINm has a 52 bp deletion between the ATG translation initiation codon and the intron of the FBAIN promoter (thereby including only 22 amino acids of the N-terminus) and a new translation consensus motif after the intron. Table 7 summarizes the components of pDMW263 (SEQ ID NO:17).
TABLE-US-00007 TABLE 7 Components of Plasmid pDMW263 (SEQ ID NO: 17) RE Sites and Nucleotides Within Description of Fragment and SEQ ID NO: 17 Chimeric Gene Components 4992-4296 ARS18 sequence (GenBank Accession No. A17608) SalI/SacII FBAINm::GUS::XPR, comprising: (8505-2014) FBAINm: FBAINm promoter (WO2005/049805) GUS: E. coli gene encoding β-glucuronidase (Jefferson, R. A. Nature. 14: 342: 837-838 (1989) XPR: ~100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741) 6303-8505 Yarrowia Leu2 gene (GenBank Accession No. AF260230)
[0343] The NcoI/SalI DNA fragment from pDMW263 (SEQ ID NO:17), containing the Yarrowia lipolytica FBAINm promoter, was cloned into the NcoI/SalI DNA fragment of pDMW237 (SEQ ID NO:18), previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), containing a synthetic delta-9 elongase gene derived from Isochrysis galbana and codon-optimized for expression in Yarrowia lipolytica (IgD9eS), to produce pY115 (SEQ ID NO:19; FIG. 3). In FIG. 3, the modified FBAINm promoter is called FBA1+Intron. It is also FBA1+Intron in other figures, as well as YAR FBA1 PRO+Intron and these terms are used interchangeably with FBAINm.
[0344] The FBAINm promoter was amplified from plasmid pY115 (SEQ ID NO:19), using PCR with oligonucleotide primers oYFBA1 (SEQ ID NO:20) and oYFBA1-6 (SEQ ID NO:21). Primer oYFBA1 (SEQ ID NO:20) was designed to introduce an BglII site at the 5' end of the promoter and primer oYFBA1-6 (SEQ ID NO:21) was designed to introduce a NotI site at the 3' end of the promoter while removing the NcoI site and thus, the ATG start codon. The resulting PCR fragment was digested with BglII and NotI and cloned into the BglII/NotI fragment of pY115, containing the vector backbone, to form pY158 (SEQ ID NO:22).
[0345] Plasmid pY158 (SEQ ID NO:22) was digested with NotI and the resulting DNA ends were filled. After filling to form blunt ends, the DNA fragments were treated with calf intestinal alkaline phosphatase and separated using agarose gel electrophoresis. The 6992 bp fragment containing the Yarrowia lipolytica FBAINm promoter was excised from the agarose gel and purified using the QIAquick® Gel Extraction Kit (Qiagen Inc., Valencia, Calif.) following the manufacturer's protocol. The purified 6992 bp fragment was ligated with cassette rfA using the Gateway Vector Conversion System (Cat. No. 11823-029, Invitrogen Corporation) following the manufacturer's protocol to form Yarrowia lipolytica Gateway® destination vector pY159 (SEQ ID NO:23; FIG. 4).
Construction of Yarrowia Expression Vectors pY169
[0346] Using the Gateway® LR Clonase® II enzyme mix (Cat. No. 11791-020, Invitrogen Corporation) and following the manufacturer's protocol, the cDNA insert from pLF119 (SEQ ID NO:11) was transferred to pY159 (SEQ ID NO:23) to form pY169 (SEQ ID NO:24, FIG. 5). In FIG. 5, EaD5Des1 is identified as EaD5-1 but they are identical.
Functional Analysis of EaD5Des1 in Yarrowia lipolytica
[0347] Strain Y2224 was isolated in the following manner: Yarrowia lipolytica ATCC #20362 cells from a YPD agar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28° C. and four of the resulting colonies were patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3 auxotrophy.
[0348] Strain Y2224 was transformed with pY169 (SEQ ID NO:24, FIG. 5) as described in the General Methods.
[0349] Single colonies of transformant Yarrowia lipolytica containing pY169 were grown in 3 mL minimal media lacking uracil supplemented with 0.2% tergitol at 30° C. for 1 day. After this, 0.1 mL was transferred to 3 mL of the same medium supplemented with either ALA, EDA, ERA, DGLA, ETA, EPA, DPA or no fatty acid. These were incubated for 16 h at 30° C., 250 rpm and then pellets were obtained by centrifugation. Cells were washed once with water, pelleted by centrifugation and air dried. Pellets were transesterified (Roughan, G. and Nishida, I., Arch. Biochem. Biophys. 276(1):38-46 (1990)) with 500 μL of 1% sodium methoxide for 30 min. at 50° C. after which 500 μL of 1M sodium chloride and 100 μL of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GC. FAMEs (5 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Cat. No. 24152, Supelco Inc.). The oven temperature was programmed to hold at 220° C. for 2.6 min, increase to 240° C. at 20° C./min and then hold for an additional 2.4 min. Carrier gas was supplied by a Whatman hydrogen generator. In the case of DPA feeding, GC analysis was carried out in a similar way except that the oven temperature was programmed to hold at 170° C. for 1.0 min, increase to 240° C. at 5° C./min and then hold for an additional 1.0 min. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).
[0350] The fatty acid profiles for Yarrowia lipolytica expressing pY169 and fed various substrates are shown in FIG. 6. Substrates (either LA--when no fatty acid fed, ALA, EDA, ERA, DGLA, ETA or DPA) were fed to assess delta-4 (DPA to DHA), delta-5 (DGLA to ARA, DTA to EPA, EDA to SCI, ERA to JUP), delta-6 (LA to GLA, ALA to STA), delta-8 (EDA to DGLA, ERA to ETA) or omega-3 (LA to ALA, EDA to ERA, DGLA to ETA) desaturase activities. Percent desaturation (% desat) was calculated by dividing the wt. % for substrate (either LA-when no fatty acid fed, ALA, EDA, ERA, DGLA, ETA or DPA) by the sum of the wt. % for the substrate (either LA--when no fatty acid fed, ALA, EDA, ERA, DGLA, ETA or DPA) and product (either GLA, STA, DGLA, ETA, ARA, EPA or DHA, respectively) and multiplying by 100 to express as a %, depending on which substrate was fed. In FIG. 6. shading indicates the substrates fed and products produced. Averages are indicated by Ave. followed by appropriate header. From the results in FIG. 6, it is clear that EaD5Des1 functions as a delta-5 desaturase with preference for DGLA and ETA over EDA and ERA. The ratio of desaturation of omega-6 substrate to omega-3 substrate (Ratio n-6/n-3) is calculated by dividing the Ave. % desat for either DGLA by ETA or EDA by ERA. In both cases, EaD5Des1 prefers n-6 substrates over n-3 substrates. The ratio of desaturation of the preferred substrate to that of the non-preferred substrate (Ratio Prod/By-Prod) is calculated by dividing the Ave. % desat for either DGLA by EDA or ETA by ERA. In both cases, EaD5Des1 has an approximately 3.5-fold preference for DGLA or ETA over EDA or ERA, respectively.
Example 5
Construction of Soybean Expression Vector pKR1153 for Co-Expression of the Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1) with a Delta-9 Elongase Derived from Euglena gracilis (EgD9e) and a Delta-8 Desaturase Derived from Euglena gracilis (EgD8)
[0351] The present Example describes construction of a soybean vector for co-expression of EaD5Des1 with EgD9e (SEQ ID NO:25; which is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562) and EgD8 (SEQ ID NO:26; described as Eg5 in PCT Publication No. WO 2006/012325)
Euglena gracilis Delta-9 Elongase (EgD9e):
[0352] A clone from the Euglena cDNA library (eeg1c), called eeg1c.pk001.n5f, containing the Euglena gracilis delta-9 elongase (EgD9e; SEQ ID NO:25; which is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562) the contents of which are hereby incorporated by reference) was used as template to amplify EgD9e with oligonucleotide primers oEugEL1-1 (SEQ ID NO:27) and oEugEL1-2 (SEQ ID NO:28) using the VentR® DNA Polymerase (Cat. No. M0254S, New England Biolabs Inc., Beverly, Mass.) following the manufacturer's protocol. The resulting DNA fragment was cloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR906 (SEQ ID NO:29).
[0353] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:30, 7085 bp sequence), a derivative of pKS123 which was previously described in PCT Publication No. WO 02/008269 (the contents of which are hereby incorporated by reference), contains the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene 25:179-188 (1983)), flanked by the T7 promoter and transcription terminator (T7prom/HPT/T7term cassette), and a bacterial origin of replication (ori) for selection and replication in bacteria (e.g., E. coli). In addition, pKR72 also contains HPT, flanked by the 35S promoter (Odell et al., Nature 313:810-812 (1985)) and NOS 3' transcription terminator (Depicker et al., J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/HPT/NOS3' cassette) for selection in plants such as soybean. pKR72 also contains a NotI restriction site, flanked by the promoter for the a' subunit of β-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3' transcription termination region of the phaseolin gene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)), thus allowing for strong tissue-specific expression in the seeds of soybean of genes cloned into the NotI site.
[0354] The AscI fragment from plasmid pKS102 (SEQ ID NO:31), previously described in PCT Publication No. WO 02/00905 (the contents of which are hereby incorporated by reference), containing the T7prom/hpt/T7term cassette and bacterial ori, was combined with the Asc1 fragment of plasmid pKR72 (SEQ ID NO:30), containing the βcon/NotI/Phas cassette to produce pKR197 (SEQ ID NO:32), previously described in PCT Publication No. WO 04/071467 (the contents of which are hereby incorporated by reference).
[0355] The gene for the Euglena gracilis delta-9 elongase was released from pKR906 (SEQ ID NO:29) by digestion with NotI and cloned into the NotI site of pKR197 to produce intermediate cloning vector pKR911 (SEQ ID NO:33).
Euglena gracilis Delta-8 Desaturase (EgD8):
[0356] Plasmid pKR680 (SEQ ID NO:34), which was previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), contains the Euglena gracilis delta-8 desaturase (EgD8; SEQ ID NO:26; described as Eg5 in WO 2006/012325) flanked by the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell 1:1079-1093 (1989)) and the KTi 3' termination region, the isolation of which is described in U.S. Pat. No. 6,372,965, followed by the soy albumin transcription terminator, which was previously described in PCT Publication No. WO 2004/071467 (Kti/NotI/Kti3'Salb3' cassette).
[0357] Plasmid pKR680 (SEQ ID NO:34) was digested with BsiWI and the fragment containing EgD8 was cloned into the BsiWI site of pKR911 (SEQ ID NO:33) to produce pKR913 (SEQ ID NO:35).
Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1):
[0358] In order to introduce NotI sites at the 5' and 3' ends of the coding sequence, EaD5Des1 was PCR amplified from pLF119 (SEQ ID NO:11) with oligonucleotide primers oEAd5-1-1 (SEQ ID NO:36) and oEAd5-1-2 (SEQ ID NO:37) using the Phusion® High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. The resulting DNA fragment was cloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR1136 (SEQ ID NO:38).
[0359] Plasmid pKR767 (SEQ ID NO:39), which was previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), contains the Mortierella alpina delta-5 desaturase (MaD5; SEQ ID NO:40, which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and WO 2005/047479) flanked by the promoter for the soybean glycinin Gy1 gene and the pea leguminA2 3' transcription termination region (Gy1/MaD5/legA2 cassette; the construction of which is described in WO 2006/012325). Plasmid pKR974 (SEQ ID NO:41) is identical to pKR767 (SEQ ID NO:40) except the NotI fragment containing MaD5 has been replaced with a NotI fragment containing the Saprolegnia diclina delta-5 desaturase (SaD5; SEQ ID NO:42, which is described in PCT Publication No. WO 2004/071467. In addition, an MfeI site in the legA2 terminator of pKR974 (SEQ ID NO:41) was removed by digestion with MfeI, filling the MfeI site and religating (i.e., CAATTG converted to CAATTAATTG) and therefore, the legA2 terminator of pKR974 (SEQ ID NO:41) is 770 bp versus 766 bp for pKR767 (SEQ ID NO:40).
[0360] The gene for the Euglena anabaena delta-5 desaturase was released from pKR1136 (SEQ ID NO:38) by digestion with NotI and cloned into the NotI site of pKR974 (SEQ ID NO:41) to produce pKR1139 (SEQ ID NO:43).
[0361] Plasmid pKR1139 (SEQ ID NO:43) was digested with SbfI and the fragment containing the Euglena anabaena delta-5 desaturase was cloned into the SbfI site of pKR913 (SEQ ID NO:35) to produce pKR1153 (SEQ ID NO:44, FIG. 7). In this way, the Euglena anabaena delta-5 desaturase (EaD5Des1) could be co-expressed with the Euglena gracilis delta-8 desaturase (EgD8) and the Euglena gracilis delta-9 elongase (EgD9e) behind strong, seed-specific promoters. In FIG. 7, EaD5Des1, EgD8 and EgD9e are referred to as EA d5 DS, eug d8-sq5 and eug ell, respectively.
Example 6
Production and Model System Transformation of Somatic Soybean Embryo Cultures with Soybean Expression Vectors and Plant Regeneration Culture Conditions
[0362] Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the preferred subculture interval is every 7 days).
Soybean embryogenic suspension cultures are transformed with the soybean expression plasmids by the method of particle gun bombardment (Klein et al., Nature 327:70 (1987)) using a DuPont Biolistic PDS1000/HE instrument (helium retrofit) for all transformations.
Soybean Embryogenic Suspension Culture Initiation:
[0363] Soybean cultures are initiated twice each month with 5-7 days between each initiation. Pods with immature seeds from available soybean plants are picked 45-55 days after planting. Seeds are removed from the pods and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 min in a 5% Clorox solution with 1 drop of Ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of soap, mixed well). Seeds are rinsed using 2 1-liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed is cut and the cotyledons pressed out of the seed coat. When cultures are being prepared for production transformation, cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and are maintained at 26° C. with cool white fluorescent lights on 16:8 h day/night photoperiod at light intensity of 60-80 μE/m2/s for eight weeks, with a media change after 4 weeks. When cultures are being prepared for model system experiments, cotyledons are transferred to plates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks, and then transferred to SB1 for 2-4 weeks. Light and temperature conditions are the same as described above. After incubation on SB1 medium, secondary embryos are cut and placed into SB196 liquid media for 7 days.
Preparation of DNA for Bombardment:
[0364] Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene are used for bombardment. Fragments from soybean expression plasmids are obtained by gel isolation of digested plasmids. In each case, 100 μg of plasmid DNA is used in 0.5 mL of the specific enzyme mix described below. Plasmids are digested with AscI (100 units) in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, pH 7.9), 100 μg/mL BSA, and 5 mM beta-mercaptoethanol at 37° C. for 1.5 hr. The resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing gene cassettes are cut from the agarose gel. DNA is purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.
[0365] A 50 μL aliquot of sterile distilled water containing 3 mg of gold particles (3 mg gold) is added to 30 μL of a 10 ng/μL DNA solution (either intact plasmid or DNA fragment prepared as described herein), 25 μL 5M CaCl2 and 20 μL of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. The supernatant is removed, followed by a wash with 400 μL 100% ethanol and another brief centrifugation. The 400 ul ethanol is removed and the pellet is resuspended in 40 μL of 100% ethanol. Five μL of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μL aliquot contains approximately 0.375 mg gold per bombardment (e.g., per disk).
[0366] For model system transformations, the protocol is identical except for a few minor changes (ie, 1 mg of gold particles is added to 5 μL of a 1 μg/μL DNA solution, 50 μL of a 2.5M CaCl2 is used and the pellet is ultimately resuspended in 85 μL of 100% ethanol thus providing 0.058 mg of gold particles per bombardment).
Tissue Preparation and Bombardment with DNA:
[0367] Approximately 150-200 mg of seven day old embryogenic suspension cultures is placed in an empty, sterile 60×15 mm petri dish and the dish is covered with plastic mesh. The chamber is evacuated to a vacuum of 27-28 inches of mercury, and tissue is bombarded one or two shots per plate with membrane rupture pressure set at 1100 PSI. Tissue is placed approximately 3.5 inches from the retaining/stopping screen. Model system transformation conditions are identical except 100-150 mg of embryogenic tissue is used, rupture pressure is set at 650 PSI and tissue is place approximately 2.5 inches from the retaining screen.
Selection of Transformed Embryos:
[0368] Transformed embryos are selected either using hygromycin (when the hygromycin B phosphotransferase (HPT) gene is used as the selectable marker) or chlorsulfuron (when the acetolactate synthase (ALS) gene is used as the selectable marker).
[0369] Following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six to eight days post-bombardment, the SB196 is exchanged with fresh SB196 containing either 30 mg/L hygromycin or 100 ng/mL chlorsulfuron, depending on the selectable marker used. The selection media is refreshed weekly. Four to six weeks post-selection, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters.
Embryo Maturation:
[0370] For production transformations, isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures. Transformed embryogenic clusters are cultured for four-six weeks in multiwell plates at 26° C. in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 μE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for one-two weeks and then subcultured to SB103 medium for 3-4 weeks to mature embryos. After maturation on plates in SB103, individual embryos are removed from the clusters, dried and screened for alterations in their fatty acid compositions as described in Example 7.
[0371] For model system transformations, embryos are matured in soybean histodifferentiation and maturation liquid medium (SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis 24:393 (2005)) using a modified procedure. Briefly, after 4 weeks of selection in SB196 as described above, embryo clusters are removed to 35 mL of SB228 (SHaM liquid media) in a 250 mL Erlenmeyer flask. Tissue is maintained in SHaM liquid media on a rotary shaker at 130 rpm and 26° C. with cool white fluorescent lights on a 16:8 hr day/night photoperiod at a light intensity of 60-85 μE/m2/s for 2 weeks as embryos mature. Embryos grown for 2 weeks in SHaM liquid media are equivalent in size and fatty acid content to embryos cultured on SB166/SB103 for 5-8 weeks.
[0372] After maturation in SHaM liquid media, individual embryos are removed from the clusters, dried and screened for alterations in their fatty acid compositions as described in Example 7.
Media Recipes:
SB 196--FN Lite Liquid Proliferation Medium (Per Liter)
TABLE-US-00008
[0373] MS FeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO3 2.83 gm (NH4)2SO4 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00009
[0374] Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na2 EDTA* 3.724 g 1.862 g FeSO4--7H2O 2.784 g 1.392 g 2 MS Sulfate 100x stock MgSO4--7H2O 37.0 g 18.5 g MnSO4--H2O 1.69 g 0.845 g ZnSO4--7H2O 0.86 g 0.43 g CuSO4--5H2O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl2--2H2O 30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl2--6H2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x Stock KH2PO4 18.5 g 9.25 g H3BO3 0.62 g 0.31 g Na2MoO4--2H2O 0.025 g 0.0125 g *Add first, dissolve in dark bottle while stirring
SB1 Solid Medium (Per Liter)
TABLE-US-00010
[0375] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 31.5 g glucose 2 mL 2,4-D (20 mg/L final concentration) pH 5.7 8 g TC agar
SB199 Solid Medium (Per Liter)
TABLE-US-00011
[0376] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 30 g Sucrose 4 ml 2,4-D (40 mg/L final concentration) pH 7.0 2 gm Gelrite
SB 166 Solid Medium (Per Liter)
TABLE-US-00012
[0377] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl2 hexahydrate 5 g activated charcoal pH 5.7 2 g gelrite
SB 103 Solid Medium (Per Liter)
TABLE-US-00013
[0378] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl2 hexahydrate pH 5.7 2 g gelrite
SB 71-4 Solid Medium (Per Liter)
TABLE-US-00014
[0379] 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL - Cat. No. 21153-036) pH 5.7 5 g TC agar
2,4-D Stock
TABLE-US-00015
[0380] Obtain premade from Phytotech Cat. No. D 295 - concentration 1 mg/mL
B5 Vitamins Stock (Per 100 mL)
TABLE-US-00016
[0381] Store aliquots at -20° C. 10 g myo-inositol 100 mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine
If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.
SB 228--Soybean Histodifferentiation & Maturation (SHaM) (Per Liter)
TABLE-US-00017
[0382] DDI H2O 600 mL FN-Lite Macro Salts for SHaM 10X 100 mL MS Micro Salts 1000x 1 mL MS FeEDTA 100x 10 mL CaCl 100x 6.82 mL B5 Vitamins 1000x 1 mL L-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mL pH 5.8 Autoclave Add to cooled media (≦30° C.): *Glutamine (final concentration 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL after glutamine addition.
Since glutamine degrades relatively rapidly, it may be preferable to add immediately prior to using media. Expiration 2 weeks after glutamine is added; base media can be kept longer w/o glutamine.
FN-Lite Macro for SHAM 10×--Stock #1 (Per Liter)
TABLE-US-00018
[0383] (NH4)2SO4 (ammonium sulfate) 4.63 g KNO3 (potassium nitrate) 28.3 g MgSO4*7H20 (magnesium sulfate heptahydrate) 3.7 g KH2PO4 (potassium phosphate, monobasic) 1.85 g Bring to volume Autoclave
MS Micro 1000×--Stock #2 (Per 1 Liter)
TABLE-US-00019
[0384] H3BO3 (boric acid) 6.2 g MnSO4*H2O (manganese sulfate monohydrate) 16.9 g ZnSO4*7H20 (zinc sulfate heptahydrate) 8.6 g Na2MoO4*2H20 (sodium molybdate dihydrate) 0.25 g CuSO4*5H20 (copper sulfate pentahydrate) 0.025 g CoCl2*6H20 (cobalt chloride hexahydrate) 0.025 g KI (potassium iodide) 0.8300 g Bring to volume Autoclave
FeEDTA 100×--Stock #3 (Per Liter)
TABLE-US-00020
[0385] Na2EDTA* (sodium EDTA) 3.73 g FeSO4*7H20 (iron sulfate heptahydrate) 2.78 g *EDTA must be completely dissolved before adding iron. Bring to Volume
Solution is photosensitive. Bottle(s) should be wrapped in foil to omit light. Autoclave
Ca 100×--Stock #4 (Per Liter)
TABLE-US-00021
[0386] CaCl2*2H20 (calcium chloride dihydrate) 44 g Bring to Volume Autoclave
B5 Vitamin 1000×--Stock #5 (Per Liter)
TABLE-US-00022
[0387] Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store frozen
4% Glutamine Stock #6 (Per Liter)
TABLE-US-00023
[0388] DDI water heated to 30° C. 900 mL L-Glutamine 40 g Gradually add while stirring and applying low heat. Do not exceed 35° C. Bring to Volume Filter Sterilize Store frozen* *Note: Warm thawed stock in 31° C. bath to fully dissolve crystals.
Regeneration of Soybean Somatic Embryos into Plants:
[0389] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated. Embryos are matured as described in above. After subculturing on medium SB103 for 3 weeks, individual embryos can be removed from the clusters and screened for alterations in their fatty acid compositions as described in Example 7. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, could be screened at this stage. This would include, but not be limited to, alterations in fatty acid profile, protein profile and content, carbohydrate content, growth rate, viability, or the ability to develop normally into a soybean plant.
[0390] Matured individual embryos are desiccated by placing them into an empty, small petri dish (35×10 mm) for approximately 4 to 7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they are left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then are planted in Redi-Earth in 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10'' pot of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for fatty acids.
Example 7
Fatty Acid Analysis of Transgenic Somatic Soybean Embryos
[0391] Mature somatic soybean embryos are a good model for zygotic embryos. While in the globular embryo state in liquid culture, somatic soybean embryos contain very low amounts of triacylglycerol or storage proteins typical of maturing, zygotic soybean embryos. At this developmental stage, the ratio of total triacylglyceride to total polar lipid (phospholipids and glycolipid) is about 1:4, as is typical of zygotic soybean embryos at the developmental stage from which the somatic embryo culture was initiated. At the globular stage as well, the mRNAs for the prominent seed proteins, α'-subunit of β-conglycinin, kunitz trypsin inhibitor 3, and seed lectin are essentially absent. Upon transfer to hormone-free media to allow differentiation to the maturing somatic embryo state, triacylglycerol becomes the most abundant lipid class. As well, mRNAs for α'-subunit of β-conglycinin, kunitz trypsin inhibitor 3 and seed lectin become very abundant messages in the total mRNA population. On this basis, the somatic soybean embryo system behaves very similarly to maturing zygotic soybean embryos in vivo, and is thus a good and rapid model system for analyzing the phenotypic effects of modifying the expression of genes in the fatty acid biosynthesis pathway (see PCT Publication No. WO 2002/00904, Example 3). Most importantly, the model system is also predictive of the fatty acid composition of seeds from plants derived from transgenic embryos.
[0392] A subset of soybean embryos for each event generated from either production transformation or model system transformation (as described in Example 6) are harvested in the following way. Embryos (5-10 embryos) from each event are picked into glass GC vials and fatty acid methyl esters are prepared by transesterification. For transesterification, 50 μL of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane is added to the embryos in glass vials and incubated for 30 min at room temperature while shaking. Fatty acid methyl esters (5 μL injected from hexane layer) are separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Cat. No. 24152, Supelco Inc.). The oven temperature is programmed to hold at 220° C. for 2.6 min, increase to 240° C. at 20° C./min and then hold for an additional 2.4 min. Carrier gas is supplied by a Whatman hydrogen generator. Retention times are compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.). Events having good phenotype can be re-analyzed by GC using identical conditions except the oven temperature is held at 150° C. for 1 min and then increased to 240° C. at 5° C./min.
Example 8
Construction of Alternate Soybean Expression Vectors for Expression of Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1)
[0393] In addition to the genes, promoters, terminators and gene cassettes described herein, one skilled in the art can appreciate that other promoter/gene/terminator cassette combinations can be synthesized in a way similar to, but not limited to, that described herein for expression of EaD5Des1. Similarly, it may be desirable to express other PUFA genes (such as those described below in Table 10), for co-expression with the delta-5 desaturase of the present invention.
[0394] For instance, PCT Publication Nos. WO 2004/071467 and WO 2004/071178 describe the isolation of a number of promoter and transcription terminator sequences for use in embryo-specific expression in soybean. Furthermore, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO 2006/012325 describe the synthesis of multiple promoter/gene/terminator cassette combinations by ligating individual promoters, genes and transcription terminators together in unique combinations. Generally, a NotI site flanked by the suitable promoter (such as those listed in, but not limited to, Table 8) and a transcription terminator (such as those listed in, but not limited to, Table 9) is used to clone the desired gene. NotI sites can be added to a gene of interest such as those listed in, but not limited to, Table 7 using PCR amplification with oligonucleotides designed to introduce NotI sites at the 5' and 3' ends of the gene. The resulting PCR product is then digested with NotI and cloned into a suitable promoter/NotI/terminator cassette.
[0395] In addition, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO 2006/012325 describe the further linking together of individual gene cassettes in unique combinations, along with suitable selectable marker cassettes, in order to obtain the desired phenotypic expression. Although this is done mainly using different restriction enzymes sites, one skilled in the art can appreciate that a number of techniques can be utilized to achieve the desired promoter/gene/transcription terminator combination. In so doing, any combination of embryo-specific promoter/gene/transcription terminator cassettes can be achieved. One skilled in the art can also appreciate that these cassettes can be located on individual DNA fragments or on multiple fragments where co-expression of genes is the outcome of co-transformation of multiple DNA fragments.
TABLE-US-00024 TABLE 8 Seed-specific Promoters Promoter Organism Promoter Reference β-conglycinin α'-subunit soybean Beachy et al., EMBO J. 4: 3047-3053 (1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell 1: 1079-1093 (1989) Annexin soybean WO 2004/071467 glycinin Gy1 soybean WO 2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613 legumin A1 pea Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991) β-conglycinin β-subunit soybean WO 2004/071467 BD30 (also called P34) soybean WO 2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991)
TABLE-US-00025 TABLE 9 Transcription Terminators Transcription Terminator Organism Reference phaseolin 3' bean WO 2004/071467 kunitz trypsin inhibitor 3' soybean WO 2004/071467 BD30 (also called P34) 3' soybean WO 2004/071467 legumin A2 3' pea WO 2004/071467 albumin 2S 3' soybean WO 2004/071467
TABLE-US-00026 TABLE 10 PUFA Biosynthetic Pathway Genes Gene Organism Reference delta-6 desaturase Saprolegnia diclina WO 2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No. 5,968,809 elongase Mortierella alpina WO 2000/12720 U.S. Pat. No. 6,403,349 delta-5 desaturase Mortierella alpina U.S. Pat. No. 6,075,183 delta-5 desaturase Saprolegnia diclina WO 2002/081668 delta-5 desaturase Peridinium sp. U.S. Provisional Application No. 60/801,119 delta-5 desaturase Euglena gracilis U.S. Provisional Application No. 60/801,172 delta-15 desaturase Fusarium WO 2005/047479 moniliforme delta-17 desaturase Saprolegnia diclina WO 2002/081668 elongase Thraustochytrium WO 2002/08401 aureum U.S. Pat. No. 6,677,145 elongase Pavlova sp. Pereira et al., Biochem. J. 384: 357-366 (2004) delta-4 desaturase Schizochytrium WO 2002/090493 aggregatum U.S. Pat. No. 7,045,683 delta-4 desaturase Isochrysis galbana WO 2002/090493 U.S. Pat. No. 7,045,683 delta-4 desaturase Thraustochytrium WO 2002/090493 aureum U.S. Pat. No. 7,045,683 delta-4 desaturase Euglena gracilis U.S. patent application No. 10/552,127 delta-9 elongase Isochrysis galbana WO 2002/077213 delta-9 elongase Euglena gracilis U.S. patent application No. 11/601,563 delta-9 elongase Eutreptiella sp. U.S. patent application No. CCMP389 11/601,564 delta-8 desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No. 6,825,017 WO 2004/057001 WO 2006/012325 delta-8 desaturase Acanthamoeba Sayanova et al., FEBS Lett. castellanii 580: 1946-1952 (2006) delta-8 desaturase Pavlova salina WO 2005/103253 delta-8 desaturase Pavlova lutheri U.S. Provisional Application No. 60/795,810 delta-8 desaturase Tetruetreptia U.S. Provisional Application pomquetensis No. 60/853,563 CCMP1491 delta-8 desaturase Eutreptiella sp. U.S. Provisional Application CCMP389 No. 60/853,563 delta-8 desaturase Eutreptiella U.S. Provisional Application cf_gymnastica No. 60/853,563 CCMP1594
Example 9
Synthesis of a Codon-Optimized Delta-5 Desaturase Gene for Yarrowia lipolytica (EaD5S)
[0396] The codon usage of the delta-5 desaturase gene (EaD5) of Euglena anabaena was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753. Specifically, a codon-optimized delta-5 desaturase gene (designated "EaD5S", SEQ ID NO:45) was designed based on the coding sequence of EaD5Des1 (SEQ ID NOs:12 and 13), according to the Yarrowia codon usage pattern (PCT Publication No. WO 2004/101753), the consensus sequence around the `ATG` translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)). In addition to modification of the translation initiation site, 183 bp of the 1362 bp coding region were modified (13.4%) and 174 codons were optimized (38.3%). The GC content was reduced from 57.6% within the wild type gene (i.e., EaD5Des1) to 54.6% within the synthetic gene (i.e., EaD5S). A NcoI site and NotI sites were incorporated around the translation initiation codon and after the stop codon of EaD5S (SEQ ID NO:45), respectively. FIGS. 8A, 8B and 8C show a comparison of the nucleotide sequences of EaD5Des1 (SEQ ID NO:12) and EaD5S (SEQ ID NO:45). The codon optimized EaD5S gene did not change any amino acid sequence of EaD5Des1 (SEQ ID NO:13). The designed EaD5S gene was synthesized by GenScript Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pEaD5S (SEQ ID NO:46; FIG. 9).
[0397] Based on the teachings herein concerning vector construction and suitable promoter and terminators for use in Yarrowia lipolytica, one of skill in the art will be able to construct additional plasmids suitable for expression of EaD5S (SEQ ID NO:45).
Example 10
Functional Analysis of the Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1) Co-Expressed with a Delta-9 Elongase Derived from Euglena gracilis (EgD9e) a Delta-8 Desaturase Derived from Euglena gracilis (EgD8) and a Delta-17 Desaturase from Saprolegnia Diclina in Soy Somatic Embryos
[0398] The present example describes the transformation and expression in soybean somatic embryos of pKR1153 (SEQ ID NO:44; Example 5) comprising EaD5Des1, EgD9e and EgD8 along with pKR328 (described in PCT Publication No. WO 04/071467) comprising the Saprolegnia diclina delta-17 desaturase SdD17 under control of the annexin promoter and having a hygromycin resistance gene for selection in plants.
[0399] Soybean embryogenic suspension culture (cv. Jack) was transformed with pKR1153 (SEQ ID NO:44) and pKR328, and embryos were matured in soybean histodifferentiation and maturation liquid medium (SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)) as described in Example 6 and previously described in PCT Publication No. WO 2007/136877 (the contents of which are hereby incorporated by reference).
[0400] After maturation in SHaM liquid media, a subset of transformed soybean embryos (i.e., 5 embryos per event) were harvested and analyzed for fatty acid profiles by GC as described in Example 7 and herein.
[0401] In this way, approximately 30 events transformed with pKR1153 and pKR328 (Experiment MSE2140) were analyzed, and the ten events having the highest average correct delta-5 desaturase activities (average of the 5 embryos analyzed) are shown in FIG. 11.
[0402] In FIG. 11, fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA and EPA. Fatty acid compositions for an individual embryo were expressed as the weight percent (wt. %) of total fatty acids and the average fatty acid composition is an average of six individual embryos for each event.
[0403] The activity of the delta-5 desaturase is expressed as percent delta-5 desaturation ("% delta-5 desat"), calculated according to the following formula: ([product]/[substrate+product])*100. More specifically, the percent delta-5 desaturation was determined as: ([ARA+EPA]/[DGLA+ETA+ARA+EPA])*100.
Sequence CWU
1
1
4611524DNAEuglena anabaena 1acccagtgta cacctttgac agtcccttcg cccaggatgt
caagcagagc gttcgggagg 60tcatgaaggg gcgcaactgg tacgccacgc ccggcttttg
gctgcggacc gcgctgatca 120tcgcgtgcac tgccataggc gaatggtatt ggatcactac
cggggcagtg atgtggggca 180tcttcaccgg gtacttccac agccagattg ggttggcgat
tcaacacgat gcctctcacg 240gagccatcag caaaaagccc tgggtgaacg cctttttcgc
ctacggcatc gacgccattg 300gatcctcccg ctggatctgg ctgcagtccc acattatgcg
ccaccacacc tacaccaacc 360agcatggcct ggacctggac gctgcctcgg cggagccgtt
cattttgttc cactcctacc 420cggcaacaaa tgcgtcacga aagtggtacc atcggttcca
ggcgtggtac atgtacatcg 480ttttggggat gtatggtgtg tcgatggtgt acaatccgat
gtacttgttc acgatgcagc 540acaacgacac aatcccagag gccacctctc ttagaccagg
cagctttttc aaccggcagc 600gcgccttcgc cgtttccctc cgcctactgt tcatcttccg
caacgccttc ctcccctggt 660acatcgcggg cgcctctccg ctgctcacca tcctgctggt
gccaacggtc acaggcatct 720tcttgacatt tgtttttgtg ctgtcccata actttgaagg
cgctgagcgg acccccgaaa 780agaactgcaa ggccaaaagg gccaaggagg ggaaggaggt
ccgcgatgta gaggaggacc 840gggtggactg gtaccgggcg caggccgaga ccgcggcgac
ctacgggggc agcgtcggga 900tgatgctgac cggcggtttg aacctgcaga tcgagcacca
cttgttcccc cgcatgtcct 960cttggcacta ccccttcatc caagatacgg tgcgggaatg
ttgcaagcgc catggcgtgc 1020gctacacata ctacccgacc atcctggaga atataatgtc
cacgctccgc tacatgcaga 1080aggtgggcgt ggcccacaca attcaggatg cccaggaatt
ctgagtgagt tcgatccgca 1140tcgacgtcta ccatttttga tgctgtctat tcctgttttc
agtcacctcc agcattctca 1200tggctggtga ccactgcccc tctaacccat tgtgacacac
cgccaaagac tttgcctctt 1260ttttttccct ttcttttgtc ctcggggtgc tttggccggt
gtttactcgc cttgcagtcc 1320ccgcaaacga ccgacgttta agctccgttg ttgactgggc
cgctcgtaaa cccatctgca 1380ggttgaggct cccatggaga attgtgatgg ctgattagga
ggtggcgggg catacatgcc 1440tcgacactca aagccgggcg gcttctggat tcgaaaacgc
aaatgggcgc tttggaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaa
152421347DNAEuglena gracilis 2atggctctca gtcttaccac
agaacagctg ttagaacgcc ctgatttggt tgcgattgat 60ggcatcctct acgaccttga
agggcttgcc aaagttcatc caggaggaga tttgattctc 120gcttctggtg cctctgatgc
ctcccctctc ttttattcaa tgcatccata cgtcaaaccg 180gagaattcca aattgcttca
acagttcgtc cgagggaagc atgaccgcac ctcgaaggac 240attgtctaca cgtatgattc
tcccttcgca caagacgtta agcggacaat gcgcgaggtg 300atgaaaggga ggaactggta
cgcaacccct ggcttctggc tgcgcaccgt tgggatcatc 360gccgtgacgg ccttttgcga
gtggcactgg gctaccacgg ggatggtgct gtggggcctg 420ttgactggat tcatgcacat
gcagatcggc ttatccatcc agcatgatgc gtcccacggg 480gccatcagca agaagccttg
ggtcaacgcc ctcttcgcct acggcattga cgtcatcgga 540tcgtcccggt ggatttggct
gcagtcgcac atcatgcggc accacaccta caccaaccag 600cacggcctcg acctggatgc
ggagtcggca gagccgttcc tggtgttcca caactacccc 660gccgcaaaca ccgcccgaaa
gtggttccac cgcttccaag cttggtacat gtaccttgtg 720ctgggggcat acggggtatc
gctggtgtac aacccgctct acattttccg gatgcagcac 780aatgacacca tcccagagtc
tgtcacggcc atgcgggaaa atggctttct gcggcgctac 840cgcacacttg cattcgtgat
gcgagctttc ttcatcttcc ggaccgcatt cttgccctgg 900tacctcactg ggacctcatt
gctgatcacc attcctctgg tgcccaccgc aactggtgcc 960ttcttgacgt tcttcttcat
tttgtcccac aattttgatg gctccgaacg gatccccgac 1020aagaactgca aggttaagag
atctgagaag gacgttgagg ctgaccaaat tgactggtat 1080cgggcgcagg tggagacgtc
ctccacatac ggtggcccca tcgccatgtt cttcactggc 1140ggtctcaatt tccagatcga
gcaccacctc tttccccgga tgtcgtcttg gcactacccc 1200ttcgtccagc aggcggtccg
ggagtgttgc gaacgccatg gagtgcgata tgttttctac 1260cctaccatcg tcggcaacat
catctccacc ctgaagtaca tgcataaggt gggtgtcgtc 1320cactgcgtga aggacgcaca
ggattcc 1347332DNAArtificial
SequenceEuglena gracilis delta-5 desaturase oligonucleotide YL794
3tttccatggc tctcagtctt accacagaac ag
32427DNAArtificial SequenceEuglena gracilis delta-5 desaturase
oligonucleotide YL797 4gtacatgtac caagcttgga agcggtg
27527DNAArtificial SequenceEuglena gracilis delta-5
desaturase oligonucleotide YL796 5caccgcttcc aagcttggta catgtac
27639DNAArtificial SequenceEuglena
gracilis delta-5 desaturase oligonucleotide YL795 6tttgcggccg
cttaggaatc ctgtgcgtcc ttcacgcag
3978165DNAArtificial Sequenceplasmid pZUF17 7gtacgagccg gaagcataaa
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60ttaattgcgt tgcgctcact
gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120taatgaatcg gccaacgcgc
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa catgtgagca 300aaaggccagc aaaaggccag
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360ctccgccccc ctgacgagca
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420acaggactat aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540tctcatagct cacgctgtag
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600tgtgtgcacg aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660gagtccaacc cggtaagaca
cgacttatcg ccactggcag cagccactgg taacaggatt 720agcagagcga ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc 780tacactagaa ggacagtatt
tggtatctgc gctctgctga agccagttac cttcggaaaa 840agagttggta gctcttgatc
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900tgcaagcagc agattacgcg
cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960acggggtctg acgctcagtg
gaacgaaaac tcacgttaag ggattttggt catgagatta 1020tcaaaaagga tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080agtatatatg agtaaacttg
gtctgacagt taccaatgct taatcagtga ggcacctatc 1140tcagcgatct gtctatttcg
ttcatccata gttgcctgac tccccgtcgt gtagataact 1200acgatacggg agggcttacc
atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260tcaccggctc cagatttatc
agcaataaac cagccagccg gaagggccga gcgcagaagt 1320ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga agctagagta 1380agtagttcgc cagttaatag
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440tcacgctcgt cgtttggtat
ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500acatgatccc ccatgttgtg
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560agaagtaagt tggccgcagt
gttatcactc atggttatgg cagcactgca taattctctt 1620actgtcatgc catccgtaag
atgcttttct gtgactggtg agtactcaac caagtcattc 1680tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740gcgccacata gcagaacttt
aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800ctctcaagga tcttaccgct
gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860tgatcttcag catcttttac
tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920aatgccgcaa aaaagggaat
aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980tttcaatatt attgaagcat
ttatcagggt tattgtctca tgagcggata catatttgaa 2040tgtatttaga aaaataaaca
aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220acgttcgccg gctttccccg
tcaagctcta aatcgggggc tccctttagg gttccgattt 2280agtgctttac ggcacctcga
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400ggactcttgt tccaaactgg
aacaacactc aaccctatct cggtctattc ttttgattta 2460taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520aacgcgaatt ttaacaaaat
attaacgctt acaatttcca ttcgccattc aggctgcgca 2580actgttggga agggcgatcg
gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640gatgtgctgc aaggcgatta
agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700aaacgacggc cagtgaattg
taatacgact cactataggg cgaattgggt accgggcccc 2760ccctcgaggt cgatggtgtc
gataagcttg atatcgaatt catgtcacac aaaccgatct 2820tcgcctcaag gaaacctaat
tctacatccg agagactgcc gagatccagt ctacactgat 2880taattttcgg gccaataatt
taaaaaaatc gtgttatata atattatatg tattatatat 2940atacatcatg atgatactga
cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000gcctccaact gatgttctca
atatttaagg ggtcatctcg cattgtttaa taataaacag 3060actccatcta ccgcctccaa
atgatgttct caaaatatat tgtatgaact tatttttatt 3120acttagtatt attagacaac
ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180tttataatgg cagttcgttc
atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240gggaaatctt aaatatggat
agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300gcaacgaaaa aaatcccttg
tacaacataa atagtcatcg agaaatatca actatcaaag 3360aacagctatt cacacgttac
tattgagatt attattggac gagaatcaca cactcaactg 3420tctttctctc ttctagaaat
acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480agtcatttca tcccacatat
tccttggatt tctctccaat gaatgacatt ctatcttgca 3540aattcaacaa ttataataag
atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600tctggtgtgc ttctcgtatt
tatttttatt ctaatgatcc attaaaggta tatatttatt 3660tcttgttata taatcctttt
gtttattaca tgggctggat acataaaggt attttgattt 3720aattttttgc ttaaattcaa
tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780ccatactttt gaagaagcaa
aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840cgttccgcag aatctagaat
gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900ctccctgaga tattgtacat
ttttgctttt acaagtacaa gtacatcgta caactatgta 3960ctactgttga tgcatccaca
acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020gattcattac cgctatgtat
acctacttgt acttgtagta agccgggtta ttggcgttca 4080attaatcata gacttatgaa
tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140tgctacttgg gtgtaatatt
gggatctgtt cggaaatcaa cggatgctca atcgatttcg 4200acagtaatta attaagtcat
acacaagtca gctttcttcg agcctcatat aagtataagt 4260agttcaacgt attagcactg
tacccagcat ctccgtatcg agaaacacaa caacatgccc 4320cattggacag atcatgcgga
tacacaggtt gtgcagtatc atacatactc gatcagacag 4380gtcgtctgac catcatacaa
gctgaacaag cgctccatac ttgcacgctc tctatataca 4440cagttaaatt acatatccat
agtctaacct ctaacagtta atcttctggt aagcctccca 4500gccagccttc tggtatcgct
tggcctcctc aataggatct cggttctggc cgtacagacc 4560tcggccgaca attatgatat
ccgttccggt agacatgaca tcctcaacag ttcggtactg 4620ctgtccgaga gcgtctccct
tgtcgtcaag acccaccccg ggggtcagaa taagccagtc 4680ctcagagtcg cccttaggtc
ggttctgggc aatgaagcca accacaaact cggggtcgga 4740tcgggcaagc tcaatggtct
gcttggagta ctcgccagtg gccagagagc ccttgcaaga 4800cagctcggcc agcatgagca
gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860ctccttgtac tgggagttct
cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt 4920ttcctcggca ccagctcgca
ggccagcaat gattccggtt ccgggtacac cgtgggcgtt 4980ggtgatatcg gaccactcgg
cgattcggtg acaccggtac tggtgcttga cagtgttgcc 5040aatatctgcg aactttctgt
cctcgaacag gaagaaaccg tgcttaagag caagttcctt 5100gagggggagc acagtgccgg
cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160catgcacaca taaggtccga
ccttatcggc aagctcaatg agctccttgg tggtggtaac 5220atccagagaa gcacacaggt
tggttttctt ggctgccacg agcttgagca ctcgagcggc 5280aaaggcggac ttgtggacgt
tagctcgagc ttcgtaggag ggcattttgg tggtgaagag 5340gagactgaaa taaatttagt
ctgcagaact ttttatcgga accttatctg gggcagtgaa 5400gtatatgtta tggtaatagt
tacgagttag ttgaacttat agatagactg gactatacgg 5460ctatcggtcc aaattagaaa
gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa 5520aatgtgatca tgatgaaagc
cagcaatgac gttgcagctg atattgttgt cggccaaccg 5580cgccgaaaac gcagctgtca
gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat 5640ccaagcacac tcatagttgg
agtcgtactc caaaggcggc aatgacgagt cagacagata 5700ctcgtcgact caggcgacga
cggaattcct gcagcccatc tgcagaattc aggagagacc 5760gggttggcgg cgtatttgtg
tcccaaaaaa cagccccaat tgccccggag aagacggcca 5820ggccgcctag atgacaaatt
caacaactca cagctgactt tctgccattg ccactagggg 5880ggggcctttt tatatggcca
agccaagctc tccacgtcgg ttgggctgca cccaacaata 5940aatgggtagg gttgcaccaa
caaagggatg ggatgggggg tagaagatac gaggataacg 6000gggctcaatg gcacaaataa
gaacgaatac tgccattaag actcgtgatc cagcgactga 6060caccattgca tcatctaagg
gcctcaaaac tacctcggaa ctgctgcgct gatctggaca 6120ccacagaggt tccgagcact
ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa 6180cgctggaaca gcgtgtacag
tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg 6240gtggtgtgac ttgttatagc
ctttagagct gcgaaagcgc gtatggattt ggctcatcag 6300gccagattga gggtctgtgg
acacatgtca tgttagtgta cttcaatcgc cccctggata 6360tagccccgac aataggccgt
ggcctcattt ttttgccttc cgcacatttc cattgctcgg 6420tacccacacc ttgcttctcc
tgcacttgcc aaccttaata ctggtttaca ttgaccaaca 6480tcttacaagc ggggggcttg
tctagggtat atataaacag tggctctccc aatcggttgc 6540cagtctcttt tttcctttct
ttccccacag attcgaaatc taaactacac atcacacaat 6600gcctgttact gacgtcctta
agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660cgtgagtatc cacgacaaga
tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 6720gaaagtcgct agcaacacac
actctctaca caaactaacc cagctctcca tggctgagga 6780taagaccaag gtcgagttcc
ctaccctgac tgagctgaag cactctatcc ctaacgcttg 6840ctttgagtcc aacctcggac
tctcgctcta ctacactgcc cgagcgatct tcaacgcatc 6900tgcctctgct gctctgctct
acgctgcccg atctactccc ttcattgccg ataacgttct 6960gctccacgct ctggtttgcg
ccacctacat ctacgtgcag ggtgtcatct tctggggttt 7020ctttaccgtc ggtcacgact
gtggtcactc tgccttctcc cgataccact ccgtcaactt 7080catcattggc tgcatcatgc
actctgccat tctgactccc ttcgagtcct ggcgagtgac 7140ccaccgacac catcacaaga
acactggcaa cattgataag gacgagatct tctaccctca 7200tcggtccgtc aaggacctcc
aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260gtttgtctac ctgaaggtcg
gatatgctcc tcgaaccatg tcccactttg acccctggga 7320ccctctcctg cttcgacgag
cctccgctgt catcgtgtcc ctcggagtct gggctgcctt 7380cttcgctgcc tacgcctacc
tcacatactc gctcggcttt gccgtcatgg gcctctacta 7440ctatgctcct ctctttgtct
ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa 7500cgacgaagct actccctggt
acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560ctccgtcgac cgatcgtacg
gagctttcgt ggacaacctg tctcaccaca ttggcaccca 7620ccaggtccat cacttgttcc
ctatcattcc ccactacaag ctcaacgaag ccaccaagca 7680ctttgctgcc gcttaccctc
acctcgtgag acgtaacgac gagcccatca ttactgcctt 7740cttcaagacc gctcacctct
ttgtcaacta cggagctgtg cccgagactg ctcagatttt 7800caccctcaaa gagtctgccg
ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860ggatggggaa gtgagtgccc
ggttctgtgt gcacaattgg caatccaaga tggatggatt 7920caacacaggg atatagcgag
ctacgtggtg gtgcgaggat atagcaacgg atatttatgt 7980ttgacacttg agaatgtacg
atacaagcac tgtccaagta caatactaaa catactgtac 8040atactcatac tcgtacccgg
gcaacggttt cacttgagtg cagtggctag tgctcttact 8100cgtacagtgt gcaatactgc
gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160gttgc
816588438DNAArtificial
Sequenceplasmid pDMW367 8catggctctc agtcttacca cagaacagct gttagaacgc
cctgatttgg ttgcgattga 60tggcatcctc tacgaccttg aagggcttgc caaagttcat
ccaggaggag atttgattct 120cgcttctggt gcctctgatg cctcccctct cttttattca
atgcatccat acgtcaaacc 180ggagaattcc aaattgcttc aacagttcgt ccgagggaag
catgaccgca cctcgaagga 240cattgtctac acgtatgatt ctcccttcgc acaagacgtt
aagcggacaa tgcgcgaggt 300gatgaaaggg aggaactggt acgcaacccc tggcttctgg
ctgcgcaccg ttgggatcat 360cgccgtgacg gccttttgcg agtggcactg ggctaccacg
gggatggtgc tgtggggcct 420gttgactgga ttcatgcaca tgcagatcgg cttatccatc
cagcatgatg cgtcccacgg 480ggccatcagc aagaagcctt gggtcaacgc cctcttcgcc
tacggcattg acgtcatcgg 540atcgtcccgg tggatttggc tgcagtcgca catcatgcgg
caccacacct acaccaacca 600gcacggcctc gacctggatg cggagtcggc agagccgttc
ctggtgttcc acaactaccc 660cgccgcaaac accgcccgaa agtggttcca ccgcttccaa
gcttggtaca tgtaccttgt 720gctgggggca tacggggtat cgctggtgta caacccgctc
tacattttcc ggatgcagca 780caatgacacc atcccagagt ctgtcacggc catgcgggaa
aatggctttc tgcggcgcta 840ccgcacactt gcattcgtga tgcgagcttt cttcatcttc
cggaccgcat tcttgccctg 900gtacctcact gggacctcat tgctgatcac cattcctctg
gtgcccaccg caactggtgc 960cttcttgacg ttcttcttca ttttgtccca caattttgat
ggctccgaac ggatccccga 1020caagaactgc aaggttaaga gatctgagaa ggacgttgag
gctgaccaaa ttgactggta 1080tcgggcgcag gtggagacgt cctccacata cggtggcccc
atcgccatgt tcttcactgg 1140cggtctcaat ttccagatcg agcaccacct ctttccccgg
atgtcgtctt ggcactaccc 1200cttcgtccag caggcggtcc gggagtgttg cgaacgccat
ggagtgcgat atgttttcta 1260ccctaccatc gtcggcaaca tcatctccac cctgaagtac
atgcataagg tgggtgtcgt 1320ccactgcgtg aaggacgcac aggattccta agcggccgca
agtgtggatg gggaagtgag 1380tgcccggttc tgtgtgcaca attggcaatc caagatggat
ggattcaaca cagggatata 1440gcgagctacg tggtggtgcg aggatatagc aacggatatt
tatgtttgac acttgagaat 1500gtacgataca agcactgtcc aagtacaata ctaaacatac
tgtacatact catactcgta 1560cccgggcaac ggtttcactt gagtgcagtg gctagtgctc
ttactcgtac agtgtgcaat 1620actgcgtatc atagtctttg atgtatatcg tattcattca
tgttagttgc gtacgagccg 1680gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
ctaactcaca ttaattgcgt 1740tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
ccagctgcat taatgaatcg 1800gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
ttccgcttcc tcgctcactg 1860actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
agctcactca aaggcggtaa 1920tacggttatc cacagaatca ggggataacg caggaaagaa
catgtgagca aaaggccagc 1980aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg ctccgccccc 2040ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
gcgaaacccg acaggactat 2100aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt ccgaccctgc 2160cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
cgtggcgctt tctcatagct 2220cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc tgtgtgcacg 2280aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt gagtccaacc 2340cggtaagaca cgacttatcg ccactggcag cagccactgg
taacaggatt agcagagcga 2400ggtatgtagg cggtgctaca gagttcttga agtggtggcc
taactacggc tacactagaa 2460ggacagtatt tggtatctgc gctctgctga agccagttac
cttcggaaaa agagttggta 2520gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt tgcaagcagc 2580agattacgcg cagaaaaaaa ggatctcaag aagatccttt
gatcttttct acggggtctg 2640acgctcagtg gaacgaaaac tcacgttaag ggattttggt
catgagatta tcaaaaagga 2700tcttcaccta gatcctttta aattaaaaat gaagttttaa
atcaatctaa agtatatatg 2760agtaaacttg gtctgacagt taccaatgct taatcagtga
ggcacctatc tcagcgatct 2820gtctatttcg ttcatccata gttgcctgac tccccgtcgt
gtagataact acgatacggg 2880agggcttacc atctggcccc agtgctgcaa tgataccgcg
agacccacgc tcaccggctc 2940cagatttatc agcaataaac cagccagccg gaagggccga
gcgcagaagt ggtcctgcaa 3000ctttatccgc ctccatccag tctattaatt gttgccggga
agctagagta agtagttcgc 3060cagttaatag tttgcgcaac gttgttgcca ttgctacagg
catcgtggtg tcacgctcgt 3120cgtttggtat ggcttcattc agctccggtt cccaacgatc
aaggcgagtt acatgatccc 3180ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
gatcgttgtc agaagtaagt 3240tggccgcagt gttatcactc atggttatgg cagcactgca
taattctctt actgtcatgc 3300catccgtaag atgcttttct gtgactggtg agtactcaac
caagtcattc tgagaatagt 3360gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
ggataatacc gcgccacata 3420gcagaacttt aaaagtgctc atcattggaa aacgttcttc
ggggcgaaaa ctctcaagga 3480tcttaccgct gttgagatcc agttcgatgt aacccactcg
tgcacccaac tgatcttcag 3540catcttttac tttcaccagc gtttctgggt gagcaaaaac
aggaaggcaa aatgccgcaa 3600aaaagggaat aagggcgaca cggaaatgtt gaatactcat
actcttcctt tttcaatatt 3660attgaagcat ttatcagggt tattgtctca tgagcggata
catatttgaa tgtatttaga 3720aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
agtgccacct gacgcgccct 3780gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
cagcgtgacc gctacacttg 3840ccagcgccct agcgcccgct cctttcgctt tcttcccttc
ctttctcgcc acgttcgccg 3900gctttccccg tcaagctcta aatcgggggc tccctttagg
gttccgattt agtgctttac 3960ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
acgtagtggg ccatcgccct 4020gatagacggt ttttcgccct ttgacgttgg agtccacgtt
ctttaatagt ggactcttgt 4080tccaaactgg aacaacactc aaccctatct cggtctattc
ttttgattta taagggattt 4140tgccgatttc ggcctattgg ttaaaaaatg agctgattta
acaaaaattt aacgcgaatt 4200ttaacaaaat attaacgctt acaatttcca ttcgccattc
aggctgcgca actgttggga 4260agggcgatcg gtgcgggcct cttcgctatt acgccagctg
gcgaaagggg gatgtgctgc 4320aaggcgatta agttgggtaa cgccagggtt ttcccagtca
cgacgttgta aaacgacggc 4380cagtgaattg taatacgact cactataggg cgaattgggt
accgggcccc ccctcgaggt 4440cgatggtgtc gataagcttg atatcgaatt catgtcacac
aaaccgatct tcgcctcaag 4500gaaacctaat tctacatccg agagactgcc gagatccagt
ctacactgat taattttcgg 4560gccaataatt taaaaaaatc gtgttatata atattatatg
tattatatat atacatcatg 4620atgatactga cagtcatgtc ccattgctaa atagacagac
tccatctgcc gcctccaact 4680gatgttctca atatttaagg ggtcatctcg cattgtttaa
taataaacag actccatcta 4740ccgcctccaa atgatgttct caaaatatat tgtatgaact
tatttttatt acttagtatt 4800attagacaac ttacttgctt tatgaaaaac acttcctatt
taggaaacaa tttataatgg 4860cagttcgttc atttaacaat ttatgtagaa taaatgttat
aaatgcgtat gggaaatctt 4920aaatatggat agcataaatg atatctgcat tgcctaattc
gaaatcaaca gcaacgaaaa 4980aaatcccttg tacaacataa atagtcatcg agaaatatca
actatcaaag aacagctatt 5040cacacgttac tattgagatt attattggac gagaatcaca
cactcaactg tctttctctc 5100ttctagaaat acaggtacaa gtatgtacta ttctcattgt
tcatacttct agtcatttca 5160tcccacatat tccttggatt tctctccaat gaatgacatt
ctatcttgca aattcaacaa 5220ttataataag atataccaaa gtagcggtat agtggcaatc
aaaaagcttc tctggtgtgc 5280ttctcgtatt tatttttatt ctaatgatcc attaaaggta
tatatttatt tcttgttata 5340taatcctttt gtttattaca tgggctggat acataaaggt
attttgattt aattttttgc 5400ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg
taggaaatta ccatactttt 5460gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt
ccaggttaga cgttccgcag 5520aatctagaat gcggtatgcg gtacattgtt cttcgaacgt
aaaagttgcg ctccctgaga 5580tattgtacat ttttgctttt acaagtacaa gtacatcgta
caactatgta ctactgttga 5640tgcatccaca acagtttgtt ttgttttttt ttgttttttt
tttttctaat gattcattac 5700cgctatgtat acctacttgt acttgtagta agccgggtta
ttggcgttca attaatcata 5760gacttatgaa tctgcacggt gtgcgctgcg agttactttt
agcttatgca tgctacttgg 5820gtgtaatatt gggatctgtt cggaaatcaa cggatgctca
atcgatttcg acagtaatta 5880attaagtcat acacaagtca gctttcttcg agcctcatat
aagtataagt agttcaacgt 5940attagcactg tacccagcat ctccgtatcg agaaacacaa
caacatgccc cattggacag 6000atcatgcgga tacacaggtt gtgcagtatc atacatactc
gatcagacag gtcgtctgac 6060catcatacaa gctgaacaag cgctccatac ttgcacgctc
tctatataca cagttaaatt 6120acatatccat agtctaacct ctaacagtta atcttctggt
aagcctccca gccagccttc 6180tggtatcgct tggcctcctc aataggatct cggttctggc
cgtacagacc tcggccgaca 6240attatgatat ccgttccggt agacatgaca tcctcaacag
ttcggtactg ctgtccgaga 6300gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa
taagccagtc ctcagagtcg 6360cccttaggtc ggttctgggc aatgaagcca accacaaact
cggggtcgga tcgggcaagc 6420tcaatggtct gcttggagta ctcgccagtg gccagagagc
ccttgcaaga cagctcggcc 6480agcatgagca gacctctggc cagcttctcg ttgggagagg
ggactaggaa ctccttgtac 6540tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt
cagagacagt ttcctcggca 6600ccagctcgca ggccagcaat gattccggtt ccgggtacac
cgtgggcgtt ggtgatatcg 6660gaccactcgg cgattcggtg acaccggtac tggtgcttga
cagtgttgcc aatatctgcg 6720aactttctgt cctcgaacag gaagaaaccg tgcttaagag
caagttcctt gagggggagc 6780acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat
gggttttgat catgcacaca 6840taaggtccga ccttatcggc aagctcaatg agctccttgg
tggtggtaac atccagagaa 6900gcacacaggt tggttttctt ggctgccacg agcttgagca
ctcgagcggc aaaggcggac 6960ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg
tggtgaagag gagactgaaa 7020taaatttagt ctgcagaact ttttatcgga accttatctg
gggcagtgaa gtatatgtta 7080tggtaatagt tacgagttag ttgaacttat agatagactg
gactatacgg ctatcggtcc 7140aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt
tgccgacaaa aatgtgatca 7200tgatgaaagc cagcaatgac gttgcagctg atattgttgt
cggccaaccg cgccgaaaac 7260gcagctgtca gacccacagc ctccaacgaa gaatgtatcg
tcaaagtgat ccaagcacac 7320tcatagttgg agtcgtactc caaaggcggc aatgacgagt
cagacagata ctcgtcgact 7380caggcgacga cggaattcct gcagcccatc tgcagaattc
aggagagacc gggttggcgg 7440cgtatttgtg tcccaaaaaa cagccccaat tgccccggag
aagacggcca ggccgcctag 7500atgacaaatt caacaactca cagctgactt tctgccattg
ccactagggg ggggcctttt 7560tatatggcca agccaagctc tccacgtcgg ttgggctgca
cccaacaata aatgggtagg 7620gttgcaccaa caaagggatg ggatgggggg tagaagatac
gaggataacg gggctcaatg 7680gcacaaataa gaacgaatac tgccattaag actcgtgatc
cagcgactga caccattgca 7740tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct
gatctggaca ccacagaggt 7800tccgagcact ttaggttgca ccaaatgtcc caccaggtgc
aggcagaaaa cgctggaaca 7860gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag
gtcgagcagg gtggtgtgac 7920ttgttatagc ctttagagct gcgaaagcgc gtatggattt
ggctcatcag gccagattga 7980gggtctgtgg acacatgtca tgttagtgta cttcaatcgc
cccctggata tagccccgac 8040aataggccgt ggcctcattt ttttgccttc cgcacatttc
cattgctcgg tacccacacc 8100ttgcttctcc tgcacttgcc aaccttaata ctggtttaca
ttgaccaaca tcttacaagc 8160ggggggcttg tctagggtat atataaacag tggctctccc
aatcggttgc cagtctcttt 8220tttcctttct ttccccacag attcgaaatc taaactacac
atcacacaat gcctgttact 8280gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg
atgtccgagc cgtgagtatc 8340cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat
gacacaatcc gaaagtcgct 8400agcaacacac actctctaca caaactaacc cagctctc
8438918DNAArtificial SequenceM13F universal primer
9tgtaaaacga cggccagt
181022DNAArtificial Sequenceprimer M13-28Rev 10gtaatacgac tcactatagg gc
22114276DNAArtificial
Sequenceplasmid pLF119 11gtacaaagtt ggcattataa gaaagcattg cttatcaatt
tgttgcaacg aacaggtcac 60tatcagtcaa aataaaatca ttatttgcca tccagctgat
atcccctata gtgagtcgta 120ttacatggtc atagctgttt cctggcagct ctggcccgtg
tctcaaaatc tctgatgtta 180cattgcacaa gataaaaata tatcatcatg ttagaaaaac
tcatcgagca tcaaatgaaa 240ctgcaattta ttcatatcag gattatcaat accatatttt
tgaaaaagcc gtttctgtaa 300tgaaggagaa aactcaccga ggcagttcca taggatggca
agatcctggt atcggtctgc 360gattccgact cgtccaacat caatacaacc tattaatttc
ccctcgtcaa aaataaggtt 420atcaagtgag aaatcaccat gagtgacgac tgaatccggt
gagaatggca aaagcttatg 480catttctttc cagacttgtt caacaggcca gccattacgc
tcgtcatcaa aatcactcgc 540atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
agacgaaata cgcgatcgct 600gttaaaagga caattacaaa caggaatcga atgcaaccgg
cgcaggaaca ctgccagcgc 660atcaacaata ttttcacctg aatcaggata ttcttctaat
acctggaatg ctgttttccc 720ggggatcgca gtggtgagta accatgcatc atcaggagta
cggataaaat gcttgatggt 780cggaagaggc ataaattccg tcagccagtt tagtctgacc
atctcatctg taacatcatt 840ggcaacgcta cctttgccat gtttcagaaa caactctggc
gcatcgggct tcccatacaa 900tcgatagatt gtcgcacctg attgcccgac attatcgcga
gcccatttat acccatataa 960atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa
gacgtttccc gttgaatatg 1020gctcatagat cttttctcca tcactgatag ggagtggtaa
aataactcca tcaatgatag 1080agtgtcaaca acatgaccaa aatcccttaa cgtgagttac
gcgtattaat tgcgttgcgc 1140tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
tgcattaatg aatcggccaa 1200cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
cttcctcgct cactgactcg 1260ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
actcaaaggc ggtaatacgg 1320ttatccacag aatcagggga taacgcagga aagaacatgt
gagcaaaagg ccagcaaaag 1380gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
ataggctccg cccccctgac 1440gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
acccgacagg actataaaga 1500taccaggcgt ttccccctgg aagctccctc gtgcgctctc
ctgttccgac cctgccgctt 1560accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
cgctttctca atgctcacgc 1620tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
tgggctgtgt gcacgaaccc 1680cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
gtcttgagtc caacccggta 1740agacacgact tatcgccact ggcagcagcc actggtaaca
ggattagcag agcgaggtat 1800gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
acggttacac tagaagaaca 1860gtatttggta tctgcgctct gctgaagcca gttaccttcg
gaaaaagagt tggtagctct 1920tgatccggca aacaaaccac cgctggtagc ggtggttttt
ttgtttgcaa gcagcagatt 1980acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
tttctacggg gtctgacgct 2040cagggaacga cgcgtaccgc tagccaggaa gagtttgtag
aaacgcaaaa aggccatccg 2100tcaggatggc cttctgctta gtttgatgcc tggcagttta
tggcgggcgt cctgcccgcc 2160accctccggg ccgttgcttc acaacgttca aatccgctcc
cggcggattt gtcctactca 2220ggagagcgtt caccgacaaa caacagataa aacgaaaggc
ccagtcttcc gactgagcct 2280ttcgttttat ttgatgcctg gcagttccct actctcgcgt
taacgctagc atggatgttt 2340tcccagtcac gacgttgtaa aacgacggcc agtcttaagc
tcgggcccca aataatgatt 2400ttattttgac tgatagtgac ctgttcgttg caacaaattg
atgagcaatg cttttttata 2460atgccaactt tgtacaaaaa agttggattt tttttcggga
tggccaccat ctctttgact 2520actgagcaac ttttagaaca cccagaactg gttgcaattg
atggggtgtt gtacgatctc 2580ttcggactgg cgaaagtgca tccaggtggc aacctcattg
aagccgccgg tgcctccgac 2640ggaaccgccc tgttctactc catgcaccct ggagtgaagc
cagagaattc gaagctgctg 2700cagcaatttg cccgaggcaa acacgaacga agctcgaagg
acccagtgta cacctttgac 2760agtcccttcg cccaggatgt caagcagagc gttcgggagg
tcatgaaggg gcgcaactgg 2820tacgccacgc ccggcttttg gctgcggacc gcgctgatca
tcgcgtgcac tgccataggc 2880gaatggtatt ggatcactac cggggcagtg atgtggggca
tcttcaccgg gtacttccac 2940agccagattg ggttggcgat tcaacacgat gcctctcacg
gagccatcag caaaaagccc 3000tgggtgaacg cctttttcgc ctacggcatc gacgccattg
gatcctcccg ctggatctgg 3060ctgcagtccc acattatgcg ccaccacacc tacaccaacc
agcatggcct ggacctggac 3120gctgcctcgg cggagccgtt cattttgttc cactcctacc
cggcaacaaa tgcgtcacga 3180aagtggtacc atcggttcca ggcgtggtac atgtacatcg
ttttggggat gtatggtgtg 3240tcgatggtgt acaatccgat gtacttgttc acgatgcagc
acaacgacac aatcccagag 3300gccacctctc ttagaccagg cagctttttc aaccggcagc
gcgccttcgc cgtttccctc 3360cgcctactgt tcatcttccg caacgccttc ctcccctggt
acatcgcggg cgcctctccg 3420ctgctcacca tcctgctggt gccaacggtc acaggcatct
tcttgacatt tgtttttgtg 3480ctgtcccata actttgaagg cgctgagcgg acccccgaaa
agaactgcaa ggccaaaagg 3540gccaaggagg ggaaggaggt ccgcgatgta gaggaggacc
gggtggactg gtaccgggcg 3600caggccgaga ccgcggcgac ctacgggggc agcgtcggga
tgatgctgac cggcggtttg 3660aacctgcaga tcgagcacca cttgttcccc cgcatgtcct
cttggcacta ccccttcatc 3720caagatacgg tgcgggaatg ttgcaagcgc catggcgtgc
gctacacata ctacccgacc 3780atcctggaga atataatgtc cacgctccgc tacatgcaga
aggtgggcgt ggcccacaca 3840attcaggatg cccaggaatt ctgagtgagt tcgatccgca
tcgacgtcta ccatttttga 3900tgctgtctat tcctgttttc agtcacctcc agcattctca
tggctggtga ccactgcccc 3960tctaacccat tgtgacacac cgccaaagac tttgcctctt
ttttttccct ttcttttgtc 4020ctcggggtgc tttggccggt gtttactcgc cttgcagtcc
ccgcaaacga ccgacgttta 4080agctccgttg ttgactgggc cgctcgtaaa cccatctgca
ggttgaggct cccatggaga 4140attgtgatgg ctgattagga ggtggcgggg catacatgcc
tcgacactca aagccgggcg 4200gcttctggat tcgaaaacgc aaatgggcgc tttggaaaaa
aaaaaaaaaa aaaaaaaaaa 4260aaaacccaac tttctt
4276121362DNAEuglena anabaena 12atggccacca
tctctttgac tactgagcaa cttttagaac acccagaact ggttgcaatt 60gatggggtgt
tgtacgatct cttcggactg gcgaaagtgc atccaggtgg caacctcatt 120gaagccgccg
gtgcctccga cggaaccgcc ctgttctact ccatgcaccc tggagtgaag 180ccagagaatt
cgaagctgct gcagcaattt gcccgaggca aacacgaacg aagctcgaag 240gacccagtgt
acacctttga cagtcccttc gcccaggatg tcaagcagag cgttcgggag 300gtcatgaagg
ggcgcaactg gtacgccacg cccggctttt ggctgcggac cgcgctgatc 360atcgcgtgca
ctgccatagg cgaatggtat tggatcacta ccggggcagt gatgtggggc 420atcttcaccg
ggtacttcca cagccagatt gggttggcga ttcaacacga tgcctctcac 480ggagccatca
gcaaaaagcc ctgggtgaac gcctttttcg cctacggcat cgacgccatt 540ggatcctccc
gctggatctg gctgcagtcc cacattatgc gccaccacac ctacaccaac 600cagcatggcc
tggacctgga cgctgcctcg gcggagccgt tcattttgtt ccactcctac 660ccggcaacaa
atgcgtcacg aaagtggtac catcggttcc aggcgtggta catgtacatc 720gttttgggga
tgtatggtgt gtcgatggtg tacaatccga tgtacttgtt cacgatgcag 780cacaacgaca
caatcccaga ggccacctct cttagaccag gcagcttttt caaccggcag 840cgcgccttcg
ccgtttccct ccgcctactg ttcatcttcc gcaacgcctt cctcccctgg 900tacatcgcgg
gcgcctctcc gctgctcacc atcctgctgg tgccaacggt cacaggcatc 960ttcttgacat
ttgtttttgt gctgtcccat aactttgaag gcgctgagcg gacccccgaa 1020aagaactgca
aggccaaaag ggccaaggag gggaaggagg tccgcgatgt agaggaggac 1080cgggtggact
ggtaccgggc gcaggccgag accgcggcga cctacggggg cagcgtcggg 1140atgatgctga
ccggcggttt gaacctgcag atcgagcacc acttgttccc ccgcatgtcc 1200tcttggcact
accccttcat ccaagatacg gtgcgggaat gttgcaagcg ccatggcgtg 1260cgctacacat
actacccgac catcctggag aatataatgt ccacgctccg ctacatgcag 1320aaggtgggcg
tggcccacac aattcaggat gcccaggaat tc
136213454PRTEuglena anabaena 13Met Ala Thr Ile Ser Leu Thr Thr Glu Gln
Leu Leu Glu His Pro Glu 1 5 10
15 Leu Val Ala Ile Asp Gly Val Leu Tyr Asp Leu Phe Gly Leu Ala
Lys 20 25 30 Val
His Pro Gly Gly Asn Leu Ile Glu Ala Ala Gly Ala Ser Asp Gly 35
40 45 Thr Ala Leu Phe Tyr Ser
Met His Pro Gly Val Lys Pro Glu Asn Ser 50 55
60 Lys Leu Leu Gln Gln Phe Ala Arg Gly Lys His
Glu Arg Ser Ser Lys 65 70 75
80 Asp Pro Val Tyr Thr Phe Asp Ser Pro Phe Ala Gln Asp Val Lys Gln
85 90 95 Ser Val
Arg Glu Val Met Lys Gly Arg Asn Trp Tyr Ala Thr Pro Gly 100
105 110 Phe Trp Leu Arg Thr Ala Leu
Ile Ile Ala Cys Thr Ala Ile Gly Glu 115 120
125 Trp Tyr Trp Ile Thr Thr Gly Ala Val Met Trp Gly
Ile Phe Thr Gly 130 135 140
Tyr Phe His Ser Gln Ile Gly Leu Ala Ile Gln His Asp Ala Ser His 145
150 155 160 Gly Ala Ile
Ser Lys Lys Pro Trp Val Asn Ala Phe Phe Ala Tyr Gly 165
170 175 Ile Asp Ala Ile Gly Ser Ser Arg
Trp Ile Trp Leu Gln Ser His Ile 180 185
190 Met Arg His His Thr Tyr Thr Asn Gln His Gly Leu Asp
Leu Asp Ala 195 200 205
Ala Ser Ala Glu Pro Phe Ile Leu Phe His Ser Tyr Pro Ala Thr Asn 210
215 220 Ala Ser Arg Lys
Trp Tyr His Arg Phe Gln Ala Trp Tyr Met Tyr Ile 225 230
235 240 Val Leu Gly Met Tyr Gly Val Ser Met
Val Tyr Asn Pro Met Tyr Leu 245 250
255 Phe Thr Met Gln His Asn Asp Thr Ile Pro Glu Ala Thr Ser
Leu Arg 260 265 270
Pro Gly Ser Phe Phe Asn Arg Gln Arg Ala Phe Ala Val Ser Leu Arg
275 280 285 Leu Leu Phe Ile
Phe Arg Asn Ala Phe Leu Pro Trp Tyr Ile Ala Gly 290
295 300 Ala Ser Pro Leu Leu Thr Ile Leu
Leu Val Pro Thr Val Thr Gly Ile 305 310
315 320 Phe Leu Thr Phe Val Phe Val Leu Ser His Asn Phe
Glu Gly Ala Glu 325 330
335 Arg Thr Pro Glu Lys Asn Cys Lys Ala Lys Arg Ala Lys Glu Gly Lys
340 345 350 Glu Val Arg
Asp Val Glu Glu Asp Arg Val Asp Trp Tyr Arg Ala Gln 355
360 365 Ala Glu Thr Ala Ala Thr Tyr Gly
Gly Ser Val Gly Met Met Leu Thr 370 375
380 Gly Gly Leu Asn Leu Gln Ile Glu His His Leu Phe Pro
Arg Met Ser 385 390 395
400 Ser Trp His Tyr Pro Phe Ile Gln Asp Thr Val Arg Glu Cys Cys Lys
405 410 415 Arg His Gly Val
Arg Tyr Thr Tyr Tyr Pro Thr Ile Leu Glu Asn Ile 420
425 430 Met Ser Thr Leu Arg Tyr Met Gln Lys
Val Gly Val Ala His Thr Ile 435 440
445 Gln Asp Ala Gln Glu Phe 450
14476PRTThalassiosira pseudonana 14Met Pro Pro Asn Ala Asp Ile Ser Arg
Ile Arg Asn Arg Ile Pro Thr 1 5 10
15 Lys Thr Gly Thr Val Ala Ser Ala Asp Asn Asn Asp Pro Ala
Thr Gln 20 25 30
Ser Val Arg Thr Leu Lys Ser Leu Lys Gly Asn Glu Val Val Ile Asn
35 40 45 Gly Thr Ile Tyr
Asp Ile Ala Asp Phe Val His Pro Gly Gly Glu Val 50
55 60 Val Lys Phe Phe Gly Gly Asn Asp
Val Thr Ile Gln Tyr Asn Met Ile 65 70
75 80 His Pro Tyr His Thr Gly Lys His Leu Glu Lys Met
Lys Ala Val Gly 85 90
95 Lys Val Val Asp Trp Gln Ser Asp Tyr Lys Phe Asp Thr Pro Phe Glu
100 105 110 Arg Glu Ile
Lys Ser Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu 115
120 125 Phe Gly Thr Thr Gly Tyr Phe Leu
Arg Ala Phe Phe Tyr Ile Ala Leu 130 135
140 Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr Cys Thr Thr
Phe Thr Thr 145 150 155
160 Tyr Asp His Trp Tyr Gln Ser Gly Val Phe Ile Ala Ile Val Phe Gly
165 170 175 Ile Ser Gln Ala
Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn His 180
185 190 Gly Ala Ala Ser Lys Arg Pro Trp Val
Asn Asp Leu Leu Gly Phe Gly 195 200
205 Thr Asp Leu Ile Gly Ser Asn Lys Trp Asn Trp Met Ala Gln
His Trp 210 215 220
Thr His His Ala Tyr Thr Asn His Ser Glu Lys Asp Pro Asp Ser Phe 225
230 235 240 Ser Ser Glu Pro Met
Phe Ala Phe Asn Asp Tyr Pro Ile Gly His Pro 245
250 255 Lys Arg Lys Trp Trp His Arg Phe Gln Gly
Gly Tyr Phe Leu Phe Met 260 265
270 Leu Gly Leu Tyr Trp Leu Pro Thr Val Phe Asn Pro Gln Phe Ile
Asp 275 280 285 Leu
Arg Gln Arg Gly Ala Gln Tyr Val Gly Ile Gln Met Glu Asn Asp 290
295 300 Phe Ile Val Lys Arg Arg
Lys Tyr Ala Val Ala Leu Arg Met Met Tyr 305 310
315 320 Ile Tyr Leu Asn Ile Val Ser Pro Phe Met Asn
Asn Gly Leu Ser Trp 325 330
335 Ser Thr Phe Gly Ile Ile Met Leu Met Gly Ile Ser Glu Ser Leu Thr
340 345 350 Leu Ser
Val Leu Phe Ser Leu Ser His Asn Phe Ile Asn Ser Asp Arg 355
360 365 Asp Pro Thr Ala Asp Phe Lys
Lys Thr Gly Glu Gln Val Cys Trp Phe 370 375
380 Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly
Phe Ile Ser Gly 385 390 395
400 Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe Pro
405 410 415 Arg Met Ser
Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg Glu 420
425 430 Val Cys Lys Lys His Gly Met Ser
Tyr Ala Tyr Tyr Pro Trp Ile Gly 435 440
445 Gln Asn Leu Val Ser Thr Phe Lys Tyr Met His Arg Ala
Gly Ser Gly 450 455 460
Ala Asn Trp Glu Leu Lys Pro Leu Ser Gly Ser Ala 465 470
475 15469PRTPhaeodactylum tricornutum 15Met Ala Pro
Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val 1 5
10 15 Ala Lys His Asn Ala Ala Thr Ile
Ser Thr Gln Glu Arg Leu Cys Ser 20 25
30 Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly
Ile Ile Tyr 35 40 45
Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe 50
55 60 Gly Gly Asn Asp
Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His 65 70
75 80 Thr Glu Lys His Leu Glu Lys Met Lys
Arg Val Gly Lys Val Thr Asp 85 90
95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu
Ile Lys 100 105 110
Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu
115 120 125 Gly Trp Phe Phe
Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130
135 140 Gln Tyr His Trp Val Thr Thr Gly
Thr Ser Trp Leu Leu Ala Val Ala 145 150
155 160 Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val
Gln His Asp Ala 165 170
175 Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly
180 185 190 Leu Gly Ala
Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195
200 205 His Trp Thr His His Ala Tyr Thr
Asn His Ala Glu Met Asp Pro Asp 210 215
220 Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr
Pro Leu Asp 225 230 235
240 His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met
245 250 255 Pro Val Leu Ala
Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260
265 270 Leu Asp Leu Gln Gln Arg Gly Ala Leu
Ser Val Gly Ile Arg Leu Asp 275 280
285 Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp
Arg Ala 290 295 300
Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 305
310 315 320 Leu Glu Trp Ser Trp
Arg Val Phe Gly Asn Ile Met Leu Met Gly Val 325
330 335 Ala Glu Ser Leu Ala Leu Ala Val Leu Phe
Ser Leu Ser His Asn Phe 340 345
350 Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly
Glu 355 360 365 Pro
Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370
375 380 Gly Phe Leu Ser Gly Cys
Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390
395 400 His His Leu Phe Pro Arg Met Ser Ser Ala Trp
Tyr Pro Tyr Ile Ala 405 410
415 Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr
420 425 430 Tyr Pro
Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435
440 445 Ala Ala Gly Thr Gly Ala Asn
Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455
460 Leu Thr Gly Arg Ala 465
16449PRTEuglena gracilis 16Met Ala Leu Ser Leu Thr Thr Glu Gln Leu Leu
Glu Arg Pro Asp Leu 1 5 10
15 Val Ala Ile Asp Gly Ile Leu Tyr Asp Leu Glu Gly Leu Ala Lys Val
20 25 30 His Pro
Gly Gly Asp Leu Ile Leu Ala Ser Gly Ala Ser Asp Ala Ser 35
40 45 Pro Leu Phe Tyr Ser Met His
Pro Tyr Val Lys Pro Glu Asn Ser Lys 50 55
60 Leu Leu Gln Gln Phe Val Arg Gly Lys His Asp Arg
Thr Ser Lys Asp 65 70 75
80 Ile Val Tyr Thr Tyr Asp Ser Pro Phe Ala Gln Asp Val Lys Arg Thr
85 90 95 Met Arg Glu
Val Met Lys Gly Arg Asn Trp Tyr Ala Thr Pro Gly Phe 100
105 110 Trp Leu Arg Thr Val Gly Ile Ile
Ala Val Thr Ala Phe Cys Glu Trp 115 120
125 His Trp Ala Thr Thr Gly Met Val Leu Trp Gly Leu Leu
Thr Gly Phe 130 135 140
Met His Met Gln Ile Gly Leu Ser Ile Gln His Asp Ala Ser His Gly 145
150 155 160 Ala Ile Ser Lys
Lys Pro Trp Val Asn Ala Leu Phe Ala Tyr Gly Ile 165
170 175 Asp Val Ile Gly Ser Ser Arg Trp Ile
Trp Leu Gln Ser His Ile Met 180 185
190 Arg His His Thr Tyr Thr Asn Gln His Gly Leu Asp Leu Asp
Ala Glu 195 200 205
Ser Ala Glu Pro Phe Leu Val Phe His Asn Tyr Pro Ala Ala Asn Thr 210
215 220 Ala Arg Lys Trp Phe
His Arg Phe Gln Ala Trp Tyr Met Tyr Leu Val 225 230
235 240 Leu Gly Ala Tyr Gly Val Ser Leu Val Tyr
Asn Pro Leu Tyr Ile Phe 245 250
255 Arg Met Gln His Asn Asp Thr Ile Pro Glu Ser Val Thr Ala Met
Arg 260 265 270 Glu
Asn Gly Phe Leu Arg Arg Tyr Arg Thr Leu Ala Phe Val Met Arg 275
280 285 Ala Phe Phe Ile Phe Arg
Thr Ala Phe Leu Pro Trp Tyr Leu Thr Gly 290 295
300 Thr Ser Leu Leu Ile Thr Ile Pro Leu Val Pro
Thr Ala Thr Gly Ala 305 310 315
320 Phe Leu Thr Phe Phe Phe Ile Leu Ser His Asn Phe Asp Gly Ser Glu
325 330 335 Arg Ile
Pro Asp Lys Asn Cys Lys Val Lys Arg Ser Glu Lys Asp Val 340
345 350 Glu Ala Asp Gln Ile Asp Trp
Tyr Arg Ala Gln Val Glu Thr Ser Ser 355 360
365 Thr Tyr Gly Gly Pro Ile Ala Met Phe Phe Thr Gly
Gly Leu Asn Phe 370 375 380
Gln Ile Glu His His Leu Phe Pro Arg Met Ser Ser Trp His Tyr Pro 385
390 395 400 Phe Val Gln
Gln Ala Val Arg Glu Cys Cys Glu Arg His Gly Val Arg 405
410 415 Tyr Val Phe Tyr Pro Thr Ile Val
Gly Asn Ile Ile Ser Thr Leu Lys 420 425
430 Tyr Met His Lys Val Gly Val Val His Cys Val Lys Asp
Ala Gln Asp 435 440 445
Ser 179472DNAArtificial Sequenceplasmid pDMW263 17catggcatgg atggtacgtc
ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg 60cctgtgggca ttcagtctgg
atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag 120cgcgttacaa gaaagccggg
caattgctgt gccaggcagt tttaacgatc agttcgccga 180tgcagatatt cgtaattatg
cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa 240aggttgggca ggccagcgta
tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt 300gtgggtcaat aatcaggaag
tgatggagca tcagggcggc tatacgccat ttgaagccga 360tgtcacgccg tatgttattg
ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga 420actgaactgg cagactatcc
cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa 480gcagtcttac ttccatgatt
tctttaacta tgccgggatc catcgcagcg taatgctcta 540caccacgccg aacacctggg
tggacgatat caccgtggtg acgcatgtcg cgcaagactg 600taaccacgcg tctgttgact
ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg 660tgatgcggat caacaggtgg
ttgcaactgg acaaggcact agcgggactt tgcaagtggt 720gaatccgcac ctctggcaac
cgggtgaagg ttatctctat gaactgtgcg tcacagccaa 780aagccagaca gagtgtgata
tctacccgct tcgcgtcggc atccggtcag tggcagtgaa 840gggcgaacag ttcctgatta
accacaaacc gttctacttt actggctttg gtcgtcatga 900agatgcggac ttacgtggca
aaggattcga taacgtgctg atggtgcacg accacgcatt 960aatggactgg attggggcca
actcctaccg tacctcgcat tacccttacg ctgaagagat 1020gctcgactgg gcagatgaac
atggcatcgt ggtgattgat gaaactgctg ctgtcggctt 1080taacctctct ttaggcattg
gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga 1140agaggcagtc aacggggaaa
ctcagcaagc gcacttacag gcgattaaag agctgatagc 1200gcgtgacaaa aaccacccaa
gcgtggtgat gtggagtatt gccaacgaac cggatacccg 1260tccgcaagtg cacgggaata
tttcgccact ggcggaagca acgcgtaaac tcgacccgac 1320gcgtccgatc acctgcgtca
atgtaatgtt ctgcgacgct cacaccgata ccatcagcga 1380tctctttgat gtgctgtgcc
tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt 1440ggaaacggca gagaaggtac
tggaaaaaga acttctggcc tggcaggaga aactgcatca 1500gccgattatc atcaccgaat
acggcgtgga tacgttagcc gggctgcact caatgtacac 1560cgacatgtgg agtgaagagt
atcagtgtgc atggctggat atgtatcacc gcgtctttga 1620tcgcgtcagc gccgtcgtcg
gtgaacaggt atggaatttc gccgattttg cgacctcgca 1680aggcatattg cgcgttggcg
gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa 1740gtcggcggct tttctgctgc
aaaaacgctg gactggcatg aacttcggtg aaaaaccgca 1800gcagggaggc aaacaatgat
taattaacta gagcggccgc caccgcggcc cgagattccg 1860gcctcttcgg ccgccaagcg
acccgggtgg acgtctagag gtacctagca attaacagat 1920agtttgccgg tgataattct
cttaacctcc cacactcctt tgacataacg atttatgtaa 1980cgaaactgaa atttgaccag
atattgtgtc cgcggtggag ctccagcttt tgttcccttt 2040agtgagggtt aatttcgagc
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 2100gttatccgct cacaattcca
cacaacatac gagccggaag cataaagtgt aaagcctggg 2160gtgcctaatg agtgagctaa
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 2220cgggaaacct gtcgtgccag
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 2280tgcgtattgg gcgctcttcc
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 2340tgcggcgagc ggtatcagct
cactcaaagg cggtaatacg gttatccaca gaatcagggg 2400ataacgcagg aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 2460ccgcgttgct ggcgtttttc
cataggctcc gcccccctga cgagcatcac aaaaatcgac 2520gctcaagtca gaggtggcga
aacccgacag gactataaag ataccaggcg tttccccctg 2580gaagctccct cgtgcgctct
cctgttccga ccctgccgct taccggatac ctgtccgcct 2640ttctcccttc gggaagcgtg
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 2700tgtaggtcgt tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 2760gcgccttatc cggtaactat
cgtcttgagt ccaacccggt aagacacgac ttatcgccac 2820tggcagcagc cactggtaac
aggattagca gagcgaggta tgtaggcggt gctacagagt 2880tcttgaagtg gtggcctaac
tacggctaca ctagaaggac agtatttggt atctgcgctc 2940tgctgaagcc agttaccttc
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3000ccgctggtag cggtggtttt
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3060ctcaagaaga tcctttgatc
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 3120gttaagggat tttggtcatg
agattatcaa aaaggatctt cacctagatc cttttaaatt 3180aaaaatgaag ttttaaatca
atctaaagta tatatgagta aacttggtct gacagttacc 3240aatgcttaat cagtgaggca
cctatctcag cgatctgtct atttcgttca tccatagttg 3300cctgactccc cgtcgtgtag
ataactacga tacgggaggg cttaccatct ggccccagtg 3360ctgcaatgat accgcgagac
ccacgctcac cggctccaga tttatcagca ataaaccagc 3420cagccggaag ggccgagcgc
agaagtggtc ctgcaacttt atccgcctcc atccagtcta 3480ttaattgttg ccgggaagct
agagtaagta gttcgccagt taatagtttg cgcaacgttg 3540ttgccattgc tacaggcatc
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 3600ccggttccca acgatcaagg
cgagttacat gatcccccat gttgtgcaaa aaagcggtta 3660gctccttcgg tcctccgatc
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 3720ttatggcagc actgcataat
tctcttactg tcatgccatc cgtaagatgc ttttctgtga 3780ctggtgagta ctcaaccaag
tcattctgag aatagtgtat gcggcgaccg agttgctctt 3840gcccggcgtc aatacgggat
aataccgcgc cacatagcag aactttaaaa gtgctcatca 3900ttggaaaacg ttcttcgggg
cgaaaactct caaggatctt accgctgttg agatccagtt 3960cgatgtaacc cactcgtgca
cccaactgat cttcagcatc ttttactttc accagcgttt 4020ctgggtgagc aaaaacagga
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 4080aatgttgaat actcatactc
ttcctttttc aatattattg aagcatttat cagggttatt 4140gtctcatgag cggatacata
tttgaatgta tttagaaaaa taaacaaata ggggttccgc 4200gcacatttcc ccgaaaagtg
ccacctgacg cgccctgtag cggcgcatta agcgcggcgg 4260gtgtggtggt tacgcgcagc
gtgaccgcta cacttgccag cgccctagcg cccgctcctt 4320tcgctttctt cccttccttt
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4380gggggctccc tttagggttc
cgatttagtg ctttacggca cctcgacccc aaaaaacttg 4440attagggtga tggttcacgt
agtgggccat cgccctgata gacggttttt cgccctttga 4500cgttggagtc cacgttcttt
aatagtggac tcttgttcca aactggaaca acactcaacc 4560ctatctcggt ctattctttt
gatttataag ggattttgcc gatttcggcc tattggttaa 4620aaaatgagct gatttaacaa
aaatttaacg cgaattttaa caaaatatta acgcttacaa 4680tttccattcg ccattcaggc
tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 4740gctattacgc cagctggcga
aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 4800agggttttcc cagtcacgac
gttgtaaaac gacggccagt gaattgtaat acgactcact 4860atagggcgaa ttgggtaccg
ggccccccct cgaggtcgat ggtgtcgata agcttgatat 4920cgaattcatg tcacacaaac
cgatcttcgc ctcaaggaaa cctaattcta catccgagag 4980actgccgaga tccagtctac
actgattaat tttcgggcca ataatttaaa aaaatcgtgt 5040tatataatat tatatgtatt
atatatatac atcatgatga tactgacagt catgtcccat 5100tgctaaatag acagactcca
tctgccgcct ccaactgatg ttctcaatat ttaaggggtc 5160atctcgcatt gtttaataat
aaacagactc catctaccgc ctccaaatga tgttctcaaa 5220atatattgta tgaacttatt
tttattactt agtattatta gacaacttac ttgctttatg 5280aaaaacactt cctatttagg
aaacaattta taatggcagt tcgttcattt aacaatttat 5340gtagaataaa tgttataaat
gcgtatggga aatcttaaat atggatagca taaatgatat 5400ctgcattgcc taattcgaaa
tcaacagcaa cgaaaaaaat cccttgtaca acataaatag 5460tcatcgagaa atatcaacta
tcaaagaaca gctattcaca cgttactatt gagattatta 5520ttggacgaga atcacacact
caactgtctt tctctcttct agaaatacag gtacaagtat 5580gtactattct cattgttcat
acttctagtc atttcatccc acatattcct tggatttctc 5640tccaatgaat gacattctat
cttgcaaatt caacaattat aataagatat accaaagtag 5700cggtatagtg gcaatcaaaa
agcttctctg gtgtgcttct cgtatttatt tttattctaa 5760tgatccatta aaggtatata
tttatttctt gttatataat ccttttgttt attacatggg 5820ctggatacat aaaggtattt
tgatttaatt ttttgcttaa attcaatccc ccctcgttca 5880gtgtcaactg taatggtagg
aaattaccat acttttgaag aagcaaaaaa aatgaaagaa 5940aaaaaaaatc gtatttccag
gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac 6000attgttcttc gaacgtaaaa
gttgcgctcc ctgagatatt gtacattttt gcttttacaa 6060gtacaagtac atcgtacaac
tatgtactac tgttgatgca tccacaacag tttgttttgt 6120ttttttttgt tttttttttt
tctaatgatt cattaccgct atgtatacct acttgtactt 6180gtagtaagcc gggttattgg
cgttcaatta atcatagact tatgaatctg cacggtgtgc 6240gctgcgagtt acttttagct
tatgcatgct acttgggtgt aatattggga tctgttcgga 6300aatcaacgga tgctcaaccg
atttcgacag taataatttg aatcgaatcg gagcctaaaa 6360tgaacccgag tatatctcat
aaaattctcg gtgagaggtc tgtgactgtc agtacaaggt 6420gccttcatta tgccctcaac
cttaccatac ctcactgaat gtagtgtacc tctaaaaatg 6480aaatacagtg ccaaaagcca
aggcactgag ctcgtctaac ggacttgata tacaaccaat 6540taaaacaaat gaaaagaaat
acagttcttt gtatcatttg taacaattac cctgtacaaa 6600ctaaggtatt gaaatcccac
aatattccca aagtccaccc ctttccaaat tgtcatgcct 6660acaactcata taccaagcac
taacctacca aacaccacta aaaccccaca aaatatatct 6720taccgaatat acagtaacaa
gctaccacca cactcgttgg gtgcagtcgc cagcttaaag 6780atatctatcc acatcagcca
caactccctt cctttaataa accgactaca cccttggcta 6840ttgaggttat gagtgaatat
actgtagaca agacactttc aagaagactg tttccaaaac 6900gtaccactgt cctccactac
aaacacaccc aatctgcttc ttctagtcaa ggttgctaca 6960ccggtaaatt ataaatcatc
atttcattag cagggcaggg ccctttttat agagtcttat 7020acactagcgg accctgccgg
tagaccaacc cgcaggcgcg tcagtttgct ccttccatca 7080atgcgtcgta gaaacgactt
actccttctt gagcagctcc ttgaccttgt tggcaacaag 7140tctccgacct cggaggtgga
ggaagagcct ccgatatcgg cggtagtgat accagcctcg 7200acggactcct tgacggcagc
ctcaacagcg tcaccggcgg gcttcatgtt aagagagaac 7260ttgagcatca tggcggcaga
cagaatggtg gcaatggggt tgaccttctg cttgccgaga 7320tcgggggcag atccgtgaca
gggctcgtac agaccgaacg cctcgttggt gtcgggcaga 7380gaagccagag aggcggaggg
cagcagaccc agagaaccgg ggatgacgga ggcctcgtcg 7440gagatgatat cgccaaacat
gttggtggtg atgatgatac cattcatctt ggagggctgc 7500ttgatgagga tcatggcggc
cgagtcgatc agctggtggt tgagctcgag ctgggggaat 7560tcgtccttga ggactcgagt
gacagtcttt cgccaaagtc gagaggaggc cagcacgttg 7620gccttgtcaa gagaccacac
gggaagaggg gggttgtgct gaagggccag gaaggcggcc 7680attcgggcaa ttcgctcaac
ctcaggaacg gagtaggtct cggtgtcgga agcgacgcca 7740gatccgtcat cctcctttcg
ctctccaaag tagatacctc cgacgagctc tcggacaatg 7800atgaagtcgg tgccctcaac
gtttcggatg ggggagagat cggcgagctt gggcgacagc 7860agctggcagg gtcgcaggtt
ggcgtacagg ttcaggtcct ttcgcagctt gaggagaccc 7920tgctcgggtc gcacgtcggt
tcgtccgtcg ggagtggtcc atacggtgtt ggcagcgcct 7980ccgacagcac cgagcataat
agagtcagcc tttcggcaga tgtcgagagt agcgtcggtg 8040atgggctcgc cctccttctc
aatggcagct cctccaatga gtcggtcctc aaacacaaac 8100tcggtgccgg aggcctcagc
aacagacttg agcaccttga cggcctcggc aatcacctcg 8160gggccacaga agtcgccgcc
gagaagaaca atcttcttgg agtcagtctt ggtcttctta 8220gtttcgggtt ccattgtgga
tgtgtgtggt tgtatgtgtg atgtggtgtg tggagtgaaa 8280atctgtggct ggcaaacgct
cttgtatata tacgcacttt tgcccgtgct atgtggaaga 8340ctaaacctcc gaagattgtg
actcaggtag tgcggtatcg gctagggacc caaaccttgt 8400cgatgccgat agcgctatcg
aacgtacccc agccggccgg gagtatgtcg gaggggacat 8460acgagatcgt caagggtttg
tggccaactg gtaaataaat gatgtcgacg tttaaacagt 8520gtacgcagat ctactataga
ggaacattta aattgccccg gagaagacgg ccaggccgcc 8580tagatgacaa attcaacaac
tcacagctga ctttctgcca ttgccactag gggggggcct 8640ttttatatgg ccaagccaag
ctctccacgt cggttgggct gcacccaaca ataaatgggt 8700agggttgcac caacaaaggg
atgggatggg gggtagaaga tacgaggata acggggctca 8760atggcacaaa taagaacgaa
tactgccatt aagactcgtg atccagcgac tgacaccatt 8820gcatcatcta agggcctcaa
aactacctcg gaactgctgc gctgatctgg acaccacaga 8880ggttccgagc actttaggtt
gcaccaaatg tcccaccagg tgcaggcaga aaacgctgga 8940acagcgtgta cagtttgtct
taacaaaaag tgagggcgct gaggtcgagc agggtggtgt 9000gacttgttat agcctttaga
gctgcgaaag cgcgtatgga tttggctcat caggccagat 9060tgagggtctg tggacacatg
tcatgttagt gtacttcaat cgccccctgg atatagcccc 9120gacaataggc cgtggcctca
tttttttgcc ttccgcacat ttccattgct cgatacccac 9180accttgcttc tcctgcactt
gccaacctta atactggttt acattgacca acatcttaca 9240agcggggggc ttgtctaggg
tatatataaa cagtggctct cccaatcggt tgccagtctc 9300ttttttcctt tctttcccca
cagattcgaa atctaaacta cacatcacag aattccgagc 9360cgtgagtatc cacgacaaga
tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 9420gaaagtcgct agcaacacac
actctctaca caaactaacc cagctctggt ac 9472187879DNAArtificial
Sequenceplasmid pDMW237 18ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt
gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag ggatatagcg agctacgtgg
tggtgcgagg atatagcaac 120ggatatttat gtttgacact tgagaatgta cgatacaagc
actgtccaag tacaatacta 180aacatactgt acatactcat actcgtaccc gggcaacggt
ttcacttgag tgcagtggct 240agtgctctta ctcgtacagt gtgcaatact gcgtatcata
gtctttgatg tatatcgtat 300tcattcatgt tagttgcgta cgagccggaa gcataaagtg
taaagcctgg ggtgcctaat 360gagtgagcta actcacatta attgcgttgc gctcactgcc
cgctttccag tcgggaaacc 420tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
gagaggcggt ttgcgtattg 480ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
ggtcgttcgg ctgcggcgag 540cggtatcagc tcactcaaag gcggtaatac ggttatccac
agaatcaggg gataacgcag 600gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
ccgtaaaaag gccgcgttgc 660tggcgttttt ccataggctc cgcccccctg acgagcatca
caaaaatcga cgctcaagtc 720agaggtggcg aaacccgaca ggactataaa gataccaggc
gtttccccct ggaagctccc 780tcgtgcgctc tcctgttccg accctgccgc ttaccggata
cctgtccgcc tttctccctt 840cgggaagcgt ggcgctttct catagctcac gctgtaggta
tctcagttcg gtgtaggtcg 900ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
gcccgaccgc tgcgccttat 960ccggtaacta tcgtcttgag tccaacccgg taagacacga
cttatcgcca ctggcagcag 1020ccactggtaa caggattagc agagcgaggt atgtaggcgg
tgctacagag ttcttgaagt 1080ggtggcctaa ctacggctac actagaagga cagtatttgg
tatctgcgct ctgctgaagc 1140cagttacctt cggaaaaaga gttggtagct cttgatccgg
caaacaaacc accgctggta 1200gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
aaaaaaagga tctcaagaag 1260atcctttgat cttttctacg gggtctgacg ctcagtggaa
cgaaaactca cgttaaggga 1320ttttggtcat gagattatca aaaaggatct tcacctagat
ccttttaaat taaaaatgaa 1380gttttaaatc aatctaaagt atatatgagt aaacttggtc
tgacagttac caatgcttaa 1440tcagtgaggc acctatctca gcgatctgtc tatttcgttc
atccatagtt gcctgactcc 1500ccgtcgtgta gataactacg atacgggagg gcttaccatc
tggccccagt gctgcaatga 1560taccgcgaga cccacgctca ccggctccag atttatcagc
aataaaccag ccagccggaa 1620gggccgagcg cagaagtggt cctgcaactt tatccgcctc
catccagtct attaattgtt 1680gccgggaagc tagagtaagt agttcgccag ttaatagttt
gcgcaacgtt gttgccattg 1740ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
ttcattcagc tccggttccc 1800aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
aaaagcggtt agctccttcg 1860gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
atcactcatg gttatggcag 1920cactgcataa ttctcttact gtcatgccat ccgtaagatg
cttttctgtg actggtgagt 1980actcaaccaa gtcattctga gaatagtgta tgcggcgacc
gagttgctct tgcccggcgt 2040caatacggga taataccgcg ccacatagca gaactttaaa
agtgctcatc attggaaaac 2100gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
gagatccagt tcgatgtaac 2160ccactcgtgc acccaactga tcttcagcat cttttacttt
caccagcgtt tctgggtgag 2220caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
ggcgacacgg aaatgttgaa 2280tactcatact cttccttttt caatattatt gaagcattta
tcagggttat tgtctcatga 2340gcggatacat atttgaatgt atttagaaaa ataaacaaat
aggggttccg cgcacatttc 2400cccgaaaagt gccacctgac gcgccctgta gcggcgcatt
aagcgcggcg ggtgtggtgg 2460ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
gcccgctcct ttcgctttct 2520tcccttcctt tctcgccacg ttcgccggct ttccccgtca
agctctaaat cgggggctcc 2580ctttagggtt ccgatttagt gctttacggc acctcgaccc
caaaaaactt gattagggtg 2640atggttcacg tagtgggcca tcgccctgat agacggtttt
tcgccctttg acgttggagt 2700ccacgttctt taatagtgga ctcttgttcc aaactggaac
aacactcaac cctatctcgg 2760tctattcttt tgatttataa gggattttgc cgatttcggc
ctattggtta aaaaatgagc 2820tgatttaaca aaaatttaac gcgaatttta acaaaatatt
aacgcttaca atttccattc 2880gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
cgggcctctt cgctattacg 2940ccagctggcg aaagggggat gtgctgcaag gcgattaagt
tgggtaacgc cagggttttc 3000ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa
tacgactcac tatagggcga 3060attgggtacc gggccccccc tcgaggtcga tggtgtcgat
aagcttgata tcgaattcat 3120gtcacacaaa ccgatcttcg cctcaaggaa acctaattct
acatccgaga gactgccgag 3180atccagtcta cactgattaa ttttcgggcc aataatttaa
aaaaatcgtg ttatataata 3240ttatatgtat tatatatata catcatgatg atactgacag
tcatgtccca ttgctaaata 3300gacagactcc atctgccgcc tccaactgat gttctcaata
tttaaggggt catctcgcat 3360tgtttaataa taaacagact ccatctaccg cctccaaatg
atgttctcaa aatatattgt 3420atgaacttat ttttattact tagtattatt agacaactta
cttgctttat gaaaaacact 3480tcctatttag gaaacaattt ataatggcag ttcgttcatt
taacaattta tgtagaataa 3540atgttataaa tgcgtatggg aaatcttaaa tatggatagc
ataaatgata tctgcattgc 3600ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac
aacataaata gtcatcgaga 3660aatatcaact atcaaagaac agctattcac acgttactat
tgagattatt attggacgag 3720aatcacacac tcaactgtct ttctctcttc tagaaataca
ggtacaagta tgtactattc 3780tcattgttca tacttctagt catttcatcc cacatattcc
ttggatttct ctccaatgaa 3840tgacattcta tcttgcaaat tcaacaatta taataagata
taccaaagta gcggtatagt 3900ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat
ttttattcta atgatccatt 3960aaaggtatat atttatttct tgttatataa tccttttgtt
tattacatgg gctggataca 4020taaaggtatt ttgatttaat tttttgctta aattcaatcc
cccctcgttc agtgtcaact 4080gtaatggtag gaaattacca tacttttgaa gaagcaaaaa
aaatgaaaga aaaaaaaaat 4140cgtatttcca ggttagacgt tccgcagaat ctagaatgcg
gtatgcggta cattgttctt 4200cgaacgtaaa agttgcgctc cctgagatat tgtacatttt
tgcttttaca agtacaagta 4260catcgtacaa ctatgtacta ctgttgatgc atccacaaca
gtttgttttg tttttttttg 4320tttttttttt ttctaatgat tcattaccgc tatgtatacc
tacttgtact tgtagtaagc 4380cgggttattg gcgttcaatt aatcatagac ttatgaatct
gcacggtgtg cgctgcgagt 4440tacttttagc ttatgcatgc tacttgggtg taatattggg
atctgttcgg aaatcaacgg 4500atgctcaatc gatttcgaca gtaattaatt aagtcataca
caagtcagct ttcttcgagc 4560ctcatataag tataagtagt tcaacgtatt agcactgtac
ccagcatctc cgtatcgaga 4620aacacaacaa catgccccat tggacagatc atgcggatac
acaggttgtg cagtatcata 4680catactcgat cagacaggtc gtctgaccat catacaagct
gaacaagcgc tccatacttg 4740cacgctctct atatacacag ttaaattaca tatccatagt
ctaacctcta acagttaatc 4800ttctggtaag cctcccagcc agccttctgg tatcgcttgg
cctcctcaat aggatctcgg 4860ttctggccgt acagacctcg gccgacaatt atgatatccg
ttccggtaga catgacatcc 4920tcaacagttc ggtactgctg tccgagagcg tctcccttgt
cgtcaagacc caccccgggg 4980gtcagaataa gccagtcctc agagtcgccc ttaggtcggt
tctgggcaat gaagccaacc 5040acaaactcgg ggtcggatcg ggcaagctca atggtctgct
tggagtactc gccagtggcc 5100agagagccct tgcaagacag ctcggccagc atgagcagac
ctctggccag cttctcgttg 5160ggagagggga ctaggaactc cttgtactgg gagttctcgt
agtcagagac gtcctccttc 5220ttctgttcag agacagtttc ctcggcacca gctcgcaggc
cagcaatgat tccggttccg 5280ggtacaccgt gggcgttggt gatatcggac cactcggcga
ttcggtgaca ccggtactgg 5340tgcttgacag tgttgccaat atctgcgaac tttctgtcct
cgaacaggaa gaaaccgtgc 5400ttaagagcaa gttccttgag ggggagcaca gtgccggcgt
aggtgaagtc gtcaatgatg 5460tcgatatggg ttttgatcat gcacacataa ggtccgacct
tatcggcaag ctcaatgagc 5520tccttggtgg tggtaacatc cagagaagca cacaggttgg
ttttcttggc tgccacgagc 5580ttgagcactc gagcggcaaa ggcggacttg tggacgttag
ctcgagcttc gtaggagggc 5640attttggtgg tgaagaggag actgaaataa atttagtctg
cagaactttt tatcggaacc 5700ttatctgggg cagtgaagta tatgttatgg taatagttac
gagttagttg aacttataga 5760tagactggac tatacggcta tcggtccaaa ttagaaagaa
cgtcaatggc tctctgggcg 5820tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag
caatgacgtt gcagctgata 5880ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac
ccacagcctc caacgaagaa 5940tgtatcgtca aagtgatcca agcacactca tagttggagt
cgtactccaa aggcggcaat 6000gacgagtcag acagatactc gtcgactcag gcgacgacgg
aattcctgca gcccatctgc 6060agaattcagg agagaccggg ttggcggcgt atttgtgtcc
caaaaaacag ccccaattgc 6120cccggagaag acggccaggc cgcctagatg acaaattcaa
caactcacag ctgactttct 6180gccattgcca ctaggggggg gcctttttat atggccaagc
caagctctcc acgtcggttg 6240ggctgcaccc aacaataaat gggtagggtt gcaccaacaa
agggatggga tggggggtag 6300aagatacgag gataacgggg ctcaatggca caaataagaa
cgaatactgc cattaagact 6360cgtgatccag cgactgacac cattgcatca tctaagggcc
tcaaaactac ctcggaactg 6420ctgcgctgat ctggacacca cagaggttcc gagcacttta
ggttgcacca aatgtcccac 6480caggtgcagg cagaaaacgc tggaacagcg tgtacagttt
gtcttaacaa aaagtgaggg 6540cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt
tagagctgcg aaagcgcgta 6600tggatttggc tcatcaggcc agattgaggg tctgtggaca
catgtcatgt tagtgtactt 6660caatcgcccc ctggatatag ccccgacaat aggccgtggc
ctcatttttt tgccttccgc 6720acatttccat tgctcggtac ccacaccttg cttctcctgc
acttgccaac cttaatactg 6780gtttacattg accaacatct tacaagcggg gggcttgtct
agggtatata taaacagtgg 6840ctctcccaat cggttgccag tctctttttt cctttctttc
cccacagatt cgaaatctaa 6900actacacatc acacaatgcc tgttactgac gtccttaagc
gaaagtccgg tgtcatcgtc 6960ggcgacgatg tccgagccgt gagtatccac gacaagatca
gtgtcgagac gacgcgtttt 7020gtgtaatgac acaatccgaa agtcgctagc aacacacact
ctctacacaa actaacccag 7080ctctccatgg ctctggccaa cgacgctggc gagcgaatct
gggctgccgt caccgatccc 7140gaaatcctca ttggcacctt ctcctacctg ctcctgaagc
ctctcctgcg aaactctggt 7200ctcgtggacg agaagaaagg agcctaccga acctccatga
tctggtacaa cgtcctcctg 7260gctctcttct ctgccctgtc cttctacgtg actgccaccg
ctctcggctg ggactacggt 7320actggagcct ggctgcgaag acagaccggt gatactcccc
agcctctctt tcagtgtccc 7380tctcctgtct gggactccaa gctgttcacc tggactgcca
aggccttcta ctattctaag 7440tacgtggagt acctcgacac cgcttggctg gtcctcaagg
gcaagcgagt gtcctttctg 7500caggccttcc atcactttgg agctccctgg gacgtctacc
tcggcattcg actgcacaac 7560gagggtgtgt ggatcttcat gttctttaac tcgttcattc
acaccatcat gtacacctac 7620tatggactga ctgccgctgg ctacaagttc aaggccaagc
ctctgatcac tgccatgcag 7680atttgccagt tcgtcggtgg ctttctcctg gtctgggact
acatcaacgt tccctgcttc 7740aactctgaca agggcaagct gttctcctgg gctttcaact
acgcctacgt cggatctgtc 7800tttctcctgt tctgtcactt cttttaccag gacaacctgg
ccaccaagaa atccgctaag 7860gctggtaagc agctttagc
7879197783DNAArtificial Sequenceplasmid pY115
19catggctctg gccaacgacg ctggcgagcg aatctgggct gccgtcaccg atcccgaaat
60cctcattggc accttctcct acctgctcct gaagcctctc ctgcgaaact ctggtctcgt
120ggacgagaag aaaggagcct accgaacctc catgatctgg tacaacgtcc tcctggctct
180cttctctgcc ctgtccttct acgtgactgc caccgctctc ggctgggact acggtactgg
240agcctggctg cgaagacaga ccggtgatac tccccagcct ctctttcagt gtccctctcc
300tgtctgggac tccaagctgt tcacctggac tgccaaggcc ttctactatt ctaagtacgt
360ggagtacctc gacaccgctt ggctggtcct caagggcaag cgagtgtcct ttctgcaggc
420cttccatcac tttggagctc cctgggacgt ctacctcggc attcgactgc acaacgaggg
480tgtgtggatc ttcatgttct ttaactcgtt cattcacacc atcatgtaca cctactatgg
540actgactgcc gctggctaca agttcaaggc caagcctctg atcactgcca tgcagatttg
600ccagttcgtc ggtggctttc tcctggtctg ggactacatc aacgttccct gcttcaactc
660tgacaagggc aagctgttct cctgggcttt caactacgcc tacgtcggat ctgtctttct
720cctgttctgt cacttctttt accaggacaa cctggccacc aagaaatccg ctaaggctgg
780taagcagctt tagcggccgc aagtgtggat ggggaagtga gtgcccggtt ctgtgtgcac
840aattggcaat ccaagatgga tggattcaac acagggatat agcgagctac gtggtggtgc
900gaggatatag caacggatat ttatgtttga cacttgagaa tgtacgatac aagcactgtc
960caagtacaat actaaacata ctgtacatac tcatactcgt acccgggcaa cggtttcact
1020tgagtgcagt ggctagtgct cttactcgta cagtgtgcaa tactgcgtat catagtcttt
1080gatgtatatc gtattcattc atgttagttg cgtacgagcc ggaagcataa agtgtaaagc
1140ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
1200ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
1260cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
1320tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
1380aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
1440aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
1500tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
1560ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
1620cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
1680ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
1740ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
1800gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
1860agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
1920cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
1980aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
2040aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
2100ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
2160aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
2220ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
2280agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
2340cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
2400ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
2460gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
2520cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
2580cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
2640ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
2700catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
2760tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
2820ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
2880catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
2940cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
3000cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
3060acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
3120ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
3180tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc
3240ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
3300tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
3360aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
3420acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
3480tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
3540caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
3600gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
3660tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
3720tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
3780acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
3840tcactatagg gcgaattggg taccgggccc cccctcgagg tcgatggtgt cgataagctt
3900gatatcgaat tcatgtcaca caaaccgatc ttcgcctcaa ggaaacctaa ttctacatcc
3960gagagactgc cgagatccag tctacactga ttaattttcg ggccaataat ttaaaaaaat
4020cgtgttatat aatattatat gtattatata tatacatcat gatgatactg acagtcatgt
4080cccattgcta aatagacaga ctccatctgc cgcctccaac tgatgttctc aatatttaag
4140gggtcatctc gcattgttta ataataaaca gactccatct accgcctcca aatgatgttc
4200tcaaaatata ttgtatgaac ttatttttat tacttagtat tattagacaa cttacttgct
4260ttatgaaaaa cacttcctat ttaggaaaca atttataatg gcagttcgtt catttaacaa
4320tttatgtaga ataaatgtta taaatgcgta tgggaaatct taaatatgga tagcataaat
4380gatatctgca ttgcctaatt cgaaatcaac agcaacgaaa aaaatccctt gtacaacata
4440aatagtcatc gagaaatatc aactatcaaa gaacagctat tcacacgtta ctattgagat
4500tattattgga cgagaatcac acactcaact gtctttctct cttctagaaa tacaggtaca
4560agtatgtact attctcattg ttcatacttc tagtcatttc atcccacata ttccttggat
4620ttctctccaa tgaatgacat tctatcttgc aaattcaaca attataataa gatataccaa
4680agtagcggta tagtggcaat caaaaagctt ctctggtgtg cttctcgtat ttatttttat
4740tctaatgatc cattaaaggt atatatttat ttcttgttat ataatccttt tgtttattac
4800atgggctgga tacataaagg tattttgatt taattttttg cttaaattca atcccccctc
4860gttcagtgtc aactgtaatg gtaggaaatt accatacttt tgaagaagca aaaaaaatga
4920aagaaaaaaa aaatcgtatt tccaggttag acgttccgca gaatctagaa tgcggtatgc
4980ggtacattgt tcttcgaacg taaaagttgc gctccctgag atattgtaca tttttgcttt
5040tacaagtaca agtacatcgt acaactatgt actactgttg atgcatccac aacagtttgt
5100tttgtttttt tttgtttttt ttttttctaa tgattcatta ccgctatgta tacctacttg
5160tacttgtagt aagccgggtt attggcgttc aattaatcat agacttatga atctgcacgg
5220tgtgcgctgc gagttacttt tagcttatgc atgctacttg ggtgtaatat tgggatctgt
5280tcggaaatca acggatgctc aatcgatttc gacagtaatt aattaagtca tacacaagtc
5340agctttcttc gagcctcata taagtataag tagttcaacg tattagcact gtacccagca
5400tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt
5460tgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa
5520gcgctccata cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc
5580tctaacagtt aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct
5640caataggatc tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg
5700tagacatgac atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa
5760gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaggt cggttctggg
5820caatgaagcc aaccacaaac tcggggtcgg atcgggcaag ctcaatggtc tgcttggagt
5880actcgccagt ggccagagag cccttgcaag acagctcggc cagcatgagc agacctctgg
5940ccagcttctc gttgggagag gggactagga actccttgta ctgggagttc tcgtagtcag
6000agacgtcctc cttcttctgt tcagagacag tttcctcggc accagctcgc aggccagcaa
6060tgattccggt tccgggtaca ccgtgggcgt tggtgatatc ggaccactcg gcgattcggt
6120gacaccggta ctggtgcttg acagtgttgc caatatctgc gaactttctg tcctcgaaca
6180ggaagaaacc gtgcttaaga gcaagttcct tgagggggag cacagtgccg gcgtaggtga
6240agtcgtcaat gatgtcgata tgggttttga tcatgcacac ataaggtccg accttatcgg
6300caagctcaat gagctccttg gtggtggtaa catccagaga agcacacagg ttggttttct
6360tggctgccac gagcttgagc actcgagcgg caaaggcgga cttgtggacg ttagctcgag
6420cttcgtagga gggcattttg gtggtgaaga ggagactgaa ataaatttag tctgcagaac
6480tttttatcgg aaccttatct ggggcagtga agtatatgtt atggtaatag ttacgagtta
6540gttgaactta tagatagact ggactatacg gctatcggtc caaattagaa agaacgtcaa
6600tggctctctg ggcgtcgcct ttgccgacaa aaatgtgatc atgatgaaag ccagcaatga
6660cgttgcagct gatattgttg tcggccaacc gcgccgaaaa cgcagctgtc agacccacag
6720cctccaacga agaatgtatc gtcaaagtga tccaagcaca ctcatagttg gagtcgtact
6780ccaaaggcgg caatgacgag tcagacagat actcgtcgac gtttaaacag tgtacgcaga
6840tctactatag aggaacattt aaattgcccc ggagaagacg gccaggccgc ctagatgaca
6900aattcaacaa ctcacagctg actttctgcc attgccacta ggggggggcc tttttatatg
6960gccaagccaa gctctccacg tcggttgggc tgcacccaac aataaatggg tagggttgca
7020ccaacaaagg gatgggatgg ggggtagaag atacgaggat aacggggctc aatggcacaa
7080ataagaacga atactgccat taagactcgt gatccagcga ctgacaccat tgcatcatct
7140aagggcctca aaactacctc ggaactgctg cgctgatctg gacaccacag aggttccgag
7200cactttaggt tgcaccaaat gtcccaccag gtgcaggcag aaaacgctgg aacagcgtgt
7260acagtttgtc ttaacaaaaa gtgagggcgc tgaggtcgag cagggtggtg tgacttgtta
7320tagcctttag agctgcgaaa gcgcgtatgg atttggctca tcaggccaga ttgagggtct
7380gtggacacat gtcatgttag tgtacttcaa tcgccccctg gatatagccc cgacaatagg
7440ccgtggcctc atttttttgc cttccgcaca tttccattgc tcgataccca caccttgctt
7500ctcctgcact tgccaacctt aatactggtt tacattgacc aacatcttac aagcgggggg
7560cttgtctagg gtatatataa acagtggctc tcccaatcgg ttgccagtct cttttttcct
7620ttctttcccc acagattcga aatctaaact acacatcaca gaattccgag ccgtgagtat
7680ccacgacaag atcagtgtcg agacgacgcg ttttgtgtaa tgacacaatc cgaaagtcgc
7740tagcaacaca cactctctac acaaactaac ccagctctgg tac
77832019DNAArtificial Sequenceoligonucleotide oYFBA1 20acgcagatct
actatagag
192127DNAArtificial Sequenceoligonucleotide oYFBA1-6 21agcggccgct
ggtaccagag ctgggtt
27226992DNAArtificial Sequenceplasmid pY158 22ggccgcaagt gtggatgggg
aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag
ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120ggatatttat gtttgacact
tgagaatgta cgatacaagc actgtccaag tacaatacta 180aacatactgt acatactcat
actcgtaccc ggcaacggtt tcacttgagt gcagtggcta 240gtgctcttac tcgtacagtg
tgcaatactg cgtatcatag tctttgatgt atatcgtatt 300cattcatgtt agttgcgtac
gagccggaag cataaagtgt aaagcctggg gtgcctaatg 360agtgagctaa ctcacattaa
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 420gtcgtgccag ctgcattaat
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 480gcgctcttcc gcttcctcgc
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 540ggtatcagct cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg 600aaagaacatg tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 660ggcgtttttc cataggctcc
gcccccctga cgagcatcac aaaaatcgac gctcaagtca 720gaggtggcga aacccgacag
gactataaag ataccaggcg tttccccctg gaagctccct 780cgtgcgctct cctgttccga
ccctgccgct taccggatac ctgtccgcct ttctcccttc 840gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 900tcgctccaag ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 960cggtaactat cgtcttgagt
ccaacccggt aagacacgac ttatcgccac tggcagcagc 1020cactggtaac aggattagca
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 1080gtggcctaac tacggctaca
ctagaaggac agtatttggt atctgcgctc tgctgaagcc 1140agttaccttc ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag 1200cggtggtttt tttgtttgca
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 1260tcctttgatc ttttctacgg
ggtctgacgc tcagtggaac gaaaactcac gttaagggat 1320tttggtcatg agattatcaa
aaaggatctt cacctagatc cttttaaatt aaaaatgaag 1380ttttaaatca atctaaagta
tatatgagta aacttggtct gacagttacc aatgcttaat 1440cagtgaggca cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc 1500cgtcgtgtag ataactacga
tacgggaggg cttaccatct ggccccagtg ctgcaatgat 1560accgcgagac ccacgctcac
cggctccaga tttatcagca ataaaccagc cagccggaag 1620ggccgagcgc agaagtggtc
ctgcaacttt atccgcctcc atccagtcta ttaattgttg 1680ccgggaagct agagtaagta
gttcgccagt taatagtttg cgcaacgttg ttgccattgc 1740tacaggcatc gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca 1800acgatcaagg cgagttacat
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 1860tcctccgatc gttgtcagaa
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 1920actgcataat tctcttactg
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 1980ctcaaccaag tcattctgag
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 2040aatacgggat aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg 2100ttcttcgggg cgaaaactct
caaggatctt accgctgttg agatccagtt cgatgtaacc 2160cactcgtgca cccaactgat
cttcagcatc ttttactttc accagcgttt ctgggtgagc 2220aaaaacagga aggcaaaatg
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 2280actcatactc ttcctttttc
aatattattg aagcatttat cagggttatt gtctcatgag 2340cggatacata tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc 2400ccgaaaagtg ccacctgacg
cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 2460tacgcgcagc gtgaccgcta
cacttgccag cgccctagcg cccgctcctt tcgctttctt 2520cccttccttt ctcgccacgt
tcgccggctt tccccgtcaa gctctaaatc gggggctccc 2580tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg attagggtga 2640tggttcacgt agtgggccat
cgccctgata gacggttttt cgccctttga cgttggagtc 2700cacgttcttt aatagtggac
tcttgttcca aactggaaca acactcaacc ctatctcggt 2760ctattctttt gatttataag
ggattttgcc gatttcggcc tattggttaa aaaatgagct 2820gatttaacaa aaatttaacg
cgaattttaa caaaatatta acgcttacaa tttccattcg 2880ccattcaggc tgcgcaactg
ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 2940cagctggcga aagggggatg
tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 3000cagtcacgac gttgtaaaac
gacggccagt gaattgtaat acgactcact atagggcgaa 3060ttgggtaccg ggccccccct
cgaggtcgat ggtgtcgata agcttgatat cgaattcatg 3120tcacacaaac cgatcttcgc
ctcaaggaaa cctaattcta catccgagag actgccgaga 3180tccagtctac actgattaat
tttcgggcca ataatttaaa aaaatcgtgt tatataatat 3240tatatgtatt atatatatac
atcatgatga tactgacagt catgtcccat tgctaaatag 3300acagactcca tctgccgcct
ccaactgatg ttctcaatat ttaaggggtc atctcgcatt 3360gtttaataat aaacagactc
catctaccgc ctccaaatga tgttctcaaa atatattgta 3420tgaacttatt tttattactt
agtattatta gacaacttac ttgctttatg aaaaacactt 3480cctatttagg aaacaattta
taatggcagt tcgttcattt aacaatttat gtagaataaa 3540tgttataaat gcgtatggga
aatcttaaat atggatagca taaatgatat ctgcattgcc 3600taattcgaaa tcaacagcaa
cgaaaaaaat cccttgtaca acataaatag tcatcgagaa 3660atatcaacta tcaaagaaca
gctattcaca cgttactatt gagattatta ttggacgaga 3720atcacacact caactgtctt
tctctcttct agaaatacag gtacaagtat gtactattct 3780cattgttcat acttctagtc
atttcatccc acatattcct tggatttctc tccaatgaat 3840gacattctat cttgcaaatt
caacaattat aataagatat accaaagtag cggtatagtg 3900gcaatcaaaa agcttctctg
gtgtgcttct cgtatttatt tttattctaa tgatccatta 3960aaggtatata tttatttctt
gttatataat ccttttgttt attacatggg ctggatacat 4020aaaggtattt tgatttaatt
ttttgcttaa attcaatccc ccctcgttca gtgtcaactg 4080taatggtagg aaattaccat
acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc 4140gtatttccag gttagacgtt
ccgcagaatc tagaatgcgg tatgcggtac attgttcttc 4200gaacgtaaaa gttgcgctcc
ctgagatatt gtacattttt gcttttacaa gtacaagtac 4260atcgtacaac tatgtactac
tgttgatgca tccacaacag tttgttttgt ttttttttgt 4320tttttttttt tctaatgatt
cattaccgct atgtatacct acttgtactt gtagtaagcc 4380gggttattgg cgttcaatta
atcatagact tatgaatctg cacggtgtgc gctgcgagtt 4440acttttagct tatgcatgct
acttgggtgt aatattggga tctgttcgga aatcaacgga 4500tgctcaatcg atttcgacag
taattaatta agtcatacac aagtcagctt tcttcgagcc 4560tcatataagt ataagtagtt
caacgtatta gcactgtacc cagcatctcc gtatcgagaa 4620acacaacaac atgccccatt
ggacagatca tgcggataca caggttgtgc agtatcatac 4680atactcgatc agacaggtcg
tctgaccatc atacaagctg aacaagcgct ccatacttgc 4740acgctctcta tatacacagt
taaattacat atccatagtc taacctctaa cagttaatct 4800tctggtaagc ctcccagcca
gccttctggt atcgcttggc ctcctcaata ggatctcggt 4860tctggccgta cagacctcgg
ccgacaatta tgatatccgt tccggtagac atgacatcct 4920caacagttcg gtactgctgt
ccgagagcgt ctcccttgtc gtcaagaccc accccggggg 4980tcagaataag ccagtcctca
gagtcgccct taggtcggtt ctgggcaatg aagccaacca 5040caaactcggg gtcggatcgg
gcaagctcaa tggtctgctt ggagtactcg ccagtggcca 5100gagagccctt gcaagacagc
tcggccagca tgagcagacc tctggccagc ttctcgttgg 5160gagaggggac taggaactcc
ttgtactggg agttctcgta gtcagagacg tcctccttct 5220tctgttcaga gacagtttcc
tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg 5280gtacaccgtg ggcgttggtg
atatcggacc actcggcgat tcggtgacac cggtactggt 5340gcttgacagt gttgccaata
tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct 5400taagagcaag ttccttgagg
gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt 5460cgatatgggt tttgatcatg
cacacataag gtccgacctt atcggcaagc tcaatgagct 5520ccttggtggt ggtaacatcc
agagaagcac acaggttggt tttcttggct gccacgagct 5580tgagcactcg agcggcaaag
gcggacttgt ggacgttagc tcgagcttcg taggagggca 5640ttttggtggt gaagaggaga
ctgaaataaa tttagtctgc agaacttttt atcggaacct 5700tatctggggc agtgaagtat
atgttatggt aatagttacg agttagttga acttatagat 5760agactggact atacggctat
cggtccaaat tagaaagaac gtcaatggct ctctgggcgt 5820cgcctttgcc gacaaaaatg
tgatcatgat gaaagccagc aatgacgttg cagctgatat 5880tgttgtcggc caaccgcgcc
gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat 5940gtatcgtcaa agtgatccaa
gcacactcat agttggagtc gtactccaaa ggcggcaatg 6000acgagtcaga cagatactcg
tcgacgttta aacagtgtac gcagatctac tatagaggaa 6060catttaaatt gccccggaga
agacggccag gccgcctaga tgacaaattc aacaactcac 6120agctgacttt ctgccattgc
cactaggggg gggccttttt atatggccaa gccaagctct 6180ccacgtcggt tgggctgcac
ccaacaataa atgggtaggg ttgcaccaac aaagggatgg 6240gatggggggt agaagatacg
aggataacgg ggctcaatgg cacaaataag aacgaatact 6300gccattaaga ctcgtgatcc
agcgactgac accattgcat catctaaggg cctcaaaact 6360acctcggaac tgctgcgctg
atctggacac cacagaggtt ccgagcactt taggttgcac 6420caaatgtccc accaggtgca
ggcagaaaac gctggaacag cgtgtacagt ttgtcttagc 6480aaaaagtgaa ggcgctgagg
tcgagcaggg tggtgtgact tgttatagcc tttagagctg 6540cgaaagcgcg tatggatttg
gctcatcagg ccagattgag ggtctgtgga cacatgtcat 6600gttagtgtac ttcaatcgcc
ccctggatat agccccgaca ataggccgtg gcctcatttt 6660tttgccttcc gcacatttcc
attgctcgat acccacacct tgcttctcct gcacttgcca 6720accttaatac tggtttacat
tgaccaacat cttacaagcg gggggcttgt ctagggtata 6780tataaacagt ggctctccca
atcggttgcc agtctctttt ttcctttctt tccccacaga 6840ttcgaaatct aaactacaca
tcacagaatt ccgagccgtg agtatccacg acaagatcag 6900tgtcgagacg acgcgttttg
tgtaatgaca caatccgaaa gtcgctagca acacacactc 6960tctacacaaa ctaacccagc
tctggtacca gc 6992238707DNAArtificial
Sequenceplasmid pY159 23ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt
gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag ggatatagcg agctacgtgg
tggtgcgagg atatagcaac 120ggatatttat gtttgacact tgagaatgta cgatacaagc
actgtccaag tacaatacta 180aacatactgt acatactcat actcgtaccc ggcaacggtt
tcacttgagt gcagtggcta 240gtgctcttac tcgtacagtg tgcaatactg cgtatcatag
tctttgatgt atatcgtatt 300cattcatgtt agttgcgtac gagccggaag cataaagtgt
aaagcctggg gtgcctaatg 360agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
gctttccagt cgggaaacct 420gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg 480gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc 540ggtatcagct cactcaaagg cggtaatacg gttatccaca
gaatcagggg ataacgcagg 600aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
cgtaaaaagg ccgcgttgct 660ggcgtttttc cataggctcc gcccccctga cgagcatcac
aaaaatcgac gctcaagtca 720gaggtggcga aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct 780cgtgcgctct cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc 840gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt 900tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
cccgaccgct gcgccttatc 960cggtaactat cgtcttgagt ccaacccggt aagacacgac
ttatcgccac tggcagcagc 1020cactggtaac aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg 1080gtggcctaac tacggctaca ctagaaggac agtatttggt
atctgcgctc tgctgaagcc 1140agttaccttc ggaaaaagag ttggtagctc ttgatccggc
aaacaaacca ccgctggtag 1200cggtggtttt tttgtttgca agcagcagat tacgcgcaga
aaaaaaggat ctcaagaaga 1260tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
gaaaactcac gttaagggat 1320tttggtcatg agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag 1380ttttaaatca atctaaagta tatatgagta aacttggtct
gacagttacc aatgcttaat 1440cagtgaggca cctatctcag cgatctgtct atttcgttca
tccatagttg cctgactccc 1500cgtcgtgtag ataactacga tacgggaggg cttaccatct
ggccccagtg ctgcaatgat 1560accgcgagac ccacgctcac cggctccaga tttatcagca
ataaaccagc cagccggaag 1620ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg 1680ccgggaagct agagtaagta gttcgccagt taatagtttg
cgcaacgttg ttgccattgc 1740tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
tcattcagct ccggttccca 1800acgatcaagg cgagttacat gatcccccat gttgtgcaaa
aaagcggtta gctccttcgg 1860tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
tcactcatgg ttatggcagc 1920actgcataat tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta 1980ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
agttgctctt gcccggcgtc 2040aatacgggat aataccgcgc cacatagcag aactttaaaa
gtgctcatca ttggaaaacg 2100ttcttcgggg cgaaaactct caaggatctt accgctgttg
agatccagtt cgatgtaacc 2160cactcgtgca cccaactgat cttcagcatc ttttactttc
accagcgttt ctgggtgagc 2220aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat 2280actcatactc ttcctttttc aatattattg aagcatttat
cagggttatt gtctcatgag 2340cggatacata tttgaatgta tttagaaaaa taaacaaata
ggggttccgc gcacatttcc 2400ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta
agcgcggcgg gtgtggtggt 2460tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
cccgctcctt tcgctttctt 2520cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
gctctaaatc gggggctccc 2580tttagggttc cgatttagtg ctttacggca cctcgacccc
aaaaaacttg attagggtga 2640tggttcacgt agtgggccat cgccctgata gacggttttt
cgccctttga cgttggagtc 2700cacgttcttt aatagtggac tcttgttcca aactggaaca
acactcaacc ctatctcggt 2760ctattctttt gatttataag ggattttgcc gatttcggcc
tattggttaa aaaatgagct 2820gatttaacaa aaatttaacg cgaattttaa caaaatatta
acgcttacaa tttccattcg 2880ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
gggcctcttc gctattacgc 2940cagctggcga aagggggatg tgctgcaagg cgattaagtt
gggtaacgcc agggttttcc 3000cagtcacgac gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa 3060ttgggtaccg ggccccccct cgaggtcgat ggtgtcgata
agcttgatat cgaattcatg 3120tcacacaaac cgatcttcgc ctcaaggaaa cctaattcta
catccgagag actgccgaga 3180tccagtctac actgattaat tttcgggcca ataatttaaa
aaaatcgtgt tatataatat 3240tatatgtatt atatatatac atcatgatga tactgacagt
catgtcccat tgctaaatag 3300acagactcca tctgccgcct ccaactgatg ttctcaatat
ttaaggggtc atctcgcatt 3360gtttaataat aaacagactc catctaccgc ctccaaatga
tgttctcaaa atatattgta 3420tgaacttatt tttattactt agtattatta gacaacttac
ttgctttatg aaaaacactt 3480cctatttagg aaacaattta taatggcagt tcgttcattt
aacaatttat gtagaataaa 3540tgttataaat gcgtatggga aatcttaaat atggatagca
taaatgatat ctgcattgcc 3600taattcgaaa tcaacagcaa cgaaaaaaat cccttgtaca
acataaatag tcatcgagaa 3660atatcaacta tcaaagaaca gctattcaca cgttactatt
gagattatta ttggacgaga 3720atcacacact caactgtctt tctctcttct agaaatacag
gtacaagtat gtactattct 3780cattgttcat acttctagtc atttcatccc acatattcct
tggatttctc tccaatgaat 3840gacattctat cttgcaaatt caacaattat aataagatat
accaaagtag cggtatagtg 3900gcaatcaaaa agcttctctg gtgtgcttct cgtatttatt
tttattctaa tgatccatta 3960aaggtatata tttatttctt gttatataat ccttttgttt
attacatggg ctggatacat 4020aaaggtattt tgatttaatt ttttgcttaa attcaatccc
ccctcgttca gtgtcaactg 4080taatggtagg aaattaccat acttttgaag aagcaaaaaa
aatgaaagaa aaaaaaaatc 4140gtatttccag gttagacgtt ccgcagaatc tagaatgcgg
tatgcggtac attgttcttc 4200gaacgtaaaa gttgcgctcc ctgagatatt gtacattttt
gcttttacaa gtacaagtac 4260atcgtacaac tatgtactac tgttgatgca tccacaacag
tttgttttgt ttttttttgt 4320tttttttttt tctaatgatt cattaccgct atgtatacct
acttgtactt gtagtaagcc 4380gggttattgg cgttcaatta atcatagact tatgaatctg
cacggtgtgc gctgcgagtt 4440acttttagct tatgcatgct acttgggtgt aatattggga
tctgttcgga aatcaacgga 4500tgctcaatcg atttcgacag taattaatta agtcatacac
aagtcagctt tcttcgagcc 4560tcatataagt ataagtagtt caacgtatta gcactgtacc
cagcatctcc gtatcgagaa 4620acacaacaac atgccccatt ggacagatca tgcggataca
caggttgtgc agtatcatac 4680atactcgatc agacaggtcg tctgaccatc atacaagctg
aacaagcgct ccatacttgc 4740acgctctcta tatacacagt taaattacat atccatagtc
taacctctaa cagttaatct 4800tctggtaagc ctcccagcca gccttctggt atcgcttggc
ctcctcaata ggatctcggt 4860tctggccgta cagacctcgg ccgacaatta tgatatccgt
tccggtagac atgacatcct 4920caacagttcg gtactgctgt ccgagagcgt ctcccttgtc
gtcaagaccc accccggggg 4980tcagaataag ccagtcctca gagtcgccct taggtcggtt
ctgggcaatg aagccaacca 5040caaactcggg gtcggatcgg gcaagctcaa tggtctgctt
ggagtactcg ccagtggcca 5100gagagccctt gcaagacagc tcggccagca tgagcagacc
tctggccagc ttctcgttgg 5160gagaggggac taggaactcc ttgtactggg agttctcgta
gtcagagacg tcctccttct 5220tctgttcaga gacagtttcc tcggcaccag ctcgcaggcc
agcaatgatt ccggttccgg 5280gtacaccgtg ggcgttggtg atatcggacc actcggcgat
tcggtgacac cggtactggt 5340gcttgacagt gttgccaata tctgcgaact ttctgtcctc
gaacaggaag aaaccgtgct 5400taagagcaag ttccttgagg gggagcacag tgccggcgta
ggtgaagtcg tcaatgatgt 5460cgatatgggt tttgatcatg cacacataag gtccgacctt
atcggcaagc tcaatgagct 5520ccttggtggt ggtaacatcc agagaagcac acaggttggt
tttcttggct gccacgagct 5580tgagcactcg agcggcaaag gcggacttgt ggacgttagc
tcgagcttcg taggagggca 5640ttttggtggt gaagaggaga ctgaaataaa tttagtctgc
agaacttttt atcggaacct 5700tatctggggc agtgaagtat atgttatggt aatagttacg
agttagttga acttatagat 5760agactggact atacggctat cggtccaaat tagaaagaac
gtcaatggct ctctgggcgt 5820cgcctttgcc gacaaaaatg tgatcatgat gaaagccagc
aatgacgttg cagctgatat 5880tgttgtcggc caaccgcgcc gaaaacgcag ctgtcagacc
cacagcctcc aacgaagaat 5940gtatcgtcaa agtgatccaa gcacactcat agttggagtc
gtactccaaa ggcggcaatg 6000acgagtcaga cagatactcg tcgacgttta aacagtgtac
gcagatctac tatagaggaa 6060catttaaatt gccccggaga agacggccag gccgcctaga
tgacaaattc aacaactcac 6120agctgacttt ctgccattgc cactaggggg gggccttttt
atatggccaa gccaagctct 6180ccacgtcggt tgggctgcac ccaacaataa atgggtaggg
ttgcaccaac aaagggatgg 6240gatggggggt agaagatacg aggataacgg ggctcaatgg
cacaaataag aacgaatact 6300gccattaaga ctcgtgatcc agcgactgac accattgcat
catctaaggg cctcaaaact 6360acctcggaac tgctgcgctg atctggacac cacagaggtt
ccgagcactt taggttgcac 6420caaatgtccc accaggtgca ggcagaaaac gctggaacag
cgtgtacagt ttgtcttagc 6480aaaaagtgaa ggcgctgagg tcgagcaggg tggtgtgact
tgttatagcc tttagagctg 6540cgaaagcgcg tatggatttg gctcatcagg ccagattgag
ggtctgtgga cacatgtcat 6600gttagtgtac ttcaatcgcc ccctggatat agccccgaca
ataggccgtg gcctcatttt 6660tttgccttcc gcacatttcc attgctcgat acccacacct
tgcttctcct gcacttgcca 6720accttaatac tggtttacat tgaccaacat cttacaagcg
gggggcttgt ctagggtata 6780tataaacagt ggctctccca atcggttgcc agtctctttt
ttcctttctt tccccacaga 6840ttcgaaatct aaactacaca tcacagaatt ccgagccgtg
agtatccacg acaagatcag 6900tgtcgagacg acgcgttttg tgtaatgaca caatccgaaa
gtcgctagca acacacactc 6960tctacacaaa ctaacccagc tctggtacca gcggccatca
caagtttgta caaaaaagct 7020gaacgagaaa cgtaaaatga tataaatatc aatatattaa
attagatttt gcataaaaaa 7080cagactacat aatactgtaa aacacaacat atccagtcat
attggcggcc gcattaggca 7140ccccaggctt tacactttat gcttccggct cgtataatgt
gtggattttg agttaggatc 7200cgtcgagatt ttcaggagct aaggaagcta aaatggagaa
aaaaatcact ggatatacca 7260ccgttgatat atcccaatgg catcgtaaag aacattttga
ggcatttcag tcagttgctc 7320aatgtaccta taaccagacc gttcagctgg atattacggc
ctttttaaag accgtaaaga 7380aaaataagca caagttttat ccggccttta ttcacattct
tgcccgcctg atgaatgctc 7440atccggaatt ccgtatggca atgaaagacg gtgagctggt
gatatgggat agtgttcacc 7500cttgttacac cgttttccat gagcaaactg aaacgttttc
atcgctctgg agtgaatacc 7560acgacgattt ccggcagttt ctacacatat attcgcaaga
tgtggcgtgt tacggtgaaa 7620acctggccta tttccctaaa gggtttattg agaatatgtt
tttcgtctca gccaatccct 7680gggtgagttt caccagtttt gatttaaacg tggccaatat
ggacaacttc ttcgcccccg 7740ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
gctgatgccg ctggcgattc 7800aggttcatca tgccgtttgt gatggcttcc atgtcggcag
aatgcttaat gaattacaac 7860agtactgcga tgagtggcag ggcggggcgt aaacgcgtgg
atccggctta ctaaaagcca 7920gataacagta tgcgtatttg cgcgctgatt tttgcggtat
aagaatatat actgatatgt 7980atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
cgtattacag tgacagttga 8040cagcgacagc tatcagttgc tcaaggcata tatgatgtca
atatctccgg tctggtaagc 8100acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
gctggaaagc ggaaaatcag 8160gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
gctcttttgc tgacgagaac 8220aggggctggt gaaatgcagt ttaaggttta cacctataaa
agagagagcc gttatcgtct 8280gtttgtggat gtacagagtg atattattga cacgcccggg
cgacggatgg tgatccccct 8340ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
ctttacccgg tggtgcatat 8400cggggatgaa agctggcgca tgatgaccac cgatatggcc
agtgtgccgg tctccgttat 8460cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
atcaaaaacg ccattaacct 8520gatgttctgg ggaatataaa tgtcaggctc ccttatacac
agccagtctg caggtcgacc 8580atagtgactg gatatgttgt gttttacagc attatgtagt
ctgtttttta tgcaaaatct 8640aatttaatat attgatattt atatcatttt acgtttctcg
ttcagctttc ttgtacaaag 8700tggtgat
8707248827DNAArtificial Sequenceplasmid pY169
24cttgtacaaa gtggtgatgg ccgcaagtgt ggatggggaa gtgagtgccc ggttctgtgt
60gcacaattgg caatccaaga tggatggatt caacacaggg atatagcgag ctacgtggtg
120gtgcgaggat atagcaacgg atatttatgt ttgacacttg agaatgtacg atacaagcac
180tgtccaagta caatactaaa catactgtac atactcatac tcgtacccgg caacggtttc
240acttgagtgc agtggctagt gctcttactc gtacagtgtg caatactgcg tatcatagtc
300tttgatgtat atcgtattca ttcatgttag ttgcgtacga gccggaagca taaagtgtaa
360agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
420tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
480aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
540cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
600atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
660taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
720aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
780tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
840gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
900cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
960cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
1020atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
1080tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
1140ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
1200acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
1260aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
1320aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
1380tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
1440cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
1500catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
1560ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
1620aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
1680ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
1740caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
1800attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
1860agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
1920actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
1980ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
2040ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
2100gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
2160atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
2220cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
2280gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
2340gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
2400ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
2460cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
2520cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
2580tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
2640aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
2700ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
2760actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
2820ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
2880gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
2940gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
3000gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
3060gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgatgg tgtcgataag
3120cttgatatcg aattcatgtc acacaaaccg atcttcgcct caaggaaacc taattctaca
3180tccgagagac tgccgagatc cagtctacac tgattaattt tcgggccaat aatttaaaaa
3240aatcgtgtta tataatatta tatgtattat atatatacat catgatgata ctgacagtca
3300tgtcccattg ctaaatagac agactccatc tgccgcctcc aactgatgtt ctcaatattt
3360aaggggtcat ctcgcattgt ttaataataa acagactcca tctaccgcct ccaaatgatg
3420ttctcaaaat atattgtatg aacttatttt tattacttag tattattaga caacttactt
3480gctttatgaa aaacacttcc tatttaggaa acaatttata atggcagttc gttcatttaa
3540caatttatgt agaataaatg ttataaatgc gtatgggaaa tcttaaatat ggatagcata
3600aatgatatct gcattgccta attcgaaatc aacagcaacg aaaaaaatcc cttgtacaac
3660ataaatagtc atcgagaaat atcaactatc aaagaacagc tattcacacg ttactattga
3720gattattatt ggacgagaat cacacactca actgtctttc tctcttctag aaatacaggt
3780acaagtatgt actattctca ttgttcatac ttctagtcat ttcatcccac atattccttg
3840gatttctctc caatgaatga cattctatct tgcaaattca acaattataa taagatatac
3900caaagtagcg gtatagtggc aatcaaaaag cttctctggt gtgcttctcg tatttatttt
3960tattctaatg atccattaaa ggtatatatt tatttcttgt tatataatcc ttttgtttat
4020tacatgggct ggatacataa aggtattttg atttaatttt ttgcttaaat tcaatccccc
4080ctcgttcagt gtcaactgta atggtaggaa attaccatac ttttgaagaa gcaaaaaaaa
4140tgaaagaaaa aaaaaatcgt atttccaggt tagacgttcc gcagaatcta gaatgcggta
4200tgcggtacat tgttcttcga acgtaaaagt tgcgctccct gagatattgt acatttttgc
4260ttttacaagt acaagtacat cgtacaacta tgtactactg ttgatgcatc cacaacagtt
4320tgttttgttt ttttttgttt tttttttttc taatgattca ttaccgctat gtatacctac
4380ttgtacttgt agtaagccgg gttattggcg ttcaattaat catagactta tgaatctgca
4440cggtgtgcgc tgcgagttac ttttagctta tgcatgctac ttgggtgtaa tattgggatc
4500tgttcggaaa tcaacggatg ctcaatcgat ttcgacagta attaattaag tcatacacaa
4560gtcagctttc ttcgagcctc atataagtat aagtagttca acgtattagc actgtaccca
4620gcatctccgt atcgagaaac acaacaacat gccccattgg acagatcatg cggatacaca
4680ggttgtgcag tatcatacat actcgatcag acaggtcgtc tgaccatcat acaagctgaa
4740caagcgctcc atacttgcac gctctctata tacacagtta aattacatat ccatagtcta
4800acctctaaca gttaatcttc tggtaagcct cccagccagc cttctggtat cgcttggcct
4860cctcaatagg atctcggttc tggccgtaca gacctcggcc gacaattatg atatccgttc
4920cggtagacat gacatcctca acagttcggt actgctgtcc gagagcgtct cccttgtcgt
4980caagacccac cccgggggtc agaataagcc agtcctcaga gtcgccctta ggtcggttct
5040gggcaatgaa gccaaccaca aactcggggt cggatcgggc aagctcaatg gtctgcttgg
5100agtactcgcc agtggccaga gagcccttgc aagacagctc ggccagcatg agcagacctc
5160tggccagctt ctcgttggga gaggggacta ggaactcctt gtactgggag ttctcgtagt
5220cagagacgtc ctccttcttc tgttcagaga cagtttcctc ggcaccagct cgcaggccag
5280caatgattcc ggttccgggt acaccgtggg cgttggtgat atcggaccac tcggcgattc
5340ggtgacaccg gtactggtgc ttgacagtgt tgccaatatc tgcgaacttt ctgtcctcga
5400acaggaagaa accgtgctta agagcaagtt ccttgagggg gagcacagtg ccggcgtagg
5460tgaagtcgtc aatgatgtcg atatgggttt tgatcatgca cacataaggt ccgaccttat
5520cggcaagctc aatgagctcc ttggtggtgg taacatccag agaagcacac aggttggttt
5580tcttggctgc cacgagcttg agcactcgag cggcaaaggc ggacttgtgg acgttagctc
5640gagcttcgta ggagggcatt ttggtggtga agaggagact gaaataaatt tagtctgcag
5700aactttttat cggaacctta tctggggcag tgaagtatat gttatggtaa tagttacgag
5760ttagttgaac ttatagatag actggactat acggctatcg gtccaaatta gaaagaacgt
5820caatggctct ctgggcgtcg cctttgccga caaaaatgtg atcatgatga aagccagcaa
5880tgacgttgca gctgatattg ttgtcggcca accgcgccga aaacgcagct gtcagaccca
5940cagcctccaa cgaagaatgt atcgtcaaag tgatccaagc acactcatag ttggagtcgt
6000actccaaagg cggcaatgac gagtcagaca gatactcgtc gacgtttaaa cagtgtacgc
6060agatctacta tagaggaaca tttaaattgc cccggagaag acggccaggc cgcctagatg
6120acaaattcaa caactcacag ctgactttct gccattgcca ctaggggggg gcctttttat
6180atggccaagc caagctctcc acgtcggttg ggctgcaccc aacaataaat gggtagggtt
6240gcaccaacaa agggatggga tggggggtag aagatacgag gataacgggg ctcaatggca
6300caaataagaa cgaatactgc cattaagact cgtgatccag cgactgacac cattgcatca
6360tctaagggcc tcaaaactac ctcggaactg ctgcgctgat ctggacacca cagaggttcc
6420gagcacttta ggttgcacca aatgtcccac caggtgcagg cagaaaacgc tggaacagcg
6480tgtacagttt gtcttagcaa aaagtgaagg cgctgaggtc gagcagggtg gtgtgacttg
6540ttatagcctt tagagctgcg aaagcgcgta tggatttggc tcatcaggcc agattgaggg
6600tctgtggaca catgtcatgt tagtgtactt caatcgcccc ctggatatag ccccgacaat
6660aggccgtggc ctcatttttt tgccttccgc acatttccat tgctcgatac ccacaccttg
6720cttctcctgc acttgccaac cttaatactg gtttacattg accaacatct tacaagcggg
6780gggcttgtct agggtatata taaacagtgg ctctcccaat cggttgccag tctctttttt
6840cctttctttc cccacagatt cgaaatctaa actacacatc acagaattcc gagccgtgag
6900tatccacgac aagatcagtg tcgagacgac gcgttttgtg taatgacaca atccgaaagt
6960cgctagcaac acacactctc tacacaaact aacccagctc tggtaccagc ggccatcaca
7020agtttgtaca aaaaagttgg attttttttc gggatggcca ccatctcttt gactactgag
7080caacttttag aacacccaga actggttgca attgatgggg tgttgtacga tctcttcgga
7140ctggcgaaag tgcatccagg tggcaacctc attgaagccg ccggtgcctc cgacggaacc
7200gccctgttct actccatgca ccctggagtg aagccagaga attcgaagct gctgcagcaa
7260tttgcccgag gcaaacacga acgaagctcg aaggacccag tgtacacctt tgacagtccc
7320ttcgcccagg atgtcaagca gagcgttcgg gaggtcatga aggggcgcaa ctggtacgcc
7380acgcccggct tttggctgcg gaccgcgctg atcatcgcgt gcactgccat aggcgaatgg
7440tattggatca ctaccggggc agtgatgtgg ggcatcttca ccgggtactt ccacagccag
7500attgggttgg cgattcaaca cgatgcctct cacggagcca tcagcaaaaa gccctgggtg
7560aacgcctttt tcgcctacgg catcgacgcc attggatcct cccgctggat ctggctgcag
7620tcccacatta tgcgccacca cacctacacc aaccagcatg gcctggacct ggacgctgcc
7680tcggcggagc cgttcatttt gttccactcc tacccggcaa caaatgcgtc acgaaagtgg
7740taccatcggt tccaggcgtg gtacatgtac atcgttttgg ggatgtatgg tgtgtcgatg
7800gtgtacaatc cgatgtactt gttcacgatg cagcacaacg acacaatccc agaggccacc
7860tctcttagac caggcagctt tttcaaccgg cagcgcgcct tcgccgtttc cctccgccta
7920ctgttcatct tccgcaacgc cttcctcccc tggtacatcg cgggcgcctc tccgctgctc
7980accatcctgc tggtgccaac ggtcacaggc atcttcttga catttgtttt tgtgctgtcc
8040cataactttg aaggcgctga gcggaccccc gaaaagaact gcaaggccaa aagggccaag
8100gaggggaagg aggtccgcga tgtagaggag gaccgggtgg actggtaccg ggcgcaggcc
8160gagaccgcgg cgacctacgg gggcagcgtc gggatgatgc tgaccggcgg tttgaacctg
8220cagatcgagc accacttgtt cccccgcatg tcctcttggc actacccctt catccaagat
8280acggtgcggg aatgttgcaa gcgccatggc gtgcgctaca catactaccc gaccatcctg
8340gagaatataa tgtccacgct ccgctacatg cagaaggtgg gcgtggccca cacaattcag
8400gatgcccagg aattctgagt gagttcgatc cgcatcgacg tctaccattt ttgatgctgt
8460ctattcctgt tttcagtcac ctccagcatt ctcatggctg gtgaccactg cccctctaac
8520ccattgtgac acaccgccaa agactttgcc tctttttttt ccctttcttt tgtcctcggg
8580gtgctttggc cggtgtttac tcgccttgca gtccccgcaa acgaccgacg tttaagctcc
8640gttgttgact gggccgctcg taaacccatc tgcaggttga ggctcccatg gagaattgtg
8700atggctgatt aggaggtggc ggggcataca tgcctcgaca ctcaaagccg ggcggcttct
8760ggattcgaaa acgcaaatgg gcgctttgga aaaaaaaaaa aaaaaaaaaa aaaaaaaacc
8820caacttt
882725774DNAEuglena gracilis 25atggaggtgg tgaatgaaat agtctcaatt
gggcaggaag ttttacccaa agttgattat 60gcccaactct ggagtgatgc cagtcactgt
gaggtgcttt acttgtccat cgcatttgtc 120atcttgaagt tcactcttgg cccccttggt
ccaaaaggtc agtctcgtat gaagtttgtt 180ttcaccaatt acaaccttct catgtccatt
tattcgttgg gatcattcct ctcaatggca 240tatgccatgt acaccatcgg tgttatgtct
gacaactgcg agaaggcttt tgacaacaac 300gtcttcagga tcaccacgca gttgttctat
ttgagcaagt tcctggagta tattgactcc 360ttctatttgc cactgatggg caagcctctg
acctggttgc aattcttcca tcatttgggg 420gcaccgatgg atatgtggct gttctataat
taccgaaatg aagctgtttg gatttttgtg 480ctgttgaatg gtttcatcca ctggatcatg
tacggttatt attggaccag attgatcaag 540ctgaagttcc ccatgccaaa atccctgatt
acatcaatgc agatcattca attcaatgtt 600ggtttctaca ttgtctggaa gtacaggaac
attccctgtt atcgccaaga tgggatgagg 660atgtttggct ggttcttcaa ttacttttat
gttggcacag tcttgtgttt gttcttgaat 720ttctatgtgc aaacgtatat cgtcaggaag
cacaagggag ccaaaaagat tcag 774261263DNAEuglena gracilis
26atgaagtcaa agcgccaagc gcttcccctt acaattgatg gaacaacata tgatgtgtct
60gcctgggtca atttccaccc tggtggtgcg gaaattatag agaattacca aggaagggat
120gccactgatg ccttcatggt tatgcactct caagaagcct tcgacaagct caagcgcatg
180cccaaaatca atcccagttc tgagttgcca ccccaggctg cagtgaatga agctcaagag
240gatttccgga agctccgaga agagttgatc gcaactggca tgtttgatgc ctcccccctc
300tggtactcat acaaaatcag caccacactg ggccttggag tgctgggtta tttcctgatg
360gttcagtatc agatgtattt cattggggca gtgttgcttg ggatgcacta tcaacagatg
420ggctggcttt ctcatgacat ttgccaccac cagactttca agaaccggaa ctggaacaac
480ctcgtgggac tggtatttgg caatggtctg caaggttttt ccgtgacatg gtggaaggac
540agacacaatg cacatcattc ggcaaccaat gttcaagggc acgaccctga tattgacaac
600ctccccctct tagcctggtc tgaggatgac gtcacacggg cgtcaccgat ttcccgcaag
660ctcattcagt tccagcagta ctatttcttg gtcatctgta tcttgttgcg gttcatttgg
720tgtttccaga gcgtgttgac cgtgcgcagt ttgaaggaca gagataacca attctatcgc
780tctcagtata agaaggaggc cattggcctc gccctgcact ggaccttgaa gaccctgttc
840cacttattct ttatgcccag catcctcaca tcgctgttgg tgtttttcgt ttcggagctg
900gttggcggct tcggcattgc gatcgtggtg ttcatgaacc actacccact ggagaagatc
960ggggactcag tctgggatgg ccatggattc tcggttggcc agatccatga gaccatgaac
1020attcggcgag ggattatcac agattggttt ttcggaggct tgaattacca gattgagcac
1080catttgtggc cgaccctccc tcgccacaac ctgacagcgg ttagctacca ggtggaacag
1140ctgtgccaga agcacaacct gccgtatcgg aacccgctgc cccatgaagg gttggtcatc
1200ctgctgcgct atctggcggt gttcgcccgg atggcggaga agcaacccgc ggggaaggct
1260cta
12632730DNAArtificial SequenceEuglena gracilis elongase sense
oligonucleotide oEugEL1-1 27agcggccgca ccatggaggt ggtgaatgaa
302830DNAArtificial SequenceEuglena gracilis
elongase anti-sense oligonucleotide oEugEL1-2 28tgcggccgct
cactgaatct ttttggctcc
30294311DNAArtificial Sequenceplasmid pKR906 29agcggccgca ccatggaggt
ggtgaatgaa atagtctcaa ttgggcagga agttttaccc 60aaagttgatt atgcccaact
ctggagtgat gccagtcact gtgaggtgct ttacttgtcc 120atcgcatttg tcatcttgaa
gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180atgaagtttg ttttcaccaa
ttacaacctt ctcatgtcca tttattcgtt gggatcattc 240ctctcaatgg catatgccat
gtacaccatc ggtgttatgt ctgacaactg cgagaaggct 300tttgacaaca acgtcttcag
gatcaccacg cagttgttct atttgagcaa gttcctggag 360tatattgact ccttctattt
gccactgatg ggcaagcctc tgacctggtt gcaattcttc 420catcatttgg gggcaccgat
ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480tggatttttg tgctgttgaa
tggtttcatc cactggatca tgtacggtta ttattggacc 540agattgatca agctgaagtt
ccccatgcca aaatccctga ttacatcaat gcagatcatt 600caattcaatg ttggtttcta
cattgtctgg aagtacagga acattccctg ttatcgccaa 660gatgggatga ggatgtttgg
ctggttcttc aattactttt atgttggcac agtcttgtgt 720ttgttcttga atttctatgt
gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780attcagtgag cggccgcacc
tgaattccag cacactggcg gccgttacta gtggatccga 840gctcggtacc aagcttgatg
catagcttga gtattctaac gcgtcaccta aatagcttgg 900cgtaatcatg gtcatagctg
tttcctgtgt gaaattgtta tccgctcaca attccacaca 960acatacgagc cggaagcata
aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1020cattaattgc gttgcgctca
ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080attaatgaat cggccaacgc
gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1140cctcgctcac tgactcgctg
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1200caaaggcggt aatacggtta
tccacagaat caggggataa cgcaggaaag aacatgtgag 1260caaaaggcca gcaaaagccc
aggaaccgta aaaaggccgc gttgctggcg tttttccata 1320ggctccgccc ccctgacgag
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380cgacaggact ataaagatac
caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1440ttccgaccct gccgcttacc
ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1500tttctcatag ctcacgctgt
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1560gctgtgtgca cgaacccccc
gttcagcccg accgctgcgc cttatccggt aactatcgtc 1620ttgagtccaa cccggtaaga
cacgacttat cgccactggc agcagccact ggtaacagga 1680ttagcagagc gaggtatgta
ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1740gctacactag aaggacagta
tttggtatct gcgctctgct gaagccagtt accttcggaa 1800aaagagttgg tagctcttga
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1860tttgcaagca gcagattacg
cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1920ctacggggtc tgacgctcag
tggaacgaaa actcacgtta agggattttg gtcatgagat 1980tatcaaaaag gatcttcacc
tagatccttt taaattaaaa atgaagtttt agcacgtgtc 2040agtcctgctc ctcggccacg
aagtgcacgc agttgccggc cgggtcgcgc agggcgaact 2100cccgccccca cggctgctcg
ccgatctcgg tcatggccgg cccggaggcg tcccggaagt 2160tcgtggacac gacctccgac
cactcggcgt acagctcgtc caggccgcgc acccacaccc 2220aggccagggt gttgtccggc
accacctggt cctggaccgc gctgatgaac agggtcacgt 2280cgtcccggac cacaccggcg
aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt 2340cggtccagaa ctcgaccgct
ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg 2400tcaacttggc catggtggcc
ctcctcacgt gctattattg aagcatttat cagggttatt 2460gtctcatgag cggatacata
tttgaatgta tttagaaaaa taaacaaata ggggttccgc 2520gcacatttcc ccgaaaagtg
ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580gagaaaatac cgcatcagga
aattgtaagc gttaataatt cagaagaact cgtcaagaag 2640gcgatagaag gcgatgcgct
gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2700gtcagcccat tcgccgccaa
gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2760atagcggtcc gccacaccca
gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2820caccatgata ttcggcaagc
aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880catgctcgcc ttgagcctgg
cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2940cagatcatcc tgatcgacaa
gaccggcttc catccgagta cgtgctcgct cgatgcgatg 3000tttcgcttgg tggtcgaatg
ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 3060atcagccatg atggatactt
tctcggcagg agcaaggtga gatgacagga gatcctgccc 3120cggcacttcg cccaatagca
gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180tgcgcaagga acgcccgtcg
tggccagcca cgatagccgc gctgcctcgt cttgcagttc 3240attcagggca ccggacaggt
cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 3300ccggaacacg gcggcatcag
agcagccgat tgtctgttgt gcccagtcat agccgaatag 3360cctctccacc caagcggccg
gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3420cgatcctcat cctgtctctt
gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480cgagaaagcc atccagttta
ctttgcaggg cttcccaacc ttaccagagg gcgccccagc 3540tggcaattcc ggttcgcttg
ctgtccataa aaccgcccag tctagctatc gccatgtaag 3600cccactgcaa gctacctgct
ttctctttgc gcttgcgttt tcccttgtcc agatagccca 3660gtagctgaca ttcatccggg
gtcagcaccg tttctgcgga ctggctttct acgtgaaaag 3720gatctaggtg aagatccttt
ttgataatct catgcctgac atttatattc cccagaacat 3780caggttaatg gcgtttttga
tgtcattttc gcggtggctg agatcagcca cttcttcccc 3840gataacggag accggcacac
tggccatatc ggtggtcatc atgcgccagc tttcatcccc 3900gatatgcacc accgggtaaa
gttcacggga gactttatct gacagcagac gtgcactggc 3960cagggggatc accatccgtc
gccccggcgt gtcaataata tcactctgta catccacaaa 4020cagacgataa cggctctctc
ttttataggt gtaaacctta aactgccgta cgtataggct 4080gcgcaactgt tgggaagggc
gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4140agggggatgt gctgcaaggc
gattaagttg ggtaacgcca gggttttccc agtcacgacg 4200ttgtaaaacg acggccagtg
aattgtaata cgactcacta tagggcgaat tgggccctct 4260agatgcatgc tcgagcggcc
gccagtgtga tggatatctg cagaattcag g 4311307085DNAArtificial
Sequenceplasmid pKR72 30gtacggatcc gtcgacggcg cgcccgatca tccggatata
gttcctcctt tcagcaaaaa 60acccctcaag acccgtttag aggccccaag gggttatgct
agttattgct cagcggtggc 120agcagccaac tcagcttcct ttcgggcttt gttagcagcc
ggatcgatcc aagctgtacc 180tcactattcc tttgccctcg gacgagtgct ggggcgtcgg
tttccactat cggcgagtac 240ttctacacag ccatcggtcc agacggccgc gcttctgcgg
gcgatttgtg tacgcccgac 300agtcccggct ccggatcgga cgattgcgtc gcatcgaccc
tgcgcccaag ctgcatcatc 360gaaattgccg tcaaccaagc tctgatagag ttggtcaaga
ccaatgcgga gcatatacgc 420ccggagccgc ggcgatcctg caagctccgg atgcctccgc
tcgaagtagc gcgtctgctg 480ctccatacaa gccaaccacg gcctccagaa gaagatgttg
gcgacctcgt attgggaatc 540cccgaacatc gcctcgctcc agtcaatgac cgctgttatg
cggccattgt ccgtcaggac 600attgttggag ccgaaatccg cgtgcacgag gtgccggact
tcggggcagt cctcggccca 660aagcatcagc tcatcgagag cctgcgcgac ggacgcactg
acggtgtcgt ccatcacagt 720ttgccagtga tacacatggg gatcagcaat cgcgcatatg
aaatcacgcc atgtagtgta 780ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
gtctggctaa gatcggccgc 840agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca
gcgggcagtt cggtttcagg 900caggtcttgc aacgtgacac cctgtgcacg gcgggagatg
caataggtca ggctctcgct 960gaattcccca atgtcaagca cttccggaat cgggagcgcg
gccgatgcaa agtgccgata 1020aacataacga tctttgtaga aaccatcggc gcagctattt
acccgcagga catatccacg 1080ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
tccgagagct gcatcaggtc 1140ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
gacgtcgcgg tgagttcagg 1200cttttccatg ggtatatctc cttcttaaag ttaaacaaaa
ttatttctag agggaaaccg 1260ttgtggtctc cctatagtga gtcgtattaa tttcgcggga
tcgagatcga tccaattcca 1320atcccacaaa aatctgagct taacagcaca gttgctcctc
tcagagcaga atcgggtatt 1380caacaccctc atatcaacta ctacgttgtg tataacggtc
cacatgccgg tatatacgat 1440gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg
gagttgcaca caagaaattt 1500gccactatta cagaggcaag agcagcagct gacgcgtaca
caacaagtca gcaaacagac 1560aggttgaact tcatccccaa aggagaagct caactcaagc
ccaagagctt tgctaaggcc 1620ctaacaagcc caccaaagca aaaagcccac tggctcacgc
taggaaccaa aaggcccagc 1680agtgatccag ccccaaaaga gatctccttt gccccggaga
ttacaatgga cgatttcctc 1740tatctttacg atctaggaag gaagttcgaa ggtgaaggtg
acgacactat gttcaccact 1800gataatgaga aggttagcct cttcaatttc agaaagaatg
ctgacccaca gatggttaga 1860gaggcctacg cagcaggtct catcaagacg atctacccga
gtaacaatct ccaggagatc 1920aaataccttc ccaagaaggt taaagatgca gtcaaaagat
tcaggactaa ttgcatcaag 1980aacacagaga aagacatatt tctcaagatc agaagtacta
ttccagtatg gacgattcaa 2040ggcttgcttc ataaaccaag gcaagtaata gagattggag
tctctaaaaa ggtagttcct 2100actgaatcta aggccatgca tggagtctaa gattcaaatc
gaggatctaa cagaactcgc 2160cgtgaagact ggcgaacagt tcatacagag tcttttacga
ctcaatgaca agaagaaaat 2220cttcgtcaac atggtggagc acgacactct ggtctactcc
aaaaatgtca aagatacagt 2280ctcagaagac caaagggcta ttgagacttt tcaacaaagg
ataatttcgg gaaacctcct 2340cggattccat tgcccagcta tctgtcactt catcgaaagg
acagtagaaa aggaaggtgg 2400ctcctacaaa tgccatcatt gcgataaagg aaaggctatc
attcaagatg cctctgccga 2460cagtggtccc aaagatggac ccccacccac gaggagcatc
gtggaaaaag aagacgttcc 2520aaccacgtct tcaaagcaag tggattgatg tgacatctcc
actgacgtaa gggatgacgc 2580acaatcccac tatccttcgc aagacccttc ctctatataa
ggaagttcat ttcatttgga 2640gaggacacgc tcgagctcat ttctctatta cttcagccat
aacaaaagaa ctcttttctc 2700ttcttattaa accatgaaaa agcctgaact caccgcgacg
tctgtcgaga agtttctgat 2760cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
gagggcgaag aatctcgtgc 2820tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
gtaaatagct gcgccgatgg 2880tttctacaaa gatcgttatg tttatcggca ctttgcatcg
gccgcgctcc cgattccgga 2940agtgcttgac attggggaat tcagcgagag cctgacctat
tgcatctccc gccgtgcaca 3000gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
gctgttctgc agccggtcgc 3060ggaggccatg gatgcgatcg ctgcggccga tcttagccag
acgagcgggt tcggcccatt 3120cggaccgcaa ggaatcggtc aatacactac atggcgtgat
ttcatatgcg cgattgctga 3180tccccatgtg tatcactggc aaactgtgat ggacgacacc
gtcagtgcgt ccgtcgcgca 3240ggctctcgat gagctgatgc tttgggccga ggactgcccc
gaagtccggc acctcgtgca 3300cgcggatttc ggctccaaca atgtcctgac ggacaatggc
cgcataacag cggtcattga 3360ctggagcgag gcgatgttcg gggattccca atacgaggtc
gccaacatct tcttctggag 3420gccgtggttg gcttgtatgg agcagcagac gcgctacttc
gagcggaggc atccggagct 3480tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
ggtcttgacc aactctatca 3540gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
cagggtcgat gcgacgcaat 3600cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
gcccgcagaa gcgcggccgt 3660ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
aaccgacgcc ccagcactcg 3720tccgagggca aaggaatagt gaggtaccta aagaaggagt
gcgtcgaagc agatcgttca 3780aacatttggc aataaagttt cttaagattg aatcctgttg
ccggtcttgc gatgattatc 3840atataatttc tgttgaatta cgttaagcat gtaataatta
acatgtaatg catgacgtta 3900tttatgagat gggtttttat gattagagtc ccgcaattat
acatttaata cgcgatagaa 3960aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg
cggtgtcatc tatgttacta 4020gatcgatgtc gaatcgatca acctgcatta atgaatcggc
caacgcgcgg ggagaggcgg 4080tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac aggactataa
agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcaatgctca
cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag cagagcgagg
tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg
acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc
tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg caagcagcag
attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgacattaac ctataaaaat
aggcgtatca cgaggccctt 4980tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
cacatgcagc tcccggagac 5040ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
gcccgtcagg gcgcgtcagc 5100gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
tcagagcaga ttgtactgag 5160agtgcaccat atggacatat tgtcgttaga acgcggctac
aattaataca taaccttatg 5220tatcatacac atacgattta ggtgacacta tagaacggcg
cgccaagctt gttgaaacat 5280ccctgaagtg tctcatttta ttttatttat tctttgctga
taaaaaaata aaataaaaga 5340agctaagcac acggtcaacc attgctctac tgctaaaagg
gttatgtgta gtgttttact 5400gcataaatta tgcagcaaac aagacaactc aaattaaaaa
atttcctttg cttgtttttt 5460tgttgtctct gacttgactt tcttgtggaa gttggttgta
taaggattgg gacaccattg 5520tccttcttaa tttaatttta ttctttgctg ataaaaaaaa
aaatttcata tagtgttaaa 5580taataatttg ttaaataacc aaaaagtcaa atatgtttac
tctcgtttaa ataattgaga 5640ttcgtccagc aaggctaaac gattgtatag atttatgaca
atatttactt ttttatagat 5700aaatgttata ttataataaa tttatataca tatattatat
gttatttatt attattttaa 5760atccttcaat attttatcaa accaactcat aatttttttt
ttatctgtaa gaagcaataa 5820aattaaatag acccacttta aggatgatcc aacctttata
cagagtaaga gagttcaaat 5880agtacccttt catatacata tcaactaaaa tattagaaat
atcatggatc aaaccttata 5940aagacattaa ataagtggat aagtataata tataaatggg
tagtatataa tatataaatg 6000gatacaaact tctctcttta taattgttat gtctccttaa
catcctaata taatacataa 6060gtgggtaata tataatatat aaatggagac aaacttcttc
cattataatt gttatgtctt 6120cttaacactt atgtctcgtt cacaatgcta aggttagaat
tgtttagaaa gtcttatagt 6180acacatttgt ttttgtacta tttgaagcat tccataagcc
gtcacgattc agatgattta 6240taataataag aggaaattta tcatagaaca ataaggtgca
tagatagagt gttaatatat 6300cataacatcc tttgtttatt catagaagaa gtgagatgga
gctcagttat tatactgtta 6360catggtcgga tacaatattc catgctctcc atgagctctt
acacctacat gcattttagt 6420tcatacttgc ggccgcagta tatcttaaat tctttaatac
ggtgtactag gatattgaac 6480tggttcttga tgatgaaaac ctgggccgag attgcagcta
tttatagtca taggtcttgt 6540taacatgcat ggacatttgg ccacggggtg gcatgcagtt
tgacgggtgt tgaaataaac 6600aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg
gttagaaaca acaaatgtga 6660gggctcatga tgggttgagt tggtgaatgt tttgggctgc
tcgattgaca cctttgtgag 6720tacgtgttgt tgtgcatggc ttttggggtc cagttttttt
ttcttgacgc ggcgatcctg 6780atcagctagt ggataagtga tgtccactgt gtgtgattgc
gtttttgttt gaattttatg 6840aacttagaca ttgctatgca aaggatactc tcattgtgtt
ttgtcttctt ttgttccttg 6900gctttttctt atgatccaag agactagtca gtgttgtggc
attcgagact accaagatta 6960attatgatgg gggaaggata agtaactgat tagtacggac
tgttaccaaa ttaattaata 7020agcggcaaat gaagggcatg gatcaaaagc ttggatctcc
tgcaggatct ggccggccgg 7080atctc
7085312540DNAArtificial Sequenceplasmid KS102
31cgatcatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc
60cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg
120ggctttgtta gcagccggat cgatccaagc tgtacctcac tattcctttg ccctcggacg
180agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac
240ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat
300tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg
360atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag
420ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct
480ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc
540aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg
600cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg
660cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc
720agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa
780tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc
840gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg
900tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc
960cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc
1020atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc
1080acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat
1140cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt tccatgggta tatctccttc
1200ttaaagttaa acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg
1260tattaatttc gcgggatcga gatctgatca acctgcatta atgaatcggc caacgcgcgg
1320ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
1380cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
1440cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
1500accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
1560acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
1620cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
1680acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
1740atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
1800agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
1860acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
1920gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
1980gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
2040gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
2100gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
2160acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca
2220cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
2280tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
2340gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
2400ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca
2460taaccttatg tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt
2520ggatccgtcg acggcgcgcc
2540324359DNAArtificial Sequenceplasmid pKR197 32cgcgcccgat catccggata
tagttcctcc tttcagcaaa aaacccctca agacccgttt 60agaggcccca aggggttatg
ctagttattg ctcagcggtg gcagcagcca actcagcttc 120ctttcgggct ttgttagcag
ccggatcgat ccaagctgta cctcactatt cctttgccct 180cggacgagtg ctggggcgtc
ggtttccact atcggcgagt acttctacac agccatcggt 240ccagacggcc gcgcttctgc
gggcgatttg tgtacgcccg acagtcccgg ctccggatcg 300gacgattgcg tcgcatcgac
cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa 360gctctgatag agttggtcaa
gaccaatgcg gagcatatac gcccggagcc gcggcgatcc 420tgcaagctcc ggatgcctcc
gctcgaagta gcgcgtctgc tgctccatac aagccaacca 480cggcctccag aagaagatgt
tggcgacctc gtattgggaa tccccgaaca tcgcctcgct 540ccagtcaatg accgctgtta
tgcggccatt gtccgtcagg acattgttgg agccgaaatc 600cgcgtgcacg aggtgccgga
cttcggggca gtcctcggcc caaagcatca gctcatcgag 660agcctgcgcg acggacgcac
tgacggtgtc gtccatcaca gtttgccagt gatacacatg 720gggatcagca atcgcgcata
tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg 780tccgaatggg ccgaacccgc
tcgtctggct aagatcggcc gcagcgatcg catccatagc 840ctccgcgacc ggctgcagaa
cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac 900accctgtgca cggcgggaga
tgcaataggt caggctctcg ctgaattccc caatgtcaag 960cacttccgga atcgggagcg
cggccgatgc aaagtgccga taaacataac gatctttgta 1020gaaaccatcg gcgcagctat
ttacccgcag gacatatcca cgccctccta catcgaagct 1080gaaagcacga gattcttcgc
cctccgagag ctgcatcagg tcggagacgc tgtcgaactt 1140ttcgatcaga aacttctcga
cagacgtcgc ggtgagttca ggcttttcca tgggtatatc 1200tccttcttaa agttaaacaa
aattatttct agagggaaac cgttgtggtc tccctatagt 1260gagtcgtatt aatttcgcgg
gatcgagatc tgatcaacct gcattaatga atcggccaac 1320gcgcggggag aggcggtttg
cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1380tgcgctcggt cgttcggctg
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1440tatccacaga atcaggggat
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1500ccaggaaccg taaaaaggcc
gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1560agcatcacaa aaatcgacgc
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1620accaggcgtt tccccctgga
agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1680ccggatacct gtccgccttt
ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 1740gtaggtatct cagttcggtg
taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1800ccgttcagcc cgaccgctgc
gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1860gacacgactt atcgccactg
gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1920taggcggtgc tacagagttc
ttgaagtggt ggcctaacta cggctacact agaaggacag 1980tatttggtat ctgcgctctg
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2040gatccggcaa acaaaccacc
gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2100cgcgcagaaa aaaaggatct
caagaagatc ctttgatctt ttctacgggg tctgacgctc 2160agtggaacga aaactcacgt
taagggattt tggtcatgac attaacctat aaaaataggc 2220gtatcacgag gccctttcgt
ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 2280tgcagctccc ggagacggtc
acagcttgtc tgtaagcgga tgccgggagc agacaagccc 2340gtcagggcgc gtcagcgggt
gttggcgggt gtcggggctg gcttaactat gcggcatcag 2400agcagattgt actgagagtg
caccatatgg acatattgtc gttagaacgc ggctacaatt 2460aatacataac cttatgtatc
atacacatac gatttaggtg acactataga acggcgcgcc 2520aagcttgttg aaacatccct
gaagtgtctc attttatttt atttattctt tgctgataaa 2580aaaataaaat aaaagaagct
aagcacacgg tcaaccattg ctctactgct aaaagggtta 2640tgtgtagtgt tttactgcat
aaattatgca gcaaacaaga caactcaaat taaaaaattt 2700cctttgcttg tttttttgtt
gtctctgact tgactttctt gtggaagttg gttgtataag 2760gattgggaca ccattgtcct
tcttaattta attttattct ttgctgataa aaaaaaaaat 2820ttcatatagt gttaaataat
aatttgttaa ataaccaaaa agtcaaatat gtttactctc 2880gtttaaataa ttgagattcg
tccagcaagg ctaaacgatt gtatagattt atgacaatat 2940ttactttttt atagataaat
gttatattat aataaattta tatacatata ttatatgtta 3000tttattatta ttttaaatcc
ttcaatattt tatcaaacca actcataatt ttttttttat 3060ctgtaagaag caataaaatt
aaatagaccc actttaagga tgatccaacc tttatacaga 3120gtaagagagt tcaaatagta
ccctttcata tacatatcaa ctaaaatatt agaaatatca 3180tggatcaaac cttataaaga
cattaaataa gtggataagt ataatatata aatgggtagt 3240atataatata taaatggata
caaacttctc tctttataat tgttatgtct ccttaacatc 3300ctaatataat acataagtgg
gtaatatata atatataaat ggagacaaac ttcttccatt 3360ataattgtta tgtcttctta
acacttatgt ctcgttcaca atgctaaggt tagaattgtt 3420tagaaagtct tatagtacac
atttgttttt gtactatttg aagcattcca taagccgtca 3480cgattcagat gatttataat
aataagagga aatttatcat agaacaataa ggtgcataga 3540tagagtgtta atatatcata
acatcctttg tttattcata gaagaagtga gatggagctc 3600agttattata ctgttacatg
gtcggataca atattccatg ctctccatga gctcttacac 3660ctacatgcat tttagttcat
acttgcggcc gcagtatatc ttaaattctt taatacggtg 3720tactaggata ttgaactggt
tcttgatgat gaaaacctgg gccgagattg cagctattta 3780tagtcatagg tcttgttaac
atgcatggac atttggccac ggggtggcat gcagtttgac 3840gggtgttgaa ataaacaaaa
atgaggtggc ggaagagaat acgagtttga ggttgggtta 3900gaaacaacaa atgtgagggc
tcatgatggg ttgagttggt gaatgttttg ggctgctcga 3960ttgacacctt tgtgagtacg
tgttgttgtg catggctttt ggggtccagt ttttttttct 4020tgacgcggcg atcctgatca
gctagtggat aagtgatgtc cactgtgtgt gattgcgttt 4080ttgtttgaat tttatgaact
tagacattgc tatgcaaagg atactctcat tgtgttttgt 4140cttcttttgt tccttggctt
tttcttatga tccaagagac tagtcagtgt tgtggcattc 4200gagactacca agattaatta
tgatggggga aggataagta actgattagt acggactgtt 4260accaaattaa ttaataagcg
gcaaatgaag ggcatggatc aaaagcttgg atctcctgca 4320ggatctggcc ggccggatct
cgtacggatc cgtcgacgg 4359335147DNAArtificial
Sequenceplasmid pKR911 33ggccgcaagt atgaactaaa atgcatgtag gtgtaagagc
tcatggagag catggaatat 60tgtatccgac catgtaacag tataataact gagctccatc
tcacttcttc tatgaataaa 120caaaggatgt tatgatatat taacactcta tctatgcacc
ttattgttct atgataaatt 180tcctcttatt attataaatc atctgaatcg tgacggctta
tggaatgctt caaatagtac 240aaaaacaaat gtgtactata agactttcta aacaattcta
accttagcat tgtgaacgag 300acataagtgt taagaagaca taacaattat aatggaagaa
gtttgtctcc atttatatat 360tatatattac ccacttatgt attatattag gatgttaagg
agacataaca attataaaga 420gagaagtttg tatccattta tatattatat actacccatt
tatatattat acttatccac 480ttatttaatg tctttataag gtttgatcca tgatatttct
aatattttag ttgatatgta 540tatgaaaggg tactatttga actctcttac tctgtataaa
ggttggatca tccttaaagt 600gggtctattt aattttattg cttcttacag ataaaaaaaa
aattatgagt tggtttgata 660aaatattgaa ggatttaaaa taataataaa taacatataa
tatatgtata taaatttatt 720ataatataac atttatctat aaaaaagtaa atattgtcat
aaatctatac aatcgtttag 780ccttgctgga cgaatctcaa ttatttaaac gagagtaaac
atatttgact ttttggttat 840ttaacaaatt attatttaac actatatgaa attttttttt
ttatcagcaa agaataaaat 900taaattaaga aggacaatgg tgtcccaatc cttatacaac
caacttccac aagaaagtca 960agtcagagac aacaaaaaaa caagcaaagg aaatttttta
atttgagttg tcttgtttgc 1020tgcataattt atgcagtaaa acactacaca taaccctttt
agcagtagag caatggttga 1080ccgtgtgctt agcttctttt attttatttt tttatcagca
aagaataaat aaaataaaat 1140gagacacttc agggatgttt caacaagctt ggcgcgccgt
tctatagtgt cacctaaatc 1200gtatgtgtat gatacataag gttatgtatt aattgtagcc
gcgttctaac gacaatatgt 1260ccatatggtg cactctcagt acaatctgct ctgatgccgc
atagttaagc cagccccgac 1320acccgccaac acccgctgac gcgccctgac gggcttgtct
gctcccggca tccgcttaca 1380gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
gttttcaccg tcatcaccga 1440aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
ataggttaat gtcatgacca 1500aaatccctta acgtgagttt tcgttccact gagcgtcaga
ccccgtagaa aagatcaaag 1560gatcttcttg agatcctttt tttctgcgcg taatctgctg
cttgcaaaca aaaaaaccac 1620cgctaccagc ggtggtttgt ttgccggatc aagagctacc
aactcttttt ccgaaggtaa 1680ctggcttcag cagagcgcag ataccaaata ctgtccttct
agtgtagccg tagttaggcc 1740accacttcaa gaactctgta gcaccgccta catacctcgc
tctgctaatc ctgttaccag 1800tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
ggactcaaga cgatagttac 1860cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
cacacagccc agcttggagc 1920gaacgaccta caccgaactg agatacctac agcgtgagca
ttgagaaagc gccacgcttc 1980ccgaagggag aaaggcggac aggtatccgg taagcggcag
ggtcggaaca ggagagcgca 2040cgagggagct tccaggggga aacgcctggt atctttatag
tcctgtcggg tttcgccacc 2100tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
gcggagccta tggaaaaacg 2160ccagcaacgc ggccttttta cggttcctgg ccttttgctg
gccttttgct cacatgttct 2220ttcctgcgtt atcccctgat tctgtggata accgtattac
cgcctttgag tgagctgata 2280ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
gagcgaggaa gcggaagagc 2340gcccaatacg caaaccgcct ctccccgcgc gttggccgat
tcattaatgc aggttgatca 2400gatctcgatc ccgcgaaatt aatacgactc actataggga
gaccacaacg gtttccctct 2460agaaataatt ttgtttaact ttaagaagga gatataccca
tggaaaagcc tgaactcacc 2520gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
gcgtctccga cctgatgcag 2580ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
taggagggcg tggatatgtc 2640ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
gttatgttta tcggcacttt 2700gcatcggccg cgctcccgat tccggaagtg cttgacattg
gggaattcag cgagagcctg 2760acctattgca tctcccgccg tgcacagggt gtcacgttgc
aagacctgcc tgaaaccgaa 2820ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg
cgatcgctgc ggccgatctt 2880agccagacga gcgggttcgg cccattcgga ccgcaaggaa
tcggtcaata cactacatgg 2940cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
actggcaaac tgtgatggac 3000gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
tgatgctttg ggccgaggac 3060tgccccgaag tccggcacct cgtgcacgcg gatttcggct
ccaacaatgt cctgacggac 3120aatggccgca taacagcggt cattgactgg agcgaggcga
tgttcgggga ttcccaatac 3180gaggtcgcca acatcttctt ctggaggccg tggttggctt
gtatggagca gcagacgcgc 3240tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
ggctccgggc gtatatgctc 3300cgcattggtc ttgaccaact ctatcagagc ttggttgacg
gcaatttcga tgatgcagct 3360tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
ccgggactgt cgggcgtaca 3420caaatcgccc gcagaagcgc ggccgtctgg accgatggct
gtgtagaagt actcgccgat 3480agtggaaacc gacgccccag cactcgtccg agggcaaagg
aatagtgagg tacagcttgg 3540atcgatccgg ctgctaacaa agcccgaaag gaagctgagt
tggctgctgc caccgctgag 3600caataactag cataacccct tggggcctct aaacgggtct
tgaggggttt tttgctgaaa 3660ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg
atccgtacga gatccggccg 3720gccagatcct gcaggagatc caagcttttg atccatgccc
ttcatttgcc gcttattaat 3780taatttggta acagtccgta ctaatcagtt acttatcctt
cccccatcat aattaatctt 3840ggtagtctcg aatgccacaa cactgactag tctcttggat
cataagaaaa agccaaggaa 3900caaaagaaga caaaacacaa tgagagtatc ctttgcatag
caatgtctaa gttcataaaa 3960ttcaaacaaa aacgcaatca cacacagtgg acatcactta
tccactagct gatcaggatc 4020gccgcgtcaa gaaaaaaaaa ctggacccca aaagccatgc
acaacaacac gtactcacaa 4080aggtgtcaat cgagcagccc aaaacattca ccaactcaac
ccatcatgag ccctcacatt 4140tgttgtttct aacccaacct caaactcgta ttctcttccg
ccacctcatt tttgtttatt 4200tcaacacccg tcaaactgca tgccaccccg tggccaaatg
tccatgcatg ttaacaagac 4260ctatgactat aaatagctgc aatctcggcc caggttttca
tcatcaagaa ccagttcaat 4320atcctagtac accgtattaa agaatttaag atatactgcg
gccgcaccat ggaggtggtg 4380aatgaaatag tctcaattgg gcaggaagtt ttacccaaag
ttgattatgc ccaactctgg 4440agtgatgcca gtcactgtga ggtgctttac ttgtccatcg
catttgtcat cttgaagttc 4500actcttggcc cccttggtcc aaaaggtcag tctcgtatga
agtttgtttt caccaattac 4560aaccttctca tgtccattta ttcgttggga tcattcctct
caatggcata tgccatgtac 4620accatcggtg ttatgtctga caactgcgag aaggcttttg
acaacaacgt cttcaggatc 4680accacgcagt tgttctattt gagcaagttc ctggagtata
ttgactcctt ctatttgcca 4740ctgatgggca agcctctgac ctggttgcaa ttcttccatc
atttgggggc accgatggat 4800atgtggctgt tctataatta ccgaaatgaa gctgtttgga
tttttgtgct gttgaatggt 4860ttcatccact ggatcatgta cggttattat tggaccagat
tgatcaagct gaagttcccc 4920atgccaaaat ccctgattac atcaatgcag atcattcaat
tcaatgttgg tttctacatt 4980gtctggaagt acaggaacat tccctgttat cgccaagatg
ggatgaggat gtttggctgg 5040ttcttcaatt acttttatgt tggcacagtc ttgtgtttgt
tcttgaattt ctatgtgcaa 5100acgtatatcg tcaggaagca caagggagcc aaaaagattc
agtgagc 5147346559DNAArtificial Sequenceplasmid pKR680
34ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac
60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg
120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct
180ttgtttacgg ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg
240ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct ataacataag
300ctgtctctga gtgtgttgta tattaataaa gatcatcact ggtgaatggt gatcgtgtac
360gtaccctact tagtaggcaa tggaagcact tagagtgtgc tttgtgcatg gccttgcctc
420tgttttgaga cttttgtaat gttttcgagt ttaaatcttt gcctttgcgt acgtgggcgg
480atcccccggg ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
540ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
600gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
660gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
720ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
780ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
840ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
900gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
960agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
1020aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
1080attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
1140cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
1200aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
1260ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
1320gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
1380attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
1440tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
1500tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
1560atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
1620agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
1680aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
1740caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
1800ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
1860gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
1920tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
1980atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
2040tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
2100accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
2160gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
2220caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
2280tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
2340ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
2400tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
2460gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
2520tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
2580gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
2640gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
2700ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
2760ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
2820ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
2880tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
2940ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
3000caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
3060ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
3120atgattacgc caagcttgca tgcctgcagg tcgactcgac gtacgtcctc gaagagaagg
3180gttaataaca cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa
3240aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa tttatgattt
3300ttaataagtt ttcaccaata aaaaatgtca taaaaatatg ttaaaaagta tattatcaat
3360attctcttta tgataaataa aaagaaaaaa aaaataaaag ttaagtgaaa atgagattga
3420agtgacttta ggtgtgtata aatatatcaa ccccgccaac aatttattta atccaaatat
3480attgaagtat attattccat agcctttatt tatttatata tttattatat aaaagcttta
3540tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta agaaaatcat
3600gtgctttgtg tcgccactca ctattgcagc tttttcatgc attggtcaga ttgacggttg
3660attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct ctttatctct
3720tcatcaggtt tgatggttac ctaatatggt ccatgggtac atgcatggtt aaattaggtg
3780gccaactttg ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat
3840gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag ttaatttgtt
3900aactctataa taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct
3960ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc cttcttttaa
4020atttggaata cattatcatc atatataaac taaaatacta aaaacaggat tacacaaatg
4080ataaataata acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg
4140atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg tgctttctgg
4200actgatgatg cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt
4260agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc atttgcttcg
4320tgtcacaaat acaatttagn taggtacatg cattggtcag attcacggtt tattatgtca
4380tgacttaagt tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga
4440taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt acgaaataac
4500agtgatcaaa acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt
4560tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc atcataaaat
4620caaatactaa aaaaattaca tatttcataa ataataacac aaatattttt aaaaaatctg
4680aaataataat gaacaatatt acatattatc acgaaaattc attaataaaa atattatata
4740aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa tatatacaaa
4800aagattaaaa tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat
4860aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac acaaataata
4920aataatagta aaaaaaatta tgataaatat ttaccatctc ataagatatt taaaataatg
4980ataaaaatat agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata
5040taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt ttatatcaag
5100tggacatacg tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt
5160ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta ttcacacaac
5220taactaagaa agtcttccat agccccccaa gcggccgcgg gaattcgatt gaaatgaagt
5280caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg tctgcctggg
5340tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg gatgccactg
5400atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc atgcccaaaa
5460tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa gaggatttcc
5520ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc ctctggtact
5580catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg atggttcagt
5640atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag atgggctggc
5700tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac aacctcgtgg
5760gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag gacagacaca
5820atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac aacctccccc
5880tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc aagctcattc
5940agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt tggtgtttcc
6000agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat cgctctcagt
6060ataagaagga ggccattggc ctcgccctgc actggacctt gaagaccctg ttccacttat
6120tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag ctggttggcg
6180gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag atcggggact
6240cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg aacattcggc
6300gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag caccatttgt
6360ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa cagctgtgcc
6420agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc atcctgctgc
6480gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag gctctataag
6540gaatcactag tgaattcgc
6559359014DNAArtificial Sequenceplasmid pKR913 35gtacgagatc cggccggcca
gatcctgcag gagatccaag cttttgatcc atgcccttca 60tttgccgctt attaattaat
ttggtaacag tccgtactaa tcagttactt atccttcccc 120catcataatt aatcttggta
gtctcgaatg ccacaacact gactagtctc ttggatcata 180agaaaaagcc aaggaacaaa
agaagacaaa acacaatgag agtatccttt gcatagcaat 240gtctaagttc ataaaattca
aacaaaaacg caatcacaca cagtggacat cacttatcca 300ctagctgatc aggatcgccg
cgtcaagaaa aaaaaactgg accccaaaag ccatgcacaa 360caacacgtac tcacaaaggt
gtcaatcgag cagcccaaaa cattcaccaa ctcaacccat 420catgagccct cacatttgtt
gtttctaacc caacctcaaa ctcgtattct cttccgccac 480ctcatttttg tttatttcaa
cacccgtcaa actgcatgcc accccgtggc caaatgtcca 540tgcatgttaa caagacctat
gactataaat agctgcaatc tcggcccagg ttttcatcat 600caagaaccag ttcaatatcc
tagtacaccg tattaaagaa tttaagatat actgcggccg 660caccatggag gtggtgaatg
aaatagtctc aattgggcag gaagttttac ccaaagttga 720ttatgcccaa ctctggagtg
atgccagtca ctgtgaggtg ctttacttgt ccatcgcatt 780tgtcatcttg aagttcactc
ttggccccct tggtccaaaa ggtcagtctc gtatgaagtt 840tgttttcacc aattacaacc
ttctcatgtc catttattcg ttgggatcat tcctctcaat 900ggcatatgcc atgtacacca
tcggtgttat gtctgacaac tgcgagaagg cttttgacaa 960caacgtcttc aggatcacca
cgcagttgtt ctatttgagc aagttcctgg agtatattga 1020ctccttctat ttgccactga
tgggcaagcc tctgacctgg ttgcaattct tccatcattt 1080gggggcaccg atggatatgt
ggctgttcta taattaccga aatgaagctg tttggatttt 1140tgtgctgttg aatggtttca
tccactggat catgtacggt tattattgga ccagattgat 1200caagctgaag ttccccatgc
caaaatccct gattacatca atgcagatca ttcaattcaa 1260tgttggtttc tacattgtct
ggaagtacag gaacattccc tgttatcgcc aagatgggat 1320gaggatgttt ggctggttct
tcaattactt ttatgttggc acagtcttgt gtttgttctt 1380gaatttctat gtgcaaacgt
atatcgtcag gaagcacaag ggagccaaaa agattcagtg 1440agcggccgca agtatgaact
aaaatgcatg taggtgtaag agctcatgga gagcatggaa 1500tattgtatcc gaccatgtaa
cagtataata actgagctcc atctcacttc ttctatgaat 1560aaacaaagga tgttatgata
tattaacact ctatctatgc accttattgt tctatgataa 1620atttcctctt attattataa
atcatctgaa tcgtgacggc ttatggaatg cttcaaatag 1680tacaaaaaca aatgtgtact
ataagacttt ctaaacaatt ctaaccttag cattgtgaac 1740gagacataag tgttaagaag
acataacaat tataatggaa gaagtttgtc tccatttata 1800tattatatat tacccactta
tgtattatat taggatgtta aggagacata acaattataa 1860agagagaagt ttgtatccat
ttatatatta tatactaccc atttatatat tatacttatc 1920cacttattta atgtctttat
aaggtttgat ccatgatatt tctaatattt tagttgatat 1980gtatatgaaa gggtactatt
tgaactctct tactctgtat aaaggttgga tcatccttaa 2040agtgggtcta tttaatttta
ttgcttctta cagataaaaa aaaaattatg agttggtttg 2100ataaaatatt gaaggattta
aaataataat aaataacata taatatatgt atataaattt 2160attataatat aacatttatc
tataaaaaag taaatattgt cataaatcta tacaatcgtt 2220tagccttgct ggacgaatct
caattattta aacgagagta aacatatttg actttttggt 2280tatttaacaa attattattt
aacactatat gaaatttttt tttttatcag caaagaataa 2340aattaaatta agaaggacaa
tggtgtccca atccttatac aaccaacttc cacaagaaag 2400tcaagtcaga gacaacaaaa
aaacaagcaa aggaaatttt ttaatttgag ttgtcttgtt 2460tgctgcataa tttatgcagt
aaaacactac acataaccct tttagcagta gagcaatggt 2520tgaccgtgtg cttagcttct
tttattttat ttttttatca gcaaagaata aataaaataa 2580aatgagacac ttcagggatg
tttcaacaag cttggcgcgc cgttctatag tgtcacctaa 2640atcgtatgtg tatgatacat
aaggttatgt attaattgta gccgcgttct aacgacaata 2700tgtccatatg gtgcactctc
agtacaatct gctctgatgc cgcatagtta agccagcccc 2760gacacccgcc aacacccgct
gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2820acagacaagc tgtgaccgtc
tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2880cgaaacgcgc gagacgaaag
ggcctcgtga tacgcctatt tttataggtt aatgtcatga 2940ccaaaatccc ttaacgtgag
ttttcgttcc actgagcgtc agaccccgta gaaaagatca 3000aaggatcttc ttgagatcct
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3060caccgctacc agcggtggtt
tgtttgccgg atcaagagct accaactctt tttccgaagg 3120taactggctt cagcagagcg
cagataccaa atactgtcct tctagtgtag ccgtagttag 3180gccaccactt caagaactct
gtagcaccgc ctacatacct cgctctgcta atcctgttac 3240cagtggctgc tgccagtggc
gataagtcgt gtcttaccgg gttggactca agacgatagt 3300taccggataa ggcgcagcgg
tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3360agcgaacgac ctacaccgaa
ctgagatacc tacagcgtga gcattgagaa agcgccacgc 3420ttcccgaagg gagaaaggcg
gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3480gcacgaggga gcttccaggg
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3540acctctgact tgagcgtcga
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 3600acgccagcaa cgcggccttt
ttacggttcc tggccttttg ctggcctttt gctcacatgt 3660tctttcctgc gttatcccct
gattctgtgg ataaccgtat taccgccttt gagtgagctg 3720ataccgctcg ccgcagccga
acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3780agcgcccaat acgcaaaccg
cctctccccg cgcgttggcc gattcattaa tgcaggttga 3840tcagatctcg atcccgcgaa
attaatacga ctcactatag ggagaccaca acggtttccc 3900tctagaaata attttgttta
actttaagaa ggagatatac ccatggaaaa gcctgaactc 3960accgcgacgt ctgtcgagaa
gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 4020cagctctcgg agggcgaaga
atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 4080gtcctgcggg taaatagctg
cgccgatggt ttctacaaag atcgttatgt ttatcggcac 4140tttgcatcgg ccgcgctccc
gattccggaa gtgcttgaca ttggggaatt cagcgagagc 4200ctgacctatt gcatctcccg
ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 4260gaactgcccg ctgttctgca
gccggtcgcg gaggctatgg atgcgatcgc tgcggccgat 4320cttagccaga cgagcgggtt
cggcccattc ggaccgcaag gaatcggtca atacactaca 4380tggcgtgatt tcatatgcgc
gattgctgat ccccatgtgt atcactggca aactgtgatg 4440gacgacaccg tcagtgcgtc
cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 4500gactgccccg aagtccggca
cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 4560gacaatggcc gcataacagc
ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 4620tacgaggtcg ccaacatctt
cttctggagg ccgtggttgg cttgtatgga gcagcagacg 4680cgctacttcg agcggaggca
tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 4740ctccgcattg gtcttgacca
actctatcag agcttggttg acggcaattt cgatgatgca 4800gcttgggcgc agggtcgatg
cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 4860acacaaatcg cccgcagaag
cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 4920gatagtggaa accgacgccc
cagcactcgt ccgagggcaa aggaatagtg aggtacagct 4980tggatcgatc cggctgctaa
caaagcccga aaggaagctg agttggctgc tgccaccgct 5040gagcaataac tagcataacc
ccttggggcc tctaaacggg tcttgagggg ttttttgctg 5100aaaggaggaa ctatatccgg
atgatcgggc gcgccgtcga cggatccgta cgcaaaggca 5160aagatttaaa ctcgaaaaca
ttacaaaagt ctcaaaacag aggcaaggcc atgcacaaag 5220cacactctaa gtgcttccat
tgcctactaa gtagggtacg tacacgatca ccattcacca 5280gtgatgatct ttattaatat
acaacacact cagagacagc ttatgttata gctagctagc 5340ataaactatc acatcatgtg
ttagtacgac aagtgacaac attgctttta acttcgcggc 5400cttggatcct ctagaccgga
tataatgagc cgtaaacaaa gatgattaag tagtaattaa 5460tacgtactag taaaagtggc
aaaagataac gagaaagaac caatttcttt gcattcggcc 5520ttagcggaag gcatatataa
gctttgatta ttttatttag tgtaatgatt tcgtacaacc 5580aaagcattta tttagtactc
tcacacttgt gtcgcggccg cgaattcact agtgattcct 5640tatagagcct tccccgcggg
ttgcttctcc gccatccggg cgaacaccgc cagatagcgc 5700agcaggatga ccaacccttc
atggggcagc gggttccgat acggcaggtt gtgcttctgg 5760cacagctgtt ccacctggta
gctaaccgct gtcaggttgt ggcgagggag ggtcggccac 5820aaatggtgct caatctggta
attcaagcct ccgaaaaacc aatctgtgat aatccctcgc 5880cgaatgttca tggtctcatg
gatctggcca accgagaatc catggccatc ccagactgag 5940tccccgatct tctccagtgg
gtagtggttc atgaacacca cgatcgcaat gccgaagccg 6000ccaaccagct ccgaaacgaa
aaacaccaac agcgatgtga ggatgctggg cataaagaat 6060aagtggaaca gggtcttcaa
ggtccagtgc agggcgaggc caatggcctc cttcttatac 6120tgagagcgat agaattggtt
atctctgtcc ttcaaactgc gcacggtcaa cacgctctgg 6180aaacaccaaa tgaaccgcaa
caagatacag atgaccaaga aatagtactg ctggaactga 6240atgagcttgc gggaaatcgg
tgacgcccgt gtgacgtcat cctcagacca ggctaagagg 6300gggaggttgt caatatcagg
gtcgtgccct tgaacattgg ttgccgaatg atgtgcattg 6360tgtctgtcct tccaccatgt
cacggaaaaa ccttgcagac cattgccaaa taccagtccc 6420acgaggttgt tccagttccg
gttcttgaaa gtctggtggt ggcaaatgtc atgagaaagc 6480cagcccatct gttgatagtg
catcccaagc aacactgccc caatgaaata catctgatac 6540tgaaccatca ggaaataacc
cagcactcca aggcccagtg tggtgctgat tttgtatgag 6600taccagaggg gggaggcatc
aaacatgcca gttgcgatca actcttctcg gagcttccgg 6660aaatcctctt gagcttcatt
cactgcagcc tggggtggca actcagaact gggattgatt 6720ttgggcatgc gcttgagctt
gtcgaaggct tcttgagagt gcataaccat gaaggcatca 6780gtggcatccc ttccttggta
attctctata atttccgcac caccagggtg gaaattgacc 6840caggcagaca catcatatgt
tgttccatca attgtaaggg gaagcgcttg gcgctttgac 6900ttcatttcaa tcgaattccc
gcggccgctt ggggggctat ggaagacttt cttagttagt 6960tgtgtgaata agcaatgttg
ggagaatcgg gactacttat aggataggaa taaaacagaa 7020aagtattaag tgctaatgaa
atatttagac tgataattaa aatcttcacg tatgtccact 7080tgatataaaa acgtcaggaa
taaaggaagt acagtagaat ttaaaggtac tctttttata 7140tatacccgtg ttctcttttt
ggctagctag ttgcataaaa aataatctat atttttatca 7200ttattttaaa tatcttatga
gatggtaaat atttatcata atttttttta ctattattta 7260ttatttgtgt gtgtaataca
tatagaagtt aattacaaat tttatttact ttttcattat 7320tttgatatga ttcaccatta
atttagtgtt attatttata atagttcatt ttaatctttt 7380tgtatatatt atgcgtgcag
tacttttttc ctacatataa ctactattac attttattta 7440tataatattt ttattaatga
attttcgtga taatatgtaa tattgttcat tattatttca 7500gattttttaa aaatatttgt
gttattattt atgaaatatg taattttttt agtatttgat 7560tttatgatga taaagtgttc
taaattcaaa agaaggggga aagcgtaaac attaaaaaac 7620gtcatcaaac aaaaacaaaa
tcttgttaat aaagataaaa ctgtttgttt tgatcactgt 7680tatttcgtaa tataaaaaca
ttatttatat ttatattgtt gacaaccaaa tttgcctatc 7740aaatctaacc aatataatgc
atgcgtggca ggtaatgtac taccatgaac ttaagtcatg 7800acataataaa ccgtgaatct
gaccaatgca tgtacctanc taaattgtat ttgtgacacg 7860aagcaaatga ttcaattcac
aatggagatg ggaaacaaat aatgaagaac ccagaactaa 7920gaaagctttt ctgaaaaata
aaataaaggc aatgtcaaaa gtatactgca tcatcagtcc 7980agaaagcaca tgatattttt
ttatcagtat caatgcagct agttttattt tacaatatcg 8040atatagctag tttaaatata
ttgcagctag atttataaat atttgtgtta ttatttatca 8100tttgtgtaat cctgttttta
gtattttagt ttatatatga tgataatgta ttccaaattt 8160aaaagaaggg aaataaattt
aaacaagaaa aaaagtcatc aaacaaaaaa caaatgaaag 8220ggtggaaaga tgttaccatg
taatgtgaat gttacagtat ttcttttatt atagagttaa 8280caaattaact aatatgattt
tgttaataat gataaaatat tttttttatt attatttcat 8340aatataaaaa tagtttactt
aatataaaaa aaattctatc gttcacaaca aagttggcca 8400cctaatttaa ccatgcatgt
acccatggac catattaggt aaccatcaaa cctgatgaag 8460agataaagag atgaagactt
aagtcataac acaaaaccat aaaaaacaaa aatacaatca 8520accgtcaatc tgaccaatgc
atgaaaaagc tgcaatagtg agtggcgaca caaagcacat 8580gattttctta caacggagat
aaaaccaaaa aaatatttca tgaacaacct agaacaaata 8640aagcttttat ataataaata
tataaataaa taaaggctat ggaataatat acttcaatat 8700atttggatta aataaattgt
tggcggggtt gatatattta tacacaccta aagtcacttc 8760aatctcattt tcacttaact
tttatttttt ttttcttttt atttatcata aagagaatat 8820tgataatata ctttttaaca
tatttttatg acatttttta ttggtgaaaa cttattaaaa 8880atcataaatt ttgtaagtta
gatttattta aagagttcct cttcttattt taaatttttt 8940aataaatttt taaataacta
aaatttgtgt taaaaatgtt aaaaaatgtg ttattaaccc 9000ttctcttcga ggac
90143630DNAArtificial
Sequenceoligonucleotide oEAd5-1-1 36agcggccgca ccatggccac catctctttg
303730DNAArtificial
Sequenceolignonucleotide oEAd5-1-2 37tgcggccgct cagaattcct gggcatcctg
30384899DNAArtificial Sequenceplasmid
pKR1136 38aattccagca cactggcggc cgttactagt ggatccgagc tcggtaccaa
gcttgatgca 60tagcttgagt attctaacgc gtcacctaaa tagcttggcg taatcatggt
catagctgtt 120tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
gaagcataaa 180gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
tgcgctcact 240gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
gccaacgcgc 300ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
actcgctgcg 360ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
tacggttatc 420cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
aaaagcccag 480gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
ctgacgagca 540tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
aaagatacca 600ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
cgcttaccgg 660atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
cacgctgtag 720gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
aaccccccgt 780tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
cggtaagaca 840cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
ggtatgtagg 900cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
ggacagtatt 960tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
gctcttgatc 1020cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
agattacgcg 1080cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
acgctcagtg 1140gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
tcttcaccta 1200gatcctttta aattaaaaat gaagttttag cacgtgtcag tcctgctcct
cggccacgaa 1260gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg
gctgctcgcc 1320gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga
cctccgacca 1380ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt
tgtccggcac 1440cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca
caccggcgaa 1500gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact
cgaccgctcc 1560ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca
tggtggccct 1620cctcacgtgc tattattgaa gcatttatca gggttattgt ctcatgagcg
gatacatatt 1680tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
gaaaagtgcc 1740acctgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
catcaggaaa 1800ttgtaagcgt taataattca gaagaactcg tcaagaaggc gatagaaggc
gatgcgctgc 1860gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
gccgccaagc 1920tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
cacacccagc 1980cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
cggcaagcag 2040gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt
gagcctggcg 2100aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
atcgacaaga 2160ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
gtcgaatggg 2220caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
ggatactttc 2280tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
caatagcagc 2340cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
gcccgtcgtg 2400gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc
ggacaggtcg 2460gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
ggcatcagag 2520cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
agcggccgga 2580gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
tgtctcttga 2640tcagagcttg atcccctgcg ccatcagatc cttggcggcg agaaagccat
ccagtttact 2700ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg
ttcgcttgct 2760gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc
tacctgcttt 2820ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
catccggggt 2880cagcaccgtt tctgcggact ggctttctac gtgaaaagga tctaggtgaa
gatccttttt 2940gataatctca tgcctgacat ttatattccc cagaacatca ggttaatggc
gtttttgatg 3000tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
cggcacactg 3060gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
cgggtaaagt 3120tcacgggaga ctttatctga cagcagacgt gcactggcca gggggatcac
catccgtcgc 3180cccggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
gctctctctt 3240ttataggtgt aaaccttaaa ctgccgtacg tataggctgc gcaactgttg
ggaagggcga 3300tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
tgcaaggcga 3360ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
ggccagtgaa 3420ttgtaatacg actcactata gggcgaattg ggccctctag atgcatgctc
gagcggccgc 3480cagtgtgatg gatatctgca gaattcagga gcggccgcac catggccacc
atctctttga 3540ctactgagca acttttagaa cacccagaac tggttgcaat tgatggggtg
ttgtacgatc 3600tcttcggact ggcgaaagtg catccaggtg gcaacctcat tgaagccgcc
ggtgcctccg 3660acggaaccgc cctgttctac tccatgcacc ctggagtgaa gccagagaat
tcgaagctgc 3720tgcagcaatt tgcccgaggc aaacacgaac gaagctcgaa ggacccagtg
tacacctttg 3780acagtccctt cgcccaggat gtcaagcaga gcgttcggga ggtcatgaag
gggcgcaact 3840ggtacgccac gcccggcttt tggctgcgga ccgcgctgat catcgcgtgc
actgccatag 3900gcgaatggta ttggatcact accggggcag tgatgtgggg catcttcacc
gggtacttcc 3960acagccagat tgggttggcg attcaacacg atgcctctca cggagccatc
agcaaaaagc 4020cctgggtgaa cgcctttttc gcctacggca tcgacgccat tggatcctcc
cgctggatct 4080ggctgcagtc ccacattatg cgccaccaca cctacaccaa ccagcatggc
ctggacctgg 4140acgctgcctc ggcggagccg ttcattttgt tccactccta cccggcaaca
aatgcgtcac 4200gaaagtggta ccatcggttc caggcgtggt acatgtacat cgttttgggg
atgtatggtg 4260tgtcgatggt gtacaatccg atgtacttgt tcacgatgca gcacaacgac
acaatcccag 4320aggccacctc tcttagacca ggcagctttt tcaaccggca gcgcgccttc
gccgtttccc 4380tccgcctact gttcatcttc cgcaacgcct tcctcccctg gtacatcgcg
ggcgcctctc 4440cgctgctcac catcctgctg gtgccaacgg tcacaggcat cttcttgaca
tttgtttttg 4500tgctgtccca taactttgaa ggcgctgagc ggacccccga aaagaactgc
aaggccaaaa 4560gggccaagga ggggaaggag gtccgcgatg tagaggagga ccgggtggac
tggtaccggg 4620cgcaggccga gaccgcggcg acctacgggg gcagcgtcgg gatgatgctg
accggcggtt 4680tgaacctgca gatcgagcac cacttgttcc cccgcatgtc ctcttggcac
taccccttca 4740tccaagatac ggtgcgggaa tgttgcaagc gccatggcgt gcgctacaca
tactacccga 4800ccatcctgga gaatataatg tccacgctcc gctacatgca gaaggtgggc
gtggcccaca 4860caattcagga tgcccaggaa ttctgagcgg ccgcacctg
4899395561DNAArtificial Sequenceplasmid pKR767 39catggtcaat
caatgagacg ccaacttctt aatctattga gacctgcagg tctagaaggg 60cggatccccg
ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg 120aaaaccctgg
cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 180gtaatagcga
agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 240aatggcgcct
gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 300ggtgcactct
cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 360caacacccgc
tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 420ctgtgaccgt
ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 480cgagacgaaa
gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 540tttcttagac
gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 600ttttctaaat
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 660aataatattg
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 720tttttgcggc
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 780atgctgaaga
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 840agatccttga
gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 900tgctatgtgg
cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 960tacactattc
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1020atggcatgac
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1080ccaacttact
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1140tgggggatca
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1200acgacgagcg
tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1260ctggcgaact
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1320aagttgcagg
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 1380ctggagccgg
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 1440cctcccgtat
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1500gacagatcgc
tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1560actcatatat
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1620agatcctttt
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1680cgtcagaccc
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1740tctgctgctt
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1800agctaccaac
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1860tccttctagt
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1920acctcgctct
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1980ccgggttgga
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2040gttcgtgcac
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2100gtgagctatg
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2160gcggcagggt
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2220tttatagtcc
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2280caggggggcg
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2340tttgctggcc
ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 2400gtattaccgc
ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 2460agtcagtgag
cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2520ggccgattca
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2580gcaacgcaat
taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2640ttccggctcg
tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2700atgaccatga
ttacgccaag cttgcatgcc tgcaggctag cctaagtacg tactcaaaat 2760gccaacaaat
aaaaaaaaag ttgctttaat aatgccaaaa caaattaata aaacacttac 2820aacaccggat
tttttttaat taaaatgtgc catttaggat aaatagttaa tatttttaat 2880aattatttaa
aaagccgtat ctactaaaat gatttttatt tggttgaaaa tattaatatg 2940tttaaatcaa
cacaatctat caaaattaaa ctaaaaaaaa aataagtgta cgtggttaac 3000attagtacag
taatataaga ggaaaatgag aaattaagaa attgaaagcg agtctaattt 3060ttaaattatg
aacctgcata tataaaagga aagaaagaat ccaggaagaa aagaaatgaa 3120accatgcatg
gtcccctcgt catcacgagt ttctgccatt tgcaatagaa acactgaaac 3180acctttctct
ttgtcactta attgagatgc cgaagccacc tcacaccatg aacttcatga 3240ggtgtagcac
ccaaggcttc catagccatg catactgaag aatgtctcaa gctcagcacc 3300ctacttctgt
gacgtgtccc tcattcacct tcctctcttc cctataaata accacgcctc 3360aggttctccg
cttcacaact caaacattct ctccattggt ccttaaacac tcatcagtca 3420tcaccgcggc
cgcatgggaa cggaccaagg aaaaaccttc acctgggaag agctggcggc 3480ccataacacc
aaggacgacc tactcttggc catccgcggc agggtgtacg atgtcacaaa 3540gttcttgagc
cgccatcctg gtggagtgga cactctcctg ctcggagctg gccgagatgt 3600tactccggtc
tttgagatgt atcacgcgtt tggggctgca gatgccatta tgaagaagta 3660ctatgtcggt
acactggtct cgaatgagct gcccatcttc ccggagccaa cggtgttcca 3720caaaaccatc
aagacgagag tcgagggcta ctttacggat cggaacattg atcccaagaa 3780tagaccagag
atctggggac gatacgctct tatctttgga tccttgatcg cttcctacta 3840cgcgcagctc
tttgtgcctt tcgttgtcga acgcacatgg cttcaggtgg tgtttgcaat 3900catcatggga
tttgcgtgcg cacaagtcgg actcaaccct cttcatgatg cgtctcactt 3960ttcagtgacc
cacaacccca ctgtctggaa gattctggga gccacgcacg actttttcaa 4020cggagcatcg
tacctggtgt ggatgtacca acatatgctc ggccatcacc cctacaccaa 4080cattgctgga
gcagatcccg acgtgtcgac gtctgagccc gatgttcgtc gtatcaagcc 4140caaccaaaag
tggtttgtca accacatcaa ccagcacatg tttgttcctt tcctgtacgg 4200actgctggcg
ttcaaggtgc gcattcagga catcaacatt ttgtactttg tcaagaccaa 4260tgacgctatt
cgtgtcaatc ccatctcgac atggcacact gtgatgttct ggggcggcaa 4320ggctttcttt
gtctggtatc gcctgattgt tcccctgcag tatctgcccc tgggcaaggt 4380gctgctcttg
ttcacggtcg cggacatggt gtcgtcttac tggctggcgc tgaccttcca 4440ggcgaaccac
gttgttgagg aagttcagtg gccgttgcct gacgagaacg ggatcatcca 4500aaaggactgg
gcagctatgc aggtcgagac tacgcaggat tacgcacacg attcgcacct 4560ctggaccagc
atcactggca gcttgaacta ccaggctgtg caccatctgt tccccaacgt 4620gtcgcagcac
cattatcccg atattctggc catcatcaag aacacctgca gcgagtacaa 4680ggttccatac
cttgtcaagg atacgttttg gcaagcattt gcttcacatt tggagcactt 4740gcgtgttctt
ggactccgtc ccaaggaaga gtaggcggcc gcatttcgca ccaaatcaat 4800gaaagtaata
atgaaaagtc tgaataagaa tacttaggct tagatgcctt tgttacttgt 4860gtaaaataac
ttgagtcatg tacctttggc ggaaacagaa taaataaaag gtgaaattcc 4920aatgctctat
gtataagtta gtaatactta atgtgttcta cggttgtttc aatatcatca 4980aactctaatt
gaaactttag aaccacaaat ctcaatcttt tcttaatgaa atgaaaaatc 5040ttaattgtac
catgtttatg ttaaacacct tacaattggt tggagaggag gaccaaccga 5100tgggacaaca
ttgggagaaa gagattcaat ggagatttgg ataggagaac aacattcttt 5160ttcacttcaa
tacaagatga gtgcaacact aaggatatgt atgagacttt cagaagctac 5220gacaacatag
atgagtgagg tggtgattcc tagcaagaaa gacattagag gaagccaaaa 5280tcgaacaagg
aagacatcaa gggcaagaga caggaccatc catctcagga aaaggagctt 5340tgggatagtc
cgagaagttg tacaagaaat tttttggagg gtgagtgatg cattgctggt 5400gactttaact
caatcaaaat tgagaaagaa agaaaaggga gggggctcac atgtgaatag 5460aagggaaacg
ggagaatttt acagttttga tctaatgggc atcccagcta gtggtaacat 5520attcaccatg
tttaaccttc acgtacgtct agaggatccc c
5561401338DNAMortierella alpina 40atgggaacgg accaaggaaa aaccttcacc
tgggaagagc tggcggccca taacaccaag 60gacgacctac tcttggccat ccgcggcagg
gtgtacgatg tcacaaagtt cttgagccgc 120catcctggtg gagtggacac tctcctgctc
ggagctggcc gagatgttac tccggtcttt 180gagatgtatc acgcgtttgg ggctgcagat
gccattatga agaagtacta tgtcggtaca 240ctggtctcga atgagctgcc catcttcccg
gagccaacgg tgttccacaa aaccatcaag 300acgagagtcg agggctactt tacggatcgg
aacattgatc ccaagaatag accagagatc 360tggggacgat acgctcttat ctttggatcc
ttgatcgctt cctactacgc gcagctcttt 420gtgcctttcg ttgtcgaacg cacatggctt
caggtggtgt ttgcaatcat catgggattt 480gcgtgcgcac aagtcggact caaccctctt
catgatgcgt ctcacttttc agtgacccac 540aaccccactg tctggaagat tctgggagcc
acgcacgact ttttcaacgg agcatcgtac 600ctggtgtgga tgtaccaaca tatgctcggc
catcacccct acaccaacat tgctggagca 660gatcccgacg tgtcgacgtc tgagcccgat
gttcgtcgta tcaagcccaa ccaaaagtgg 720tttgtcaacc acatcaacca gcacatgttt
gttcctttcc tgtacggact gctggcgttc 780aaggtgcgca ttcaggacat caacattttg
tactttgtca agaccaatga cgctattcgt 840gtcaatccca tctcgacatg gcacactgtg
atgttctggg gcggcaaggc tttctttgtc 900tggtatcgcc tgattgttcc cctgcagtat
ctgcccctgg gcaaggtgct gctcttgttc 960acggtcgcgg acatggtgtc gtcttactgg
ctggcgctga ccttccaggc gaaccacgtt 1020gttgaggaag ttcagtggcc gttgcctgac
gagaacggga tcatccaaaa ggactgggca 1080gctatgcagg tcgagactac gcaggattac
gcacacgatt cgcacctctg gaccagcatc 1140actggcagct tgaactacca ggctgtgcac
catctgttcc ccaacgtgtc gcagcaccat 1200tatcccgata ttctggccat catcaagaac
acctgcagcg agtacaaggt tccatacctt 1260gtcaaggata cgttttggca agcatttgct
tcacatttgg agcacttgcg tgttcttgga 1320ctccgtccca aggaagag
1338415661DNAArtificial Sequenceplasmid
pKR974 41gtacgtctag aggatccccc atggtcaatc aatgagacgc caacttctta
atctattgag 60acctgcaggt ctagaagggc ggatccccgg gtaccgagct cgaattcact
ggccgtcgtt 120ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
tgcagcacat 180ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
ttcccaacag 240ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
gcatctgtgc 300ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
cgcatagtta 360agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
tctgctcccg 420gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
gaggttttca 480ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
tttataggtt 540aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
aaatgtgcgc 600ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
catgagacaa 660taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
tcaacatttc 720cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
tcacccagaa 780acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
ttacatcgaa 840ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
ttttccaatg 900atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
cgccgggcaa 960gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
ctcaccagtc 1020acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
tgccataacc 1080atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
gaaggagcta 1140accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
ggaaccggag 1200ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
aatggcaaca 1260acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
acaattaata 1320gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
tccggctggc 1380tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
cattgcagca 1440ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
gagtcaggca 1500actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
taagcattgg 1560taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
tcatttttaa 1620tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
cccttaacgt 1680gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
ttcttgagat 1740cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
accagcggtg 1800gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
cttcagcaga 1860gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
cttcaagaac 1920tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
tgctgccagt 1980ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
taaggcgcag 2040cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
gacctacacc 2100gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
agggagaaag 2160gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
ggagcttcca 2220gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
acttgagcgt 2280cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
caacgcggcc 2340tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
tgcgttatcc 2400cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
tcgccgcagc 2460cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
aatacgcaaa 2520ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
gtttcccgac 2580tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
ttaggcaccc 2640caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
cggataacaa 2700tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct
gcaggctagc 2760ctaagtacgt actcaaaatg ccaacaaata aaaaaaaagt tgctttaata
atgccaaaac 2820aaattaataa aacacttaca acaccggatt ttttttaatt aaaatgtgcc
atttaggata 2880aatagttaat atttttaata attatttaaa aagccgtatc tactaaaatg
atttttattt 2940ggttgaaaat attaatatgt ttaaatcaac acaatctatc aaaattaaac
taaaaaaaaa 3000ataagtgtac gtggttaaca ttagtacagt aatataagag gaaaatgaga
aattaagaaa 3060ttgaaagcga gtctaatttt taaattatga acctgcatat ataaaaggaa
agaaagaatc 3120caggaagaaa agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt
tctgccattt 3180gcaatagaaa cactgaaaca cctttctctt tgtcacttaa ttgagatgcc
gaagccacct 3240cacaccatga acttcatgag gtgtagcacc caaggcttcc atagccatgc
atactgaaga 3300atgtctcaag ctcagcaccc tacttctgtg acgtgtccct cattcacctt
cctctcttcc 3360ctataaataa ccacgcctca ggttctccgc ttcacaactc aaacattctc
tccattggtc 3420cttaaacact catcagtcat caccgcggcc gccaattcat ggccccgcag
acggagctcc 3480gccagcgcca cgccgccgtc gccgagacgc cggtggccgg caagaaggcc
tttacatggc 3540aggaggtcgc gcagcacaac acggcggcct cggcctggat cattatccgc
ggcaaggtct 3600acgacgtgac cgagtgggcc aacaagcacc ccggcggccg cgagatggtg
ctgctgcacg 3660ccggtcgcga ggccaccgac acgttcgact cgtaccaccc gttcagcgac
aaggccgagt 3720cgatcttgaa caagtatgag attggcacgt tcacgggccc gtccgagttt
ccgaccttca 3780agccggacac gggcttctac aaggagtgcc gcaagcgcgt tggcgagtac
ttcaagaaga 3840acaacctcca tccgcaggac ggcttcccgg gcctctggcg catgatggtc
gtgtttgcgg 3900tcgccggcct cgccttgtac ggcatgcact tttcgactat ctttgcgctg
cagctcgcgg 3960ccgcggcgct ctttggcgtc tgccaggcgc tgccgctgct ccacgtcatg
cacgactcgt 4020cgcacgcgtc gtacaccaac atgccgttct tccattacgt cgtcggccgc
tttgccatgg 4080actggtttgc cggcggctcg atggtgtcat ggctcaacca gcacgtcgtg
ggccaccaca 4140tctacacgaa cgtcgcgggc tcggacccgg atcttccggt caacatggac
ggcgacatcc 4200gccgcatcgt gaaccgccag gtgttccagc ccatgtacgc attccagcac
atctaccttc 4260cgccgctcta tggcgtgctt ggcctcaagt tccgcatcca ggacttcacc
gacacgttcg 4320gctcgcacac gaacggcccg atccgcgtca acccgcacgc gctctcgacg
tggatggcca 4380tgatcagctc caagtcgttc tgggccttct accgcgtgta ccttccgctt
gccgtgctcc 4440agatgcccat caagacgtac cttgcgatct tcttcctcgc cgagtttgtc
acgggctggt 4500acctcgcgtt caacttccaa gtaagccatg tctcgaccga gtgcggctac
ccatgcggcg 4560acgaggccaa gatggcgctc caggacgagt gggcagtctc gcaggtcaag
acgtcggtcg 4620actacgccca tggctcgtgg atgacgacgt tccttgccgg cgcgctcaac
taccaggtcg 4680tgcaccactt gttccccagc gtgtcgcagt accactaccc ggcgatcgcg
cccatcatcg 4740tcgacgtctg caaggagtac aacatcaagt acgccatctt gccggacttt
acggcggcgt 4800tcgttgccca cttgaagcac ctccgcaaca tgggccagca gggcatcgcc
gccacgatcc 4860acatgggcta actcgagctc agctagatcg cggccgcatt tcgcaccaaa
tcaatgaaag 4920taataatgaa aagtctgaat aagaatactt aggcttagat gcctttgtta
cttgtgtaaa 4980ataacttgag tcatgtacct ttggcggaaa cagaataaat aaaaggtgaa
attccaatgc 5040tctatgtata agttagtaat acttaatgtg ttctacggtt gtttcaatat
catcaaactc 5100taattgaaac tttagaacca caaatctcaa tcttttctta atgaaatgaa
aaatcttaat 5160tgtaccatgt ttatgttaaa caccttacaa ttaattggtt ggagaggagg
accaaccgat 5220gggacaacat tgggagaaag agattcaatg gagatttgga taggagaaca
acattctttt 5280tcacttcaat acaagatgag tgcaacacta aggatatgta tgagactttc
agaagctacg 5340acaacataga tgagtgaggt ggtgattcct agcaagaaag acattagagg
aagccaaaat 5400cgaacaagga agacatcaag ggcaagagac aggaccatcc atctcaggaa
aaggagcttt 5460gggatagtcc gagaagttgt acaagaaatt ttttggaggg tgagtgatgc
attgctggtg 5520actttaactc aatcaaaatt gagaaagaaa gaaaagggag ggggctcaca
tgtgaataga 5580agggaaacgg gagaatttta cagttttgat ctaatgggca tcccagctag
tggtaacata 5640ttcaccatgt ttaaccttca c
5661421413DNASaprolegnia diclina 42atggccccgc agacggagct
ccgccagcgc cacgccgccg tcgccgagac gccggtggcc 60ggcaagaagg cctttacatg
gcaggaggtc gcgcagcaca acacggcggc ctcggcctgg 120atcattatcc gcggcaaggt
ctacgacgtg accgagtggg ccaacaagca ccccggcggc 180cgcgagatgg tgctgctgca
cgccggtcgc gaggccaccg acacgttcga ctcgtaccac 240ccgttcagcg acaaggccga
gtcgatcttg aacaagtatg agattggcac gttcacgggc 300ccgtccgagt ttccgacctt
caagccggac acgggcttct acaaggagtg ccgcaagcgc 360gttggcgagt acttcaagaa
gaacaacctc catccgcagg acggcttccc gggcctctgg 420cgcatgatgg tcgtgtttgc
ggtcgccggc ctcgccttgt acggcatgca cttttcgact 480atctttgcgc tgcagctcgc
ggccgcggcg ctctttggcg tctgccaggc gctgccgctg 540ctccacgtca tgcacgactc
gtcgcacgcg tcgtacacca acatgccgtt cttccattac 600gtcgtcggcc gctttgccat
ggactggttt gccggcggct cgatggtgtc atggctcaac 660cagcacgtcg tgggccacca
catctacacg aacgtcgcgg gctcggaccc ggatcttccg 720gtcaacatgg acggcgacat
ccgccgcatc gtgaaccgcc aggtgttcca gcccatgtac 780gcattccagc acatctacct
tccgccgctc tatggcgtgc ttggcctcaa gttccgcatc 840caggacttca ccgacacgtt
cggctcgcac acgaacggcc cgatccgcgt caacccgcac 900gcgctctcga cgtggatggc
catgatcagc tccaagtcgt tctgggcctt ctaccgcgtg 960taccttccgc ttgccgtgct
ccagatgccc atcaagacgt accttgcgat cttcttcctc 1020gccgagtttg tcacgggctg
gtacctcgcg ttcaacttcc aagtaagcca tgtctcgacc 1080gagtgcggct acccatgcgg
cgacgaggcc aagatggcgc tccaggacga gtgggcagtc 1140tcgcaggtca agacgtcggt
cgactacgcc catggctcgt ggatgacgac gttccttgcc 1200ggcgcgctca actaccaggt
cgtgcaccac ttgttcccca gcgtgtcgca gtaccactac 1260ccggcgatcg cgcccatcat
cgtcgacgtc tgcaaggagt acaacatcaa gtacgccatc 1320ttgccggact ttacggcggc
gttcgttgcc cacttgaagc acctccgcaa catgggccag 1380cagggcatcg ccgccacgat
ccacatgggc taa 1413435592DNAArtificial
Sequenceplasmid pKR1139 43ggccgcattt cgcaccaaat caatgaaagt aataatgaaa
agtctgaata agaatactta 60ggcttagatg cctttgttac ttgtgtaaaa taacttgagt
catgtacctt tggcggaaac 120agaataaata aaaggtgaaa ttccaatgct ctatgtataa
gttagtaata cttaatgtgt 180tctacggttg tttcaatatc atcaaactct aattgaaact
ttagaaccac aaatctcaat 240cttttcttaa tgaaatgaaa aatcttaatt gtaccatgtt
tatgttaaac accttacaat 300taattggttg gagaggagga ccaaccgatg ggacaacatt
gggagaaaga gattcaatgg 360agatttggat aggagaacaa cattcttttt cacttcaata
caagatgagt gcaacactaa 420ggatatgtat gagactttca gaagctacga caacatagat
gagtgaggtg gtgattccta 480gcaagaaaga cattagagga agccaaaatc gaacaaggaa
gacatcaagg gcaagagaca 540ggaccatcca tctcaggaaa aggagctttg ggatagtccg
agaagttgta caagaaattt 600tttggagggt gagtgatgca ttgctggtga ctttaactca
atcaaaattg agaaagaaag 660aaaagggagg gggctcacat gtgaatagaa gggaaacggg
agaattttac agttttgatc 720taatgggcat cccagctagt ggtaacatat tcaccatgtt
taaccttcac gtacgtctag 780aggatccccc atggtcaatc aatgagacgc caacttctta
atctattgag acctgcaggt 840ctagaagggc ggatccccgg gtaccgagct cgaattcact
ggccgtcgtt ttacaacgtc 900gtgactggga aaaccctggc gttacccaac ttaatcgcct
tgcagcacat ccccctttcg 960ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
ttcccaacag ttgcgcagcc 1020tgaatggcga atggcgcctg atgcggtatt ttctccttac
gcatctgtgc ggtatttcac 1080accgcatatg gtgcactctc agtacaatct gctctgatgc
cgcatagtta agccagcccc 1140gacacccgcc aacacccgct gacgcgccct gacgggcttg
tctgctcccg gcatccgctt 1200acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
gaggttttca ccgtcatcac 1260cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
tttataggtt aatgtcatga 1320taataatggt ttcttagacg tcaggtggca cttttcgggg
aaatgtgcgc ggaaccccta 1380tttgtttatt tttctaaata cattcaaata tgtatccgct
catgagacaa taaccctgat 1440aaatgcttca ataatattga aaaaggaaga gtatgagtat
tcaacatttc cgtgtcgccc 1500ttattccctt ttttgcggca ttttgccttc ctgtttttgc
tcacccagaa acgctggtga 1560aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
ttacatcgaa ctggatctca 1620acagcggtaa gatccttgag agttttcgcc ccgaagaacg
ttttccaatg atgagcactt 1680ttaaagttct gctatgtggc gcggtattat cccgtattga
cgccgggcaa gagcaactcg 1740gtcgccgcat acactattct cagaatgact tggttgagta
ctcaccagtc acagaaaagc 1800atcttacgga tggcatgaca gtaagagaat tatgcagtgc
tgccataacc atgagtgata 1860acactgcggc caacttactt ctgacaacga tcggaggacc
gaaggagcta accgcttttt 1920tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
ggaaccggag ctgaatgaag 1980ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
aatggcaaca acgttgcgca 2040aactattaac tggcgaacta cttactctag cttcccggca
acaattaata gactggatgg 2100aggcggataa agttgcagga ccacttctgc gctcggccct
tccggctggc tggtttattg 2160ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
cattgcagca ctggggccag 2220atggtaagcc ctcccgtatc gtagttatct acacgacggg
gagtcaggca actatggatg 2280aacgaaatag acagatcgct gagataggtg cctcactgat
taagcattgg taactgtcag 2340accaagttta ctcatatata ctttagattg atttaaaact
tcatttttaa tttaaaagga 2400tctaggtgaa gatccttttt gataatctca tgaccaaaat
cccttaacgt gagttttcgt 2460tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
ttcttgagat cctttttttc 2520tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
accagcggtg gtttgtttgc 2580cggatcaaga gctaccaact ctttttccga aggtaactgg
cttcagcaga gcgcagatac 2640caaatactgt ccttctagtg tagccgtagt taggccacca
cttcaagaac tctgtagcac 2700cgcctacata cctcgctctg ctaatcctgt taccagtggc
tgctgccagt ggcgataagt 2760cgtgtcttac cgggttggac tcaagacgat agttaccgga
taaggcgcag cggtcgggct 2820gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
gacctacacc gaactgagat 2880acctacagcg tgagctatga gaaagcgcca cgcttcccga
agggagaaag gcggacaggt 2940atccggtaag cggcagggtc ggaacaggag agcgcacgag
ggagcttcca gggggaaacg 3000cctggtatct ttatagtcct gtcgggtttc gccacctctg
acttgagcgt cgatttttgt 3060gatgctcgtc aggggggcgg agcctatgga aaaacgccag
caacgcggcc tttttacggt 3120tcctggcctt ttgctggcct tttgctcaca tgttctttcc
tgcgttatcc cctgattctg 3180tggataaccg tattaccgcc tttgagtgag ctgataccgc
tcgccgcagc cgaacgaccg 3240agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
aatacgcaaa ccgcctctcc 3300ccgcgcgttg gccgattcat taatgcagct ggcacgacag
gtttcccgac tggaaagcgg 3360gcagtgagcg caacgcaatt aatgtgagtt agctcactca
ttaggcaccc caggctttac 3420actttatgct tccggctcgt atgttgtgtg gaattgtgag
cggataacaa tttcacacag 3480gaaacagcta tgaccatgat tacgccaagc ttgcatgcct
gcaggctagc ctaagtacgt 3540actcaaaatg ccaacaaata aaaaaaaagt tgctttaata
atgccaaaac aaattaataa 3600aacacttaca acaccggatt ttttttaatt aaaatgtgcc
atttaggata aatagttaat 3660atttttaata attatttaaa aagccgtatc tactaaaatg
atttttattt ggttgaaaat 3720attaatatgt ttaaatcaac acaatctatc aaaattaaac
taaaaaaaaa ataagtgtac 3780gtggttaaca ttagtacagt aatataagag gaaaatgaga
aattaagaaa ttgaaagcga 3840gtctaatttt taaattatga acctgcatat ataaaaggaa
agaaagaatc caggaagaaa 3900agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt
tctgccattt gcaatagaaa 3960cactgaaaca cctttctctt tgtcacttaa ttgagatgcc
gaagccacct cacaccatga 4020acttcatgag gtgtagcacc caaggcttcc atagccatgc
atactgaaga atgtctcaag 4080ctcagcaccc tacttctgtg acgtgtccct cattcacctt
cctctcttcc ctataaataa 4140ccacgcctca ggttctccgc ttcacaactc aaacattctc
tccattggtc cttaaacact 4200catcagtcat caccgcggcc gcaccatggc caccatctct
ttgactactg agcaactttt 4260agaacaccca gaactggttg caattgatgg ggtgttgtac
gatctcttcg gactggcgaa 4320agtgcatcca ggtggcaacc tcattgaagc cgccggtgcc
tccgacggaa ccgccctgtt 4380ctactccatg caccctggag tgaagccaga gaattcgaag
ctgctgcagc aatttgcccg 4440aggcaaacac gaacgaagct cgaaggaccc agtgtacacc
tttgacagtc ccttcgccca 4500ggatgtcaag cagagcgttc gggaggtcat gaaggggcgc
aactggtacg ccacgcccgg 4560cttttggctg cggaccgcgc tgatcatcgc gtgcactgcc
ataggcgaat ggtattggat 4620cactaccggg gcagtgatgt ggggcatctt caccgggtac
ttccacagcc agattgggtt 4680ggcgattcaa cacgatgcct ctcacggagc catcagcaaa
aagccctggg tgaacgcctt 4740tttcgcctac ggcatcgacg ccattggatc ctcccgctgg
atctggctgc agtcccacat 4800tatgcgccac cacacctaca ccaaccagca tggcctggac
ctggacgctg cctcggcgga 4860gccgttcatt ttgttccact cctacccggc aacaaatgcg
tcacgaaagt ggtaccatcg 4920gttccaggcg tggtacatgt acatcgtttt ggggatgtat
ggtgtgtcga tggtgtacaa 4980tccgatgtac ttgttcacga tgcagcacaa cgacacaatc
ccagaggcca cctctcttag 5040accaggcagc tttttcaacc ggcagcgcgc cttcgccgtt
tccctccgcc tactgttcat 5100cttccgcaac gccttcctcc cctggtacat cgcgggcgcc
tctccgctgc tcaccatcct 5160gctggtgcca acggtcacag gcatcttctt gacatttgtt
tttgtgctgt cccataactt 5220tgaaggcgct gagcggaccc ccgaaaagaa ctgcaaggcc
aaaagggcca aggaggggaa 5280ggaggtccgc gatgtagagg aggaccgggt ggactggtac
cgggcgcagg ccgagaccgc 5340ggcgacctac gggggcagcg tcgggatgat gctgaccggc
ggtttgaacc tgcagatcga 5400gcaccacttg ttcccccgca tgtcctcttg gcactacccc
ttcatccaag atacggtgcg 5460ggaatgttgc aagcgccatg gcgtgcgcta cacatactac
ccgaccatcc tggagaatat 5520aatgtccacg ctccgctaca tgcagaaggt gggcgtggcc
cacacaattc aggatgccca 5580ggaattctga gc
55924411920DNAArtificial Sequenceplasmid pKR1153
44ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca
60gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat
120gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa
180aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac
240gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa
300aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga
360gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac
420ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca
480aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa
540tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc
600gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct
660caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc
720actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc
780ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt
840ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta
900tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt
960tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc
1020ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct
1080ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga
1140tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc
1200tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca
1260ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact
1320tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca
1380ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat
1440gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat
1500aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac
1560tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga
1620atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt
1680tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa
1740ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata
1800ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt
1860atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga
1920tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc
1980ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt
2040acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa
2100taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa
2160gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt
2220aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata
2280tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc
2340aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca
2400aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta
2460cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta
2520tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa
2580gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg
2640tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc
2700tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
2760tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
2820tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
2880atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc
2940cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
3000cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
3060gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
3120aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
3180cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
3240tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
3300acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
3360ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
3420ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
3480tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
3540tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
3600ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
3660gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
3720cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
3780gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg
3840actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga
3900aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
3960cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
4020tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
4080tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
4140agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
4200gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
4260ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
4320cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
4380tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
4440ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
4500cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
4560ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
4620gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
4680tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
4740gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
4800cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
4860ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
4920tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg
4980aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
5040ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg
5100cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag
5160tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta
5220agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac
5280tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga
5340caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag
5400ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa
5460cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt
5520attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg
5580tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc
5640cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag
5700cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc
5760tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc
5820tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc
5880aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt
5940catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa
6000cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg
6060cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc
6120cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca
6180gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg
6240tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc
6300ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa
6360accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa
6420agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag
6480caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc
6540aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc
6600agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc
6660ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc
6720ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat
6780aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc
6840aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct
6900tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg
6960ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga
7020ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag
7080tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta
7140gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa
7200tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt
7260taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt
7320tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt
7380cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg
7440ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt
7500tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa
7560aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa
7620taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata
7680tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc
7740aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc
7800atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat
7860gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg
7920caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta
7980tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta
8040gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag
8100tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa
8160aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa
8220tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa
8280tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa
8340aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga
8400ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa
8460cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag
8520ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa
8580aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa
8640ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt
8700tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt
8760tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat
8820gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt
8880aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg
8940ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc
9000ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg
9060ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg
9120cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct
9180cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc
9240ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg
9300atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct
9360ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata
9420catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc
9480caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa
9540ccaattaatt gtaaggtgtt taacataaac atggtacaat taagattttt catttcatta
9600agaaaagatt gagatttgtg gttctaaagt ttcaattaga gtttgatgat attgaaacaa
9660ccgtagaaca cattaagtat tactaactta tacatagagc attggaattt caccttttat
9720ttattctgtt tccgccaaag gtacatgact caagttattt tacacaagta acaaaggcat
9780ctaagcctaa gtattcttat tcagactttt cattattact ttcattgatt tggtgcgaaa
9840tgcggccgct cagaattcct gggcatcctg aattgtgtgg gccacgccca ccttctgcat
9900gtagcggagc gtggacatta tattctccag gatggtcggg tagtatgtgt agcgcacgcc
9960atggcgcttg caacattccc gcaccgtatc ttggatgaag gggtagtgcc aagaggacat
10020gcgggggaac aagtggtgct cgatctgcag gttcaaaccg ccggtcagca tcatcccgac
10080gctgcccccg taggtcgccg cggtctcggc ctgcgcccgg taccagtcca cccggtcctc
10140ctctacatcg cggacctcct tcccctcctt ggcccttttg gccttgcagt tcttttcggg
10200ggtccgctca gcgccttcaa agttatggga cagcacaaaa acaaatgtca agaagatgcc
10260tgtgaccgtt ggcaccagca ggatggtgag cagcggagag gcgcccgcga tgtaccaggg
10320gaggaaggcg ttgcggaaga tgaacagtag gcggagggaa acggcgaagg cgcgctgccg
10380gttgaaaaag ctgcctggtc taagagaggt ggcctctggg attgtgtcgt tgtgctgcat
10440cgtgaacaag tacatcggat tgtacaccat cgacacacca tacatcccca aaacgatgta
10500catgtaccac gcctggaacc gatggtacca ctttcgtgac gcatttgttg ccgggtagga
10560gtggaacaaa atgaacggct ccgccgaggc agcgtccagg tccaggccat gctggttggt
10620gtaggtgtgg tggcgcataa tgtgggactg cagccagatc cagcgggagg atccaatggc
10680gtcgatgccg taggcgaaaa aggcgttcac ccagggcttt ttgctgatgg ctccgtgaga
10740ggcatcgtgt tgaatcgcca acccaatctg gctgtggaag tacccggtga agatgcccca
10800catcactgcc ccggtagtga tccaatacca ttcgcctatg gcagtgcacg cgatgatcag
10860cgcggtccgc agccaaaagc cgggcgtggc gtaccagttg cgccccttca tgacctcccg
10920aacgctctgc ttgacatcct gggcgaaggg actgtcaaag gtgtacactg ggtccttcga
10980gcttcgttcg tgtttgcctc gggcaaattg ctgcagcagc ttcgaattct ctggcttcac
11040tccagggtgc atggagtaga acagggcggt tccgtcggag gcaccggcgg cttcaatgag
11100gttgccacct ggatgcactt tcgccagtcc gaagagatcg tacaacaccc catcaattgc
11160aaccagttct gggtgttcta aaagttgctc agtagtcaaa gagatggtgg ccatggtgcg
11220gccgcggtga tgactgatga gtgtttaagg accaatggag agaatgtttg agttgtgaag
11280cggagaacct gaggcgtggt tatttatagg gaagagagga aggtgaatga gggacacgtc
11340acagaagtag ggtgctgagc ttgagacatt cttcagtatg catggctatg gaagccttgg
11400gtgctacacc tcatgaagtt catggtgtga ggtggcttcg gcatctcaat taagtgacaa
11460agagaaaggt gtttcagtgt ttctattgca aatggcagaa actcgtgatg acgaggggac
11520catgcatggt ttcatttctt ttcttcctgg attctttctt tccttttata tatgcaggtt
11580cataatttaa aaattagact cgctttcaat ttcttaattt ctcattttcc tcttatatta
11640ctgtactaat gttaaccacg tacacttatt ttttttttag tttaattttg atagattgtg
11700ttgatttaaa catattaata ttttcaacca aataaaaatc attttagtag atacggcttt
11760ttaaataatt attaaaaata ttaactattt atcctaaatg gcacatttta attaaaaaaa
11820atccggtgtt gtaagtgttt tattaatttg ttttggcatt attaaagcaa cttttttttt
11880atttgttggc attttgagta cgtacttagg ctagcctgca
11920451362DNAArtificial Sequencecodon-optimized Euglena anabaena delta-5
desaturase 45atggccacca tctccctgac taccgagcag ctcctggaac accccgagct
cgttgccatc 60gacggagtcc tgtacgatct cttcggtctg gccaaggtgc atccaggagg
caacctcatc 120gaagctgccg gtgcatccga cggaaccgct ctgttctact ccatgcatcc
tggagtcaag 180ccagagaact cgaagcttct gcagcaattt gcccgaggca agcacgaacg
aagctccaag 240gatcccgtgt acaccttcga ctctcccttt gctcaggacg tcaagcagtc
cgttcgagag 300gtcatgaagg gtcgaaactg gtacgccact cctggcttct ggctgagaac
cgcactcatc 360atcgcttgta ctgccattgg cgagtggtac tggatcacaa ccggagcagt
gatgtggggt 420atctttactg gatacttcca ctcgcagatt ggcttggcca ttcaacacga
tgcttctcac 480ggagccatca gcaaaaagcc ctgggtcaac gcctttttcg cttatggcat
cgacgccatt 540ggttcctctc gttggatctg gctgcagtcc cacattatgc gacatcacac
ttacaccaac 600cagcatggcc tcgacctgga tgctgcctcg gcagagccgt tcatcttgtt
ccactcctat 660cctgctacca acgcctctcg aaagtggtac caccgatttc aggcgtggta
catgtacatc 720gttctgggaa tgtatggtgt ctcgatggtg tacaatccca tgtacctctt
cacaatgcag 780cacaacgaca ccattcccga ggccacttct ctcagaccag gcagcttttt
caatcggcag 840cgagctttcg ccgtttccct tcgactgctc ttcatcttcc gaaacgcctt
tcttccctgg 900tacattgctg gtgcctctcc tctgctcacc attcttctgg tgcccacggt
cacaggcatc 960ttcctcacct ttgtgttcgt tctgtcccat aacttcgagg gagccgaacg
gaccccagag 1020aagaactgca aggccaaacg agctaaggaa ggcaaggagg tcagagacgt
ggaagaggat 1080cgagtcgact ggtaccgagc acaggccgag actgctgcca cctacggtgg
cagcgtggga 1140atgatgctta caggcggtct caacctgcag atcgagcatc acttgtttcc
ccgaatgtcc 1200tcttggcact atcccttcat tcaagacacc gttcgggagt gttgcaagcg
acatggcgtc 1260cgttacacat actatcctac cattctcgag aacatcatgt ccactcttcg
atacatgcag 1320aaggtgggtg ttgctcacac cattcaggat gcccaggagt tc
1362463983DNAArtificial Sequenceplasmid pEaD5S 46tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg
tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg
gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct
ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420tgcatctaga
tccatggtca agcgacccgc tctgcctctc accgtggacg gtgtcaccta 480cgacgtttct
gcctggctca accaccatcc cggaggtgcc gacattatcg agaactaccg 540aggtcgggat
gctaccgacg tcttcatggt tatgcactcc gagaacgccg tgtccaaact 600cagacgaatg
cccatcatgg aaccttcctc tcccctgact ccaacacctc ccaagccaaa 660ctccgacgaa
cctcaggagg atttccgaaa gctgcgagac gagctcattg ctgcaggcat 720gttcgatgcc
tctcccatgt ggtacgctta caagaccctg tcgactctcg gactgggtgt 780ccttgccgtg
ctgttgatga cccagtggca ctggtacctg gttggtgcta tcgtcctcgg 840cattcacttt
caacagatgg gatggctctc gcacgacatt tgccatcacc agctgttcaa 900ggaccgatcc
atcaacaatg ccattggcct gctcttcgga aacgtgcttc agggcttttc 960tgtcacttgg
tggaaggacc gacacaacgc tcatcactcc gccaccaacg tgcagggtca 1020cgatcccgac
atcgacaacc tgcctctcct ggcgtggtcc aaggaggacg tcgagcgagc 1080tggcccgttt
tctcgacgga tgatcaagta ccaacagtat tacttctttt tcatctgtgc 1140ccttctgcga
ttcatctggt gctttcagtc cattcatact gccacgggtc tcaaggatcg 1200aagcaatcag
tactatcgaa gacagtacga gaaggagtcc gtcggtctgg cactccactg 1260gggtctcaag
gccttgttct actatttcta catgccctcg tttctcaccg gactcatggt 1320gttctttgtc
tccgagctgc ttggtggctt cggaattgcc atcgttgtct tcatgaacca 1380ctaccctctg
gagaagattc aggactccgt gtgggatggt catggcttct gtgctggaca 1440gattcacgag
accatgaacg ttcagcgagg cctcgtcaca gactggtttt tcggtggcct 1500caactaccag
atcgaacatc acctgtggcc tactcttccc agacacaacc tcaccgctgc 1560ctccatcaaa
gtggagcagc tgtgcaagaa gcacaacctg ccctaccgat ctcctcccat 1620gctcgaaggt
gtcggcattc ttatctccta cctgggcacc ttcgctcgaa tggttgccaa 1680ggcagacaag
gcctaagcgg ccgcatcgga tcccgggccc gtcgactgca gaggcctgca 1740tgcaagcttg
gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 1800aattccacac
aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 1860gagctaactc
acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 1920gtgccagctg
cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 1980ctcttccgct
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 2040atcagctcac
tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 2100gaacatgtga
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 2160gtttttccat
aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2220gtggcgaaac
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2280gcgctctcct
gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2340aagcgtggcg
ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2400ctccaagctg
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2460taactatcgt
cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2520tggtaacagg
attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2580gcctaactac
ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 2640taccttcgga
aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 2700tggttttttt
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 2760tttgatcttt
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 2820ggtcatgaga
ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 2880taaatcaatc
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 2940tgaggcacct
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 3000cgtgtagata
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 3060gcgagaccca
cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 3120cgagcgcaga
agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 3180ggaagctaga
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 3240aggcatcgtg
gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 3300atcaaggcga
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 3360tccgatcgtt
gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 3420gcataattct
cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 3480aaccaagtca
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 3540acgggataat
accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 3600ttcggggcga
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 3660tcgtgcaccc
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 3720aacaggaagg
caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 3780catactcttc
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 3840atacatattt
gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 3900aaaagtgcca
cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 3960gcgtatcacg
aggccctttc gtc 3983
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