Patent application title: 21-DEOXYMACBECIN ANALOGUES USEFUL AS ANTITUMOR AGENTS
Inventors:
Christine Janet Martin (Saffron Walden, GB)
Barrie Wilkinson (Saffron Walden, GB)
Sabine Gaisser (Walden Essex, GB)
Ming-Oiang Zhang (Saffron Walden, GB)
Rose Mary Sheridan (Saffron Walden, GB)
Lesley Sarah Sheehan (Singapore, SG)
Rachel Edith Lill (Redhill, GB)
Mohammed Nur-E-Alam (Saffron Walden, GB)
William Alexander Vousden (Cambrige, GB)
IPC8 Class: AA61K3166FI
USPC Class:
514110
Class name: Designated organic active ingredient containing (doai) phosphorus containing other than solely as part of an inorganic ion in an addition salt doai phosphorus is part of a ring
Publication date: 2009-08-20
Patent application number: 20090209494
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Patent application title: 21-DEOXYMACBECIN ANALOGUES USEFUL AS ANTITUMOR AGENTS
Inventors:
Christine Janet Martin
Barrie Wilkinson
Sabine Gaisser
Ming-Oiang Zhang
Rose Mary Sheridan
Lesley Sarah Sheehan
Rachel Edith Lill
Mohammed Nur-E-Alam
William Alexander Vousden
Agents:
DANN, DORFMAN, HERRELL & SKILLMAN
Assignees:
Origin: PHILADELPHIA, PA US
IPC8 Class: AA61K3166FI
USPC Class:
514110
Abstract:
The present invention relates to 21-deoxymacbecin analogues that are
useful, e.g. in the treatment of cancer, B-cell malignancies, malaria,
fungal infection, diseases of the central nervous system and
neurodegenerative diseases, diseases dependent on angiogenesis,
autoimmune diseases or as a prophylactic pretreatment for cancer. The
present invention also provides methods for the production of these
compounds and their use in medicine, in particular in the treatment and
or prophylaxis of cancer or B-cell malignancies.Claims:
1: A 21-deoxymacbecin analogue according to the formula (I) below, or a
pharmaceutically acceptable salt thereof: ##STR00009## wherein:R1
represents H, OH or OCH3 R2 represents H or CH3 R3
and R4 either both represent H or together they represent a bond
(i.e. C4 to C5 is a double bond)R5 represents H or --C(O)--NH.sub.2.
2: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R1 represents H or OH.
3: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R1 represents H.
4: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R1 represents OH.
5: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R2 represents H.
6: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R3 and R4 both represent H.
7: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R5 represents --C(O)--NH.sub.2.
8: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R1 represents H, R2 represents H, R3 and R4 both represent H and R5 represents --C(O)--NH.sub.2.
9: The 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1 wherein R1 represents OH, R2 represents H, R3 and R4 both represent H and R5 represents --C(O)--NH.sub.2.
10: The 21-deoxymacbecin analogue according to claim 1 which is ##STR00010## or a pharmaceutically acceptable salt thereof.
11: The 21-deoxymacbecin analogue according to claim 1 which is ##STR00011## or a pharmaceutically acceptable salt thereof.
12: A pharmaceutical composition comprising a 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof according to claim 1, together with one or more pharmaceutically acceptable diluents or carriers.
13-15. (canceled)
16: A method of treatment of cancer, B-cell malignancies, malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases and/or as a prophylactic pretreatment for cancer which comprises administering to a patient in need thereof an effective amount of a 21-deoxymacbecin analogue according to claim 1.
17: The method of claim 16, wherein the 21-deoxymacbecin analogue or a pharmaceutically acceptable salt thereof is administered in combination with another treatment.
18: The method according to claim 17 where the other treatment is selected from the group consisting of: methotrexate, leukovorin, prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody, capecitabine, raloxifene hydrochloride, EGFR inhibitors, VEGF inhibitors, proteasome inhibitors radiotherapy and surgery.
19: The method according to claim 17 where the other treatment is selected from the group consisting of conventional chemotherapeutics such as cisplatin, cytarabine, cyclohexylchloroethylnitrosurea, cyclophosphamide, gemcitabine, Ifosfamid, leucovorin, mitomycin, mitoxantone, oxaliplatin and taxanes including taxol and vindesine; hormonal therapies such as anastrozole, goserelin, megestrol acetate and prednisone; monoclonal antibody therapies such as cetuximab (anti-EGFR); protein kinase inhibitors such as dasatinib, lapatinib; histone deacetylase (HDAC) inhibitors such as vorinostat; angiogenesis inhibitors such as sunitinib, sorafenib, lenalidomide; and mTOR inhibitors such as temsirolimus.
20: The method according to claim 17 where the other treatment is a cytotoxic agent.
21: The method according to claim 20 where the other treatment is selected from mitomycin C, ifosfamid, CCNU, mitoxantrone and vindesine.
22: A method for the production of a 21-deoxymacbecin analogue according to claim 1, said method comprising:a) providing a first host strain that produces macbecin when cultured under appropriate conditions,b) deleting or inactivating one or more post-PKS genes, wherein at least one of the post-PKS genes is mbcM, or a homologue thereof,c) culturing said modified host strain under suitable conditions for the production of 21-deoxymacbecin analogues; andd) optionally isolating the compounds produced.
23: A method for the production of a 21-deoxymacbecin analogue according to claim 1, said method comprising:a) providing a first host strain that produces macbecin when cultured under appropriate conditions,b) deleting or inactivating one or more post-PKS genes, wherein at least one of the post-PKS genes is mbcM, or a homologue thereof,c) re-introducing some or all of the post-PKS genes not including mbcM,d) culturing said modified host strain under suitable conditions for the production of 21-deoxymacbecin analogues; ande) optionally isolating the compounds produced.
24: The method according to claim 23 wherein in step (a) the strain is a macbecin producing strain.
25: The method according to claim 23 wherein the engineered strain which is cultured for the production of 21-deoxymacbecin analogues is based on a macbecin producing strain in which one or more of the post-PKS genes including mbcM have been deleted or inactivated.
26: The method according to claim 25 wherein the engineered strain which is cultured for the production of 21-deoxymacbecin analogues is an engineered strain based on a macbecin producing strain in which mbcM has been deleted or inactivated.
27: The method according to claim 25 wherein the engineered strain which is cultured for the production of 21-deoxymacbecin analogues is an engineered strain based on a macbecin producing strain in which mbcM, mbcMT1, mbcMT2, mbcP and mbcP450 have been deleted or inactivated.
28: A host strain which naturally produces macbecin and analogues thereof, in which the mbcM gene or a homologue thereof has been deleted or inactivated such that it thereby produces 21-deoxymacbecin or an analogue thereof.
29: An engineered strain based on a macbecin producing strain in which mbcM and optionally further post-PKS genes have been deleted or inactivated.
30: The engineered strain according to claim 29 in which mbcM has been deleted or inactivated.
31: The engineered strain according to claim 29 in which mbcM as well as 1 or more genes selected from mbcN, mbcP mbcMT1, mbcMT2, and mbcP450 have been deleted or inactivated.
32: The engineered strain according to claim 29 in which mbcM as well as 2 or more genes selected from mbcN, mbcP mbcMT1, mbcMT2, and mbcP450 have been deleted or inactivated.
33: The engineered strain according to claim 29 in which mbcM as well as 3 or more genes selected from mbcN, mbcP mbcMT1, mbcMT2, and mbcP450 have been deleted or inactivated.
34: The engineered strain according to claim 29 in which mbcM as well as 4 or more genes selected from mbcN, mbcP mbcMT1, mbcMT2, and mbcP450 have been deleted or inactivated.
35: The engineered strain according to claim 29 wherein the starting macbecin producing strain is A pretiosum or A mirum.
36-38. (canceled)
39: The method according to claim 16 for the treatment of cancer and/or B-cell malignancies.
40: A 21-deoxymacbecin analogue producible by the method according to claim 22.
41: A 21-deoxymacbecin analogue producible by the method according to claim 23.
42: The method according to claim 22 wherein in step (a) the strain is a macbecin producing strain.
43: The method according to claim 22 wherein the engineered strain which is cultured for the production of 21-deoxymacbecin analogues is based on a macbecin producing strain in which one or more of the post-PKS genes including mbcM have been deleted or inactivated.
44: The method according to claim 43 wherein the engineered strain which is cultured for the production of 21-deoxymacbecin analogues is an engineered strain based on a macbecin producing strain in which mbcM has been deleted or inactivated.
45: The method according to claim 43 wherein the engineered strain which is cultured for the production of 21-deoxymacbecin analogues is an engineered strain based on a macbecin producing strain in which mbcM, mbcMT1, mbcMT2, mbcP and mbcP450 have been deleted or inactivated.
46: The pharmaceutical composition according to claim 12, further comprising another treatment agent.
47: The pharmaceutical composition according to claim 46 where the other treatment agent is selected from the group consisting of: methotrexate, leukovorin, prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody, capecitabine, raloxifene hydrochloride, EGFR inhibitors, VEGF inhibitors, and proteasome inhibitors.
48: The pharmaceutical composition according to claim 46 where the other treatment agent is selected from the group consisting of conventional chemotherapeutics such as cisplatin, cytarabine, cyclohexylchloroethylnitrosurea, cyclophosphamide, gemcitabine, Ifosfamid, leucovorin, mitomycin, mitoxantone, oxaliplatin and taxanes including taxol and vindesine; hormonal therapies such as anastrozole, goserelin, megestrol acetate and prednisone; monoclonal antibody therapies such as cetuximab (anti-EGFR); protein kinase inhibitors such as dasatinib, lapatinib; histone deacetylase (HDAC) inhibitors such as vorinostat; angiogenesis inhibitors such as sunitinib, sorafenib, lenalidomide; and mTOR inhibitors such as temsirolimus.
49: The pharmaceutical composition according to claim 46 where the other treatment is a cytotoxic agent.
50: The pharmaceutical composition according to claim 49 where the other treatment agent is selected from mitomycin C, ifosfamid, CCNU, mitoxantrone and vindesine.
Description:
BACKGROUND OF THE INVENTION
[0001]The 90 kDa heat shock protein (Hsp90) is an abundant molecular chaperone involved in the folding and assembly of proteins, many of which are involved in signal transduction pathways (for reviews see Neckers, 2002; Sreedhar et al., 2004a; Wegele et al., 2004 and references therein). So far nearly 50 of these so-called client proteins have been identified and include steroid receptors, non-receptor tyrosine kinases e.g. src family, cyclin-dependent kinases e.g. cdk4 and cdk6, the cystic transmembrane regulator, nitric oxide synthase and others (Donze and Picard, 1999; McLaughlin et al., 2002; Chiosis et al., 2004; Wegele et al., 2004; http://www.picard.ch/downloads/Hsp90 interactors.pdf). Furthermore, Hsp90 plays a key role in stress response and protection of the cell against the effects of mutation (Bagatell and Whitesell, 2004; Chiosis et al., 2004). The function of Hsp90 is complicated and it involves the formation of dynamic multi-enzyme complexes (Bohen, 1998; Liu et al., 1999; Young et al., 2001; Takahashi et al., 2003; Sreedhar et al., 2004; Wegele et al., 2004). Hsp90 is a target for inhibitors (Fang et al., 1998; Liu et al., 1999; Blagosklonny, 2002; Neckers, 2003; Takahashi et al., 2003; Beliakoff and Whitesell, 2004; Wegele et al., 2004) resulting in degradation of client proteins, cell cycle dysregulation/or normalisation and apoptosis. More recently, Hsp90 has been identified as an important extracellular mediator for tumour invasion (Eustace et al., 2004). Hsp90 was identified as a new major therapeutic target for cancer therapy which is mirrored in the intense and detailed research about Hsp90 function (Blagosklonny et al., 1996; Neckers, 2002; Workman and Kaye, 2002; Beliakoff and Whitesell, 2004; Harris et al., 2004; Jez et al., 2003; Lee et al., 2004) and the development of high-throughput screening assays (Carreras et al., 2003; Rowlands et al., 2004). Hsp90 inhibitors include compound classes such as ansamycins, macrolides, purines, pyrazoles, coumarin antibiotics and others (for review see Bagatell and Whitesell, 2004; Chiosis et al., 2004 and references therein).
[0002]The benzenoid ansamycins are a broad class of chemical structures characterised by an aliphatic ring of varying length joined either side of an aromatic ring structure. Naturally occurring ansamycins include: macbecin and 18,21-dihydromacbecin (also known as macbecin 1 and macbecin 11 respectively) (1 & 2; Tanida et al., 1980), geldanamycin (3; DeBoer et al., 1970; DeBoer and Dietz, 1976; WO 03/106653 and references therein), and the herbimycin family (4; 5, 6, Omura et al., 1979, Iwai et al., 1980 and Shibata et al, 1986a, WO 03/106653 and references therein).
##STR00001##
[0003]Ansamycins were originally identified for their antibacterial and antiviral activity, however, recently their potential utility as anticancer agents has become of greater interest (Beliakoff and Whitesell, 2004). Many Hsp90 inhibitors are currently being assessed in clinical trials (Csermely and Soti, 2003; Workman, 2003). In particular, geldanamycin has nanomolar potency and apparent specificity for aberrant protein kinase dependent tumour cells (Chiosis et al., 2003; Workman, 2003).
[0004]It has been shown that treatment with Hsp90 inhibitors enhances the induction of tumour cell death by radiation and increased cell killing abilities (e.g. breast cancer, chronic myeloid leukaemia and non-small cell lung cancer) by combination of Hsp90 inhibitors with cytotoxic agents has also been demonstrated (Neckers, 2002; Beliakoff and Whitesell, 2004). The potential for anti-angiogenic activity is also of interest: the Hsp90 client protein HIF-1α plays a key role in the progression of solid tumours (Hur et al., 2002; Workman and Kaye, 2002; Kaur et al., 2004).
[0005]Hsp90 inhibitors also function as immunosuppressants and are involved in the complement-induced lysis of several types of tumour cells after Hsp90 inhibition (Sreedhar et al., 2004).
[0006]The use of Hsp90 inhibitors as potential anti-malaria drugs has also been discussed (Kumar et al., 2003). Furthermore, it has been shown that geldanamycin interferes with the formation of complex glycosylated mammalian prion protein PrPc (Winklhofer et al., 2003).
[0007]As described above, ansamycins are of interest as potential anticancer and anti-B-cell malignancy compounds, however the currently available ansamycins exhibit poor pharmacological or pharmaceutical properties, for example they show poor water solubility, poor metabolic stability, poor bioavailability or poor formulation ability (Goetz et al., 2003; Workman 2003; Chiosis 2004). Both herbimycin A and geldanamycin were identified as poor candidates for clinical trials due to their strong hepatotoxicity (review Workman, 2003) and geldanamycin was withdrawn from Phase I clinical trials due to hepatotoxicity (Supko et al., 1995, WO 03/106653).
[0008]Geldanamycin was isolated from culture filtrates of Streptomyces hygroscopicus and shows strong activity in vitro against protozoa and weak activity against bacteria and fungi. In 1994 the association of geldanamycin with Hsp90 was shown (Whitesell et al., 1994). The biosynthetic gene cluster for geldanamycin was cloned and sequenced (Allen and Ritchie, 1994; Rascher et al., 2003; WO 03/106653). The DNA sequence is available under the NCBI accession number AY179507. The isolation of genetically engineered geldanamycin producer strains derived from S. hygroscopicus subsp. duamyceticus JCM4427 and the isolation of 4,5-dihydro-7-O-descarbamoyl-7-hydroxygeldanamycin and 4,5-dihydro-7-O-descarbamoyl-7-hydroxy-17-O-demethylgeldanamycin were described recently (Hong et al., 2004). By feeding geldanamycin to the herbimycin producing strain Streptomyces hygroscopicus AM-3672 the compounds 15-hydroxygeldanamycin, the tricyclic geldanamycin analogue KOSN-1633 and methyl-geldanamycinate were isolated (Hu et al., 2004). The two compounds 17-formyl-17-demethoxy-18-O-21-O-dihydrogeldanamycin and 17-hydroxymethyl-17-demethoxygeldanamycin were isolated from S. hygroscopicus K279-78. S. hygroscopicus K279-78 is S. hygroscopicus NRRL 3602 containing cosmid pKOS279-78 which has a 44 kbp insert which contains various genes from the herbimycin producing strain Streptomyces hygroscopicus AM-3672 (Hu et al., 2004). Substitutions of acyltransferase (AT) domains have been made in four of the modules of the polyketide synthase of the geldanamycin biosynthetic cluster (Patel et al., 2004). AT substitutions were carried out in modules 1, 4 and 5 leading to the fully processed analogues 14-desmethyl-geldanamycin, 8-desmethyl-geldanamycin and 6-desmethoxy-geldanamycin and the not fully processed 4,5-dihydro-6-desmethoxy-geldanamycin. Substitution of the module 7 AT led to production of three 2-desmethyl compounds, KOSN1619, KOSN1558 and KOSN1559, one of which (KOSN1559), a 2-demethyl-4,5-dihydro-17-demethoxy-21-deoxy derivative of geldanamycin, binds to Hsp90 with a 4-fold greater binding affinity than geldanamycin and an 8-fold greater binding affinity than 17-AAG. However this is not reflected in an improvement in the IC50 measurement using SKBr3. Another analogue, a novel nonbenzoquinoid geldanamycin, designated KOS-1806 has a monophenolic structure (Rascher et al., 2005). No activity data was given for KOS-1806.
[0009]In 1979 the ansamycin antibiotic herbimycin A was isolated from the fermentation broth of Streptomyces hygroscopicus strain No. AM-3672 and named according to its potent herbicidal activity. The antitumour activity was established by using cells of a rat kidney line infected with a temperature sensitive mutant of Rous sarcoma virus (RSV) for screening for drugs that reverted the transformed morphology of the these cells (for review see Uehara, 2003). Herbimycin A was postulated as acting primarily through the binding to Hsp90 chaperone proteins but the direct binding to the conserved cysteine residues and subsequent inactivation of kinases was also discussed (Uehara, 2003).
[0010]Chemical derivatives have been isolated and compounds with altered substituents at C19 of the benzoquinone nucleus and halogenated compounds in the ansa chain showed less toxicity and higher antitumour activities than herbimycin A (Omura et al., 1984; Shibata et al., 1986b). The sequence of the herbimycin biosynthetic gene cluster was identified in WO 03/106653 and in a recent paper (Rascher et al., 2005).
[0011]The ansamycin compounds macbecin (1) and 18,21-dihydromacbecin (2) (C-14919E-1 and C-14919E-1), identified by their antifungal and antiprotozoal activity, were isolated from the culture supernatants of Nocardia sp No. C-14919 (Actinosynnema pretiosum subsp pretiosum ATCC 31280) (Tanida et al., 1980; Muroi et al., 1980; Muroi et al., 1981; U.S. Pat. No. 4,315,989 and U.S. Pat. No. 4,187,292). 18,21-Dihydromacbecin is characterized by containing the dihydroquinone form of the aromatic nucleus. Both macbecin and 18,21-dihydromacbecin were shown to possess similar antibacterial and antitumour activities against cancer cell lines such as the murine leukaemia P388 cell line (Ono et al., 1982). Reverse transcriptase and terminal deoxynucleotidyl transferase activities were not inhibited by macbecin (Ono et al., 1982). The Hsp90 inhibitory function of macbecin has been reported in the literature (Bohen, 1998; Liu et al., 1999). The conversion of macbecin and 18,21-dihydromacbecin after adding to a microbial culture broth into a compound with a hydroxy group instead of a methoxy group at a certain position or positions is described in U.S. Pat. No. 4,421,687 and U.S. Pat. No. 4,512,975.
[0012]During a screen of a large variety of soil microorganisms, the compounds TAN-420A to E were identified from producer strains belonging to the genus Streptomyces (7-11, EP 0 110 710).
##STR00002##
[0013]In 2000, the isolation of the geldanamycin related, non-benzoquinone ansamycin metabolite reblastin from cell cultures of Streptomyces sp. S6699 and its potential therapeutic value in the treatment of rheumatoid arthritis was described (Stead et al., 2000).
[0014]A further Hsp90 inhibitor, distinct from the chemically unrelated benzoquinone ansamycins is Radicicol (monorden) which was originally discovered for its antifungal activity from the fungus Monosporium bonorden (for review see Uehara, 2003) and the structure was found to be identical to the 14-membered macrolide isolated from Nectria radicicola. In addition to its antifungal, antibacterial, anti-protozoan and cytotoxic activity it was subsequently identified as an inhibitor of Hsp90 chaperone proteins (for review see Uehara, 2003; Schulte et al., 1999). The anti-angiogenic activity of radicicol (Hur et al., 2002) and semi-synthetic derivates thereof (Kurebayashi et al., 2001) has also been described.
[0015]Recent interest has focussed on 17-amino derivatives of geldanamycin as a new generation of ansamycin anticancer compounds (Bagatell and Whitesell, 2004), for example 17-(allylamino)-17-desmethoxy geldanamycin (17-AAG, 12) (Hostein et al., 2001; Neckers, 2002; Nimmanapalli et al., 2003; Vasilevskaya et al., 2003; Smith-Jones et al., 2004) and 17-desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG, 13) (Egorin et al., 2002; Jez et al., 2003). More recently geldanamycin was derivatised on the 17-position to create 17-geldanmycin amides, carbamates, ureas and 17-arylgeldanamycin (Le Brazidec et al., 2003). A library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogues has been reported and tested for their affinity for Hsp90 and water solubility (Tian et al., 2004). A further approach to reduce the toxicity of geldanamycin is the selective targeting and delivering of an active geldanamycin compound into malignant cells by conjugation to a tumour-targeting monoclonal antibody (Mandler et al., 2000).
##STR00003##
[0016]Whilst many of these derivatives exhibit reduced hepatotoxicity they still have only limited water solubility. For example 17-AAG requires the use of a solubilising carrier (e.g. Cremophore®, DMSO-egg lecithin), which itself may result in side-effects in some patients (Hu et al., 2004).
[0017]Most of ansamycin class of Hsp90 inhibitors bear the common structural moiety: the benzoquinone which is a Michael acceptor that can readily form covalent bonds with nucleophiles such as proteins, glutathione, etc. The benzoquinone moiety also undergoes redox equilibrium with dihydroquinone, during which oxygen radicals are formed, which give rise to further unspecific toxicity (Dikalov et al., 2002). For example treatment with geldanamycin can result in induced superoxide production (Sreedhar et al., 2004a).
[0018]Therefore, there remains a need to identify novel ansamycin derivatives devoid of the benzoquinone moiety, which may have utility in the treatment of cancer and/or B-cell malignancies, preferably such ansamycins have improved water solubility, an improved pharmacological profile and reduced side-effect profile for administration. The present invention discloses novel ansamycin analogues generated by genetic engineering of the parent producer strain. In particular the present invention discloses 21-deoxymacbecin analogues, which generally have improved pharmaceutical properties compared with the presently available ansamycins; in particular they show improvements in respect of one or more of the following properties: toxicity, conjugation with nucleophiles such as glutathione, water solubility, metabolic stability, bioavailability and formulation ability. Preferably the 21-deoxymacbecin analogues show improved toxicity and/or water solubility.
SUMMARY OF THE INVENTION
[0019]The inventors of the present invention have made significant effort to clone and elucidate the gene cluster that is responsible for the biosynthesis of macbecin. With this insight, the gene that is responsible for the production of the benzoquinone moiety has been specifically targetted, e.g. by integration into mbcM, targeted deletion of a region of the macbecin cluster including all or part of the mbcM gene optionally followed by insertion of gene(s), or other methods of rendering MbcM non-functional e.g. chemical inhibition, site-directed mutagenesis or mutagenesis of the cell for example by UV, in order to produce novel derivatives devoid of a benzoquinone moiety. Optionally targeted inactivation or deletion of further genes responsible for the post-PKS modifications of macbecin may be carried out. Additionally, some of these genes, but not mbcM may be re-introduced into the cell. The optional targeting of the post-PKS genes may occur via a variety of mechanisms, e.g. by integration, targeted deletion of a region of the macbecin cluster including all or some of the post-PKS genes optionally followed by insertion of gene(s) or other methods of rendering the post-PKS genes or their encoded enzymes non-functional e.g. chemical inhibition, site-directed mutagenesis or mutagenesis of the cell for example by the use of UV radiation. As a result, the present invention provides 21-deoxymacbecin analogues, methods for the preparation of these compounds, and methods for the use of these compounds in medicine or as intermediates in the production of further compounds.
[0020]Therefore, in a first aspect the present invention provides analogues of macbecin which are lacking the oxygen atom usually present at the C21 position, in macbecin this oxygen atom is present as a keto group, in 18,21-dihydromacbecin this oxygen atom is present as a hydroxyl group.
[0021]In a more specific aspect the present invention provides 21-deoxymacbecin analogues according to the formula (I) below, or a pharmaceutically acceptable salt thereof:
##STR00004##
wherein:
[0022]R1 represents H, OH or OCH3
[0023]R2 represents H or CH3
[0024]R3 and R4 either both represent H or together they represent a bond (i.e. C4 to C5 is a double bond)
[0025]R5 represents H or --C(O)--NH2
[0026]21-deoxymacbecin analogues are also referred to herein as "compounds of the invention" such terms are used interchangeably herein.
[0027]The above structure shows a representative tautomer and the invention embraces all tautomers of the compounds of formula (I) for example keto compounds where enol compounds are illustrated and vice versa.
[0028]The invention embraces all stereoisomers of the compounds defined by structure (I) as shown above.
[0029]In a further aspect, the present invention provides 21-deoxymacbecin analogues such as compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.
DEFINITIONS
[0030]The articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example "an analogue" means one analogue or more than one analogue.
[0031]As used herein the term "analogue(s)" refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).
[0032]As used herein, the term "homologue(s)" refers to a homologue of a gene or of a protein encoded by a gene disclosed herein from either an alternative macbecin biosynthetic cluster from a different macbecin producing strain or a homologue from an alternative ansamycin biosynthetic gene cluster e.g. from geldanamycin, herbimycin or reblastatin. Such homologue(s) encode a protein that performs the same function or can itself perform the same function as said gene or protein in the synthesis of macbecin or a related ansamycin polyketide. Preferably, such homologue(s) have at least 40% sequence identity, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the sequence of the particular gene disclosed herein (Table 3, SEQ ID NO: 17 which is a sequence of all the genes in the cluster, from which the sequences of particular genes may be deduced). Percentage identity may be calculated using any program known to a person of skill in the art such as BLASTn or BLASTp, available on the NCBI website.
[0033]As used herein, the term "cancer" refers to a benign or malignant new growth of cells in skin or in body organs, for example but without limitation, breast, prostate, lung, kidney, pancreas, brain, stomach or bowel. A cancer tends to infiltrate into adjacent tissue and spread (metastasise) to distant organs, for example to bone, liver, lung or the brain. As used herein the term cancer includes both metastatic tumour cell types, such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, gliobastoma, primary liver cancer and ovarian cancer.
[0034]As used herein the term "B-cell malignancies" includes a group of disorders that include chronic lymphocytic leukaemia (CLL), multiple myeloma, and non-Hodgkin's lymphoma (NHL). They are neoplastic diseases of the blood and blood forming organs. They cause bone marrow and immune system dysfunction, which renders the host highly susceptible to infection and bleeding.
[0035]As used herein, the term "bioavailability" refers to the degree to which or rate at which a drug or other substance is absorbed or becomes available at the site of biological activity after administration. This property is dependent upon a number of factors including the solubility of the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc. Various tests for bioavailability that would be familiar to a person of skill in the art are described herein (see also Egorin et al. (2002)).
[0036]The term "water solubility" as used in this application refers to solubility in aqueous media, e.g. phosphate buffered saline (PBS) at pH 7.3. A test for water solubility is given below in the Examples as "water solubility assay".
[0037]As used herein the term "post-PKS genes(s)" refers to the genes required for post-polyketide synthase modifications of the polyketide, for example but without limitation monooxygenases, O-methyltransferases and carbamoyltransferases. Specifically, in the macbecin system these modifying genes include mbcM, mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450.
[0038]The pharmaceutically acceptable salts of compounds of the invention such as the compounds of formula (I) include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. References hereinafter to a compound according to the invention include both compounds of formula (I) and their pharmaceutically acceptable salts.
[0039]As used herein the terms "18,21-dihydromacbecin" and "macbecin II" (the dihydroquinone form of macbecin) are used interchangeably.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040]FIG. 1: Representation of the biosynthesis of macbecin showing the first putative enzyme free intermediate, pre-macbecin and the post-PKS processing to macbecin. The list of PKS processing steps in the figure is not intended to represent the order of events. The following abbreviations are used for particular genes in the cluster: AL0-AHBA loading domain; ACP-Acyl Carrier Protein; KS-β-ketoacylsynthase; AT-acyl transferase; DH-dehydratase; ER-enoyl reductase; KR-β-ketoreductase.
[0041]FIG. 2: Depiction of the sites of post-PKS processing of pre-macbecin to give macbecin.
[0042]FIG. 3: Diagrammatic representation of generation of the engineered strain BIOT-3806 in which plasmid pLSS308 was integrated into the chromosome by homologous recombination resulting in mbcM gene disruption.
[0043]FIG. 4: Sequence alignment of gdmM (SEQ ID NO: 1) and riforf19 (SEQ ID NO: 2). Binding regions of degenerate oligos are underlined.
[0044]FIG. 5: Sequence alignment of gdmM (SEQ ID NO: 1), mbcM fragment (SEQ ID NO: 3, A. mirum) and mbcM gene (SEQ ID NO: 4, A. pretiosum). Binding regions of degenerate oligos are underlined.
[0045]FIG. 6: A: Protein sequence of GdmM (SEQ ID NO: 5) generated by translation of gdmM, B: Protein sequence of Riforf19 (SEQ ID NO: 6) generated by translation of riforf19; this demonstrates the similarity between the protein sequences.
[0046]FIG. 7: Diagrammatic representation of the construction of the in-frame deletion of mbcM described in example 4.
[0047]FIG. 8: A--shows the sequence of the PCR product PCRwv308, SEQ ID NO: 20 B--shows the sequence of the PCR product PCRwv309, SEQ ID NO: 21
[0048]FIG. 9: A: Shows the DNA sequence resulting from the in-frame deletion of 502 amino acids in mbcM as described in example 4 (SEQ ID NO: 26 and 27), Key: 1-21 bp encodes 3' end of the phosphatase of 3-amino-5-hydroxybenzoic acid biosynthesis, 136-68 bp encodes mbcM deletion protein, 161-141 bp encodes 3' end of mbcF. B: shows the amino acid sequence of the protein (SEQ ID NO: 28). The protein sequence is generated from the complement strand shown in FIG. 9A.
[0049]FIG. 10: Diagrammatic representation of the generation of an Actinosynnema pretiosum strain in which the mbcP, mbcP450, mbcMT1 and mbcMT2 genes have been deleted in frame following deletion of mbcM.
[0050]FIG. 11: Sequence of the amplified PCR product 1+2a (SEQ ID NO: 31)
[0051]FIG. 12: Sequence of the amplified PCR product 3b+4 (SEQ ID NO: 34)
[0052]FIG. 13: Graph showing the effect of in vitro combination of compound 14 at 0-80 nM with Mitomycin C at 0-3 μg/ml on the growth of cancer cell line DU145
[0053]FIG. 14: Graph showing the effect of in vitro combination of compound 14 at 0-160 nM with Cyclohexylchloroethylnitrosurea (CCNU) at 0-100 μg/ml on the growth of cancer cell line DU145
[0054]FIG. 15: Graph showing the effect of in vitro combination of compound 14 at 0-160 nM with Ifosfamid at 0-100 μg/ml on the growth of cancer cell line DU145
[0055]FIG. 16: Graph showing the effect of in vitro combination of compound 14 at 0-160 nM with Mitoxantrone at 0-10 μg/ml on the growth of cancer cell line DU145
[0056]FIG. 17: Graph showing the effect of in vitro combination of compound 14 at 0-160 nM with Vindesine at 0-1 μg/ml on the growth of cancer cell line DU145
DESCRIPTION OF THE INVENTION
[0057]The present invention provides 21-deoxymacbecin analogues, as set out above, methods for the preparation of these compounds, methods for the use of these compounds in medicine and the use of these compounds as intermediates or templates for further semi-synthetic derivatisation or derivatisation by biotransformation methods.
[0058]Preferably R1 represents H or OH. In one embodiment of the invention R1 represents H.
[0059]In another embodiment of the invention R1 represents OH.
[0060]Preferably R2 represents H
[0061]Preferably R3 and R4 both represent H
[0062]Preferably R5 represents --C(O)--NH2
[0063]In one preferred embodiment of the invention R1 represents H, R2 represents H, R3 and
[0064]R4 both represent H and R5 represents --C(O)--NH2
[0065]In another preferred embodiment of the invention R1 represents OH, R2 represents H, R3 and R4 both represent H and R5 represents --C(O)--NH2.
[0066]The preferred stereochemistry of the non-hydrogen sidechains to the ansa ring is as shown in structure (15) below and in FIGS. 1 and 2 below (that is to say the preferred stereochemistry follows that of macbecin).
[0067]The present invention also provides a pharmaceutical composition comprising a 21-deoxymacbecin analogue, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
[0068]The present invention also provides for the use of a 21-deoxymacbecin analogue as a substrate for further modification either by biotransformation or by synthetic chemistry.
[0069]Some existing ansamycin Hsp90 inhibitors that are in or have been in clinical trials, such as geldanamycin and 17-AAG, have poor pharmacological profiles, poor water solubility and poor bioavailability. The present invention provides novel 21-deoxymacbecin analogues which have improved properties such as water solubility. A person of skill in the art will be able to readily determine the water solubility of a given compound of the invention using standard methods. A representative method is shown in the examples herein.
[0070]In one aspect the present invention provides for the use of a 21-deoxymacbecin analogue in the manufacture of a medicament. In a further embodiment the present invention provides for the use of a 21-deoxymacbecin analogue in the manufacture of a medicament for the treatment of cancer and/or B-cell malignancies. In a further embodiment the present invention provides for the use of a 21-deoxymacbecin analogue in the manufacture of a medicament for the treatment of malaria, fungal infection, diseases of the central nervous system, diseases dependent on angiogenesis, autoimmune diseases and/or as a prophylactic pretreatment for cancer.
[0071]In another aspect the present invention provides for the use of a 21-deoxymacbecin analogue in medicine. In a further embodiment the present invention provides for the use of a 21-deoxymacbecin analogue in the treatment of cancer and/or B-cell malignancies. In a further embodiment the present invention provides for the use of a 21-deoxymacbecin analogue in the manufacture of a medicament for the treatment of malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases and/or as a prophylactic pretreatment for cancer.
[0072]In a further embodiment the present invention provides a method of treatment of cancer and/or B-cell malignancies, said method comprising administering to a patient in need thereof a therapeutically effective amount of a 21-deoxymacbecin analogue. In a further embodiment the present invention provides a method of treatment of malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases and/or a prophylactic pretreatment for cancer, said method comprising administering to a patient in need thereof a therapeutically effective amount of a 21-deoxymacbecin analogue.
[0073]As noted above, compounds of the invention may be expected to be useful in the treatment of cancer and/or B-cell malignancies. Compounds of the invention and especially those which may have good selectivity for Hsp90 and/or a good toxicology profile and/or good pharmacokinetics may also be effective in the treatment of other indications for example, but not limited to malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases such as rheumatoid arthritis or as a prophylactic pretreatment for cancer.
[0074]Diseases of the central nervous system and neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, prion diseases, spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS).
[0075]Diseases dependent on angiogenesis include, but are not limited to, age-related macular degeneration, diabetic retinopathy and various other ophthalmic disorders, atherosclerosis and rheumatoid arthritis.
[0076]Autoimmune diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus and psoriasis.
[0077]"Patient" embraces human and other animal (especially mammalian) subjects, preferably human subjects. Accordingly the methods and uses of the 21-deoxymacbecin analogues of the invention are of use in human and veterinary medicine, preferably human medicine.
[0078]The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method for example but without limitation they may be administered parenterally (including intravenous administration), orally, topically (including buccal, sublingual or transdermal), via a medical device (e.g. a stent), by inhalation, or via injection (subcutaneous or intramuscular). The treatment may consist of a single dose or a plurality of doses over a period of time.
[0079]Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. Thus there is provided a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable diluents or carriers. The diluents(s) or carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Examples of suitable carriers are described in more detail below.
[0080]The compounds of the invention may be administered alone or in combination with other therapeutic agents. Co-administration of two (or more) agents may allow for significantly lower doses of each to be used, thereby reducing the side effects seen. It might also allow resensitisation of a disease, such as cancer, to the effects of a prior therapy to which the disease has become resistant. There is also provided a pharmaceutical composition comprising a compound of the invention and a further therapeutic agent together with one or more pharmaceutically acceptable diluents or carriers.
[0081]In a further aspect, the present invention provides for the use of a compound of the invention in combination therapy with a second agent eg a second agent for the treatment of cancer or B-cell malignancies such as a cytotoxic or cytostatic agent.
[0082]In one embodiment, a compound of the invention is co-administered with another therapeutic agent eg a therapeutic agent such as a cytotoxic or cytostatic agent for the treatment of cancer or B-cell malignancies. Exemplary further agents include cytotoxic agents such as alkylating agents and mitotic inhibitors (including topoisomerase II inhibitors and tubulin inhibitors). Other exemplary further agents include DNA binders, antimetabolites and cytostatic agents such as protein kinase inhibitors and tyrosine kinase receptor blockers. Suitable agents include, but are not limited to, methotrexate, leukovorin, prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin (adriamycin), tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g. trastuzumab, trade name Herceptin®), capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g. gefitinib, trade name Iressa®, erlotinib, trade name Tarceva®, cetuximab, trade name Erbitux®), VEGF inhibitors (e.g. bevacizumab, trade name Avastin®), proteasome inhibitors (e.g. bortezomib, trade name Velcade®) or imatinib, trade name Glivec®. Further suitable agents include, but are not limited to, conventional chemotherapeutics such as cisplatin, cytarabine, cyclohexylchloroethylnitrosurea, gemcitabine, Ifosfamid, leucovorin, mitomycin, mitoxantone, oxaliplatin, taxanes including taxol and vindesine; hormonal therapies; monoclonal antibody therapies; protein kinase inhibitors such as dasatinib, lapatinib; histone deacetylase (HDAC) inhibitors such as vorinostat; angiogenesis inhibitors such as sunitinib, sorafenib, lenalidomide; and mTOR inhibitors such as temsirolimus. Additionally, a compound of the invention may be administered in combination with other therapies including, but not limited to, radiotherapy or surgery.
[0083]The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
[0084]The compounds of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.
[0085]For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
[0086]Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
[0087]Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
[0088]A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
[0089]Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
[0090]Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
[0091]It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
[0092]Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, dusting powders, and the like. These compositions may be prepared via conventional methods containing the active agent. Thus, they may also comprise compatible conventional carriers and additives, such as preservatives, solvents to assist drug penetration, emollient in creams or ointments and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the composition. More usually they will form up to about 80% of the composition. As an illustration only, a cream or ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.
[0093]Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active agent may be delivered from the patch by iontophoresis.
[0094]For applications to external tissues, for example the mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active agent may be employed with either a paraffinic or a water-miscible ointment base.
[0095]Alternatively, the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
[0096]For parenteral administration, fluid unit dosage forms are prepared utilizing the active ingredient and a sterile vehicle, for example but without limitation water, alcohols, polyols, glycerine and vegetable oils, water being preferred. The active ingredient, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the active ingredient can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
[0097]Advantageously, agents such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.
[0098]Parenteral suspensions are prepared in substantially the same manner as solutions, except that the active ingredient is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The active ingredient can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient.
[0099]The compounds of the invention may also be administered using medical devices known in the art. For example, in one embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163; U.S. Pat. No. 5,383,851; U.S. Pat. No. 5,312,335; U.S. Pat. No. 5,064,413; U.S. Pat. No. 4,941,880; U.S. Pat. No. 4,790,824; or U.S. Pat. No. 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.
[0100]The dosage to be administered of a compound of the invention will vary according to the particular compound, the disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration. The appropriate dosage can be readily determined by a person skilled in the art.
[0101]The compositions may contain from 0.1% by weight, preferably from 5-60%, more preferably from 10-30% by weight, of a compound of invention, depending on the method of administration.
[0102]It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.
[0103]In a further aspect the present invention provides methods for the production of 21-deoxymacbecin analogues.
[0104]Macbecin can be considered to be biosynthesised in two stages. In the first stage the core-PKS genes assemble the macrolide core by the repeated assembly of 2-carbon units which are then cyclised to form the first enzyme-free intermediate "pre-macbecin", see FIG. 1. In the second stage a series of "post-PKS" tailoring enzymes (e.g. P450 monooxygenases, methyltransferases, FAD-dependent oxygenases and a carbamoyltransferase) act to add the various additional groups to the pre-macbecin template resulting in the final parent compound structure, see FIG. 2. The 21-deoxymacbecin analogues may be biosynthesised in a similar manner.
[0105]This biosynthetic production may be exploited by genetic engineering of suitable producer strains to result in the production of novel compounds. In particular, the present invention provides a method of producing 21-deoxymacbecin analogues said method comprising:
[0106]a) providing a first host strain that produces macbecin or an analogue thereof when cultured under appropriate conditions
[0107]b) deleting or inactivating one or more post-PKS genes, wherein at least one of those post-PKS genes is mbcM, or a homologue thereof
[0108]c) culturing said modified host strain under suitable conditions for the production of 21-deoxymacbecin analogues; and
[0109]d) optionally isolating the compounds produced.
[0110]In step (a) by "macbecin or an analogue thereof" is meant macbecin or those analogues of macbecin that are embraced by the definitions of R1-R5.
[0111]In step (b), deleting or inactivating one or more post-PKS genes, wherein at least one of those post-PKS genes is mbcM, or a homologue thereof will suitably be done selectively.
[0112]In a further embodiment, step b) comprises inactivating mbcM (or a homologue thereof) by integration of DNA into the mbcM gene (or a homologue thereof) such that functional mbcM protein is not produced. In an alternative embodiment, step b) comprises making a targeted deletion of the mbcM gene, or a homologue thereof. In a further embodiment mbcM, or a homologue thereof, is inactivated by site-directed mutagenesis. In a further embodiment the host strain of step a) is subjected to mutagenesis and a modified strain is selected in which one or more of the post-PKS enzymes is not functional, wherein at least one of these is MbcM. The present invention also encompasses mutations of the regulators controlling the expression of mbcM, or a homologue thereof, a person of skill in the art will appreciate that deletion or inactivation of a regulator may have the same outcome as deletion or inactivation of the gene.
[0113]In a particular embodiment of the present invention, a method of selectively deleting or inactivating a post PKS gene comprises:
[0114](i) designing degenerate oligos based on homologue(s) of the gene of interest (e.g. from the geldanamycin PKS biosynthetic cluster and/or from the rifamycin biosynthetic cluster) and isolating the internal fragment of the gene of interest (e.g. mbcM) from a suitable macbecin producing strain, by using these primers in a PCR reaction;
[0115](ii) integrating a plasmid containing this fragment into either the same, or a different macbecin producing strain followed by homologous recombination, which results in the disruption of the targeted gene (e.g. mbcM or a homologue thereof),
[0116](iii) culturing the strain thus produced under conditions suitable for the production of the macbecin analogues, i.e. 21-deoxymacbecin analogues.
In a specific embodiment, the macbecin-producing strain in step (i) is Actinosynnema mirum (A. mirum). In a further specific embodiment the macbecin-producing strain in step (ii) is A. pretiosum
[0117]A person of skill in the art will appreciate that an equivalent strain may be achieved using alternative methods to that described above, e.g.: [0118]Degenerate oligos may be used to amplify the gene of interest from other macbecin producing strains for example, but not limited to A. pretiosum, or A. mirum [0119]Different degenerate oligos may be designed which will successfully amplify an appropriate region of the mcbM gene of a macbecin producer, or a homologue thereof. [0120]The sequence of the mbcM gene of the A. pretiosum strain may be used to generate the oligos which may be specific to the mbcM gene of A. pretiosum and then the internal fragment may be amplified from any macbecin producing strain e.g A. pretiosum or Actinosynnema mirum (A. mirum). [0121]The sequence of the mbcM gene of the A. pretiosum strain may be used along with the sequence of homologous genes to generate degenerate oligos to the mbcM gene of A. pretiosum and then the internal fragment may be amplified from any macbecin producing strain e.g A. pretiosum or A. mirum.
[0122]In further aspects of the invention, additional post-PKS genes may also be deleted or inactivated in addition to mbcM. FIG. 2 shows the activity of the post-PKS genes in the macbecin biosynthetic cluster. A person of skill in the art would thus be able to identify which additional post-PKS genes would need to be deleted or inactivated in order to arrive at a strain that will produce the compound(s) of interest.
[0123]In further aspects of the invention, an engineered strain in which one or more post-PKS genes including mbcM have been deleted or inactivated as above, has re-introduced into it one or more of the same post PKS genes not including mbcM, or homologues thereof, e.g. from an alternative macbecin producing strain, or even from the same strain.
[0124]Thus according to a further aspect of the invention there is provided a method for the production of a 21-deoxymacbecin analogue, said method comprising:
[0125]a) providing a first host strain that produces macbecin when cultured under appropriate conditions
[0126]b) deleting or inactivating one or more post-PKS genes, wherein at least one of the post-PKS genes is mbcM, or a homologue thereof,
[0127]c) re-introducing some or all of the post-PKS genes not including mbcM.
[0128]d) culturing said modified host strain under suitable conditions for the production of 21-deoxymacbecin analogues; and
[0129]e) optionally isolating the compounds produced.
[0130]In a further embodiment an engineered strain in which one or more post-PKS genes including mbcM have been deleted or inactivated is complemented by one or more of the post PKS genes from a heterologous PKS cluster including, but not limited to the clusters directing the biosynthesis of rifamycin, ansamitocin, geldanamycin or herbimycin.
[0131]Specifically, the host strain may be an engineered strain based on a macbecin producing strain in which mbcM has been deleted or inactivated. Alternatively the host strain may be an engineered strain based on a macbecin producing strain in which mbcM, mbcMT1, mbcMT2, mbcP and mbcP450 have been deleted or inactivated.
[0132]It may be observed in these systems that when a strain is generated in which mbcM, or a homologue thereof, does not function as a result of one of the methods described including inactivation or deletion, that more than one macbecin analogue may be produced. There are a number of possible reasons for this which will be appreciated by those skilled in the art. For example there may be a preferred order of post-PKS steps and removing a single activity leads to all subsequent steps being carried out on substrates that are not natural to the enzymes involved. This can lead to intermediates building up in the culture broth due to a lowered efficiency towards the novel substrates presented to the post-PKS enzymes, or to shunt products which are no longer substrates for the remaining enzymes possibly because the order of steps has been altered.
[0133]The ratio of compounds observed in a mixture can be manipulated by using variations in the growth conditions such as the setting of revolutions per minute (rpm) in the shaking incubator, and the throw of the shaking incubator. As described in the examples, incubation of production cultures of BIOT-3806 in an incubator with a smaller throw (2.5 cm) but higher rpm (300) lead to a bias towards less processed analogues whilst a parallel experiment in an incubator with a wider throw (5 cm) and lower rpm (200 or 250) lead to a bias towards more processed intermediates.
[0134]One skilled in the art will appreciate that in a biosynthetic cluster some genes are organised in operons and disruption of one gene will often have an effect on expression of subsequent genes in the same operon.
[0135]When a mixture of compounds is observed these can be readily separated using standard techniques some of which are described in the following examples.
[0136]21-Deoxymacbecin analogues may be screened by a number of methods, as described herein, and in the circumstance where a single compound shows a favourable profile a strain can be engineered to make this compound preferably. In the unusual circumstance when this is not possible, an intermediate can be generated which is then biotransformed to produce the desired compound.
[0137]The present invention provides novel macbecin analogues generated by the selected deletion or inactivation of one or more post-PKS genes from the macbecin PKS gene cluster. In particular, the present invention relates to novel 21-deoxymacbecin analogues produced by the selected deletion or inactivation of at least mbcM, or a homologue thereof, from the macbecin biosynthetic gene cluster. In one embodiment, mbcM, or a homologue thereof, alone is deleted or inactivated. In an alternative embodiment, other post-PKS genes in addition to mbcM are additionally deleted or inactivated. In a specific embodiment, additional genes selected from the group consisting of: mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are deleted or inactivated in the host strain. In a further embodiment, additionally 1 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are deleted or inactivated. In a further embodiment, additionally 2 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are deleted or inactivated. In a further embodiment, additionally 3 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are deleted or inactivated. In a further embodiment, additionally 4 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are deleted or inactivated.
[0138]A person of skill in the art will appreciate that a gene does not need to be completely deleted for it to be rendered non-functional, consequentially the term "deleted or inactivated" as used herein encompasses any method by which a gene is rendered non-functional including but not limited to: deletion of the gene in its entirety, inactivation by insertion into the target gene, site-directed mutagenesis which results in the gene either not being expressed or being expressed in an inactive form, mutagenesis of the host strain which results in the gene either not being expressed or being expressed in an inactive form (e.g. by radiation or exposure to mutagenic chemicals, protoplast fusion or transposon mutagenesis). Further it includes deletion of an internal fragment of the gene. Alternatively the function of an active gene can be impaired chemically with inhibitors, for example metapyrone (alternative name 2-methyl-1,2-di(3-pyridyl-1-propanone), EP 0 627 009) and ancymidol are inhibitors of oxygenases and these compounds can be added to the production medium to generate analogues. Additionally, sinefungin is a methyl transferase inhibitor that can be used similarly but for the inhibition of methyl transferase activity in vivo (McCammon and Parks 1981).
[0139]In an alternative embodiment, all of the post-PKS genes may be deleted or inactivated and then one or more of the genes, but not including mbcM, or a homologue thereof, may then be reintroduced by complementation (e.g. at an att site, on a self-replicating plasmid or by insertion into a homologous region of the chromosome). Therefore, in a particular embodiment the present invention relates to methods for the generation of 21-deoxymacbecin analogues, said method comprising:
[0140]a) providing a first host strain that produces macbecin when cultured under appropriate conditions
[0141]b) selectively deleting or inactivating all the post-PKS genes,
[0142]c) culturing said modified host strain under suitable conditions for the production of 21-deoxymacbecin analogues; and
[0143]d) optionally isolating the compounds produced.
[0144]In an alternative embodiment, one or more of the deleted post-PKS genes are reintroduced, provided that mbcM is not one of the genes reintroduced. In a further embodiment, 1 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are reintroduced. In a further embodiment, 2 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are reintroduced. In a further embodiment, 3 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are reintroduced. In a further embodiment, 4 or more of the post-PKS genes selected from the group consisting of mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are reintroduced. In a further alternative embodiment, mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450 are reintroduced.
[0145]Additionally, it will be apparent to a person of skill in the art that a subset of the post-PKS genes, including mbcM, or a homologue thereof, could be deleted or inactivated and a smaller subset of said post-PKS genes not including mbcM could be reintroduced to arrive at a strain producing 21-deoxymacbecin analogues.
[0146]A person of skill in the art will appreciate that there are a number of ways to generate a strain that contains the biosynthetic gene cluster for macbecin but that is lacking at least mbcM, or a homologue thereof.
[0147]It is well known to those skilled in the art that polyketide gene clusters may be expressed in heterologous hosts (Pfeifer and Khosla, 2001). Accordingly, the present invention includes the transfer of the macbecin biosynthetic gene cluster without mbcM or with a non-functional mutant of mbcM, with or without resistance and regulatory genes, either otherwise complete or containing additional deletions, into a heterologous host. Alternatively, the complete macbecin biosynthetic cluster can be transferred into a heterologous host, with or without resistance and regulatory genes, and it can then be manipulated by the methods described herein to delete or inactivate mbcM. Methods and vectors for the transfer as defined above of such large pieces of DNA are well known in the art (Rawlings, 2001; Staunton and Weissman, 2001) or are provided herein in the methods disclosed. In this context a preferred host cell strain is a prokaryote, more preferably an actinomycete or Escherichia coli, still more preferably preferred host cell strains include, but are not limited to Actinosynnema mirum (A. mirum), Actinosynnema pretiosum subsp. pretiosum (A. pretiosum), S. hygroscopicus, S. hygroscopicus sp., S. hygroscopicus var. ascomyceticus, Streptomyces tsukubaensis, Streptomyces coelicolor, Streptomyces lividans, Saccharopolyspora erythraea, Streptomyces fradiae, Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces rimosus, Streptomyces albus, Streptomyces griseofuscus, Streptomyces longisporoflavus, Streptomyces venezuelae, Streptomyces albus, Micromonospora sp., Micromonospora griseorubida, Amycolatopsis mediteffanei or Actinoplanes sp. N902-109. Further examples include Streptomyces hygroscopicus subsp. geldanus and Streptomyces violaceusniger.
[0148]In one embodiment the entire biosynthetic cluster is transferred. In an alternative embodiment the entire PKS without mbcM is transferred. In an alternative embodiment the entire PKS is transferred without any of the associated post-PKS genes, including mbcM.
[0149]In a further embodiment the entire macbecin biosynthetic cluster is transferred and then manipulated according to the description herein.
[0150]In an alternative aspect of the invention, the 21-deoxymacbecin analogue of the present invention may be further processed by biotransformation with an appropriate strain. The appropriate strain either being an available wild type strain for example, but without limitation Actinosynnema mirum, Actinosynnema pretiosum subsp. pretiosum, S. hygroscopicus, S. hygroscopicus sp. Alternatively, an appropriate strain may be a engineered to allow biotransformation with particular post-PKS enzymes for example, but without limitation, those encoded by mbcN, mbcP, mbcMT1, mbcMT2, mbcP450 (as defined herein), gdmN, gdmM, gdmL, gdmP, (Rascher et al., 2003) the geldanamycin 17-O-methyl transferase, asm7, asm10, asm11, asm12, asm19 and asm21 (Cassady et al., 2004, Spiteller et al., 2003). Where genes have yet to be identified or the sequences are not in the public domain it is routine to those skilled in the art to acquire such sequences by standard methods. For example the sequence of the gene encoding the geldanamycin 17-O-methyl transferase is not in the public domain, but one skilled in the art could generate a probe, either a heterologous probe using a similar O-methyl transferase, or a homologous probe by designing degenerate primers from available homologous genes to carry out Southern blots on a geldanamycin producing strain and thus acquire this gene to generate biotransformation systems.
[0151]In a particular embodiment the strain may have had one or more of its native polyketide clusters deleted, either entirely or in part, or otherwise inactivated, so as to prevent the production of the polyketide produced by said native polyketide cluster. Said engineered strain may be selected from the group including, for example but without limitation, Actinosynnema mirum, Actinosynnema pretiosum subsp. pretiosum, S. hygroscopicus, S. hygroscopicus sp., S. hygroscopicus var. ascomyceticus, Streptomyces tsukubaensis, Streptomyces coelicolor, Streptomyces lividans, Saccharopolyspora erythraea, Streptomyces fradiae, Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces rimosus, Streptomyces albus, Streptomyces griseofuscus, Streptomyces longisporoflavus, Streptomyces venezuelae, Micromonospora sp., Micromonospora griseorubida, Amycolatopsis mediteffanei or Actinoplanes sp. N902-109. Further possible strains include Streptomyces hygroscopicus subsp. geldanus and Streptomyces violaceusniger.
[0152]In a further aspect the present invention provides host strains which naturally produce macbecin or analogue thereof, in which the mbcM gene, or a homologue thereof, has been deleted or inactivated such that it thereby produces 21-deoxymacbecin or an analogue thereof (e.g. a 21-deoxymacbecin analogue as defined by compounds of formula (I)) and their use in the production of 21-deoxymacbecin or analogues thereof.
[0153]Therefore, in one embodiment the present invention provides a genetically engineered strain which naturally produces macbecin in its unaltered state, said strain having one or more post-PKS genes from the macbecin PKS gene cluster deleted wherein one of said deleted or inactivated post-PKS genes is mbcM, or a homologue thereof.
[0154]The invention embraces all products of the inventive processes described herein.
[0155]Although the process for preparation of the 21-deoxymacbecin analogues of the invention as described above is substantially or entirely biosynthetic, it is not ruled out to produce or interconvert 21-deoxymacbecin analogues of the invention by a process which comprises standard synthetic chemical methods.
[0156]In order to allow for the genetic manipulation of the macbecin biosynthetic gene cluster, first the gene cluster was sequenced from Actinosynnema pretiosum subsp. pretiosum however, a person of skill in the art will appreciate that there are alternative strains which produce macbecin, for example but without limitation Actinosynnema mirum. The macbecin biosynthetic gene cluster from these strains may be sequenced as described herein for Actinosynnema pretiosum subsp. pretiosum, and the information used to generate equivalent strains.
[0157]Further aspects of the invention include: [0158]An engineered strain based on a macbecin producing strain in which mbcM and optionally further post-PKS genes have been deleted or inactivated, particularly such an engineered strain in which mbcM has been deleted or inactivated or such an engineered strain in which mbcM, mbcMT1, mbcMT2, mbcP and mbcP450 have been deleted or inactivated. Suitably the macbecin producing strain is A pretiosum or A mirum. [0159]Use of such an engineered strain in the preparation of a 21-deoxymacbecin analogue.
[0160]Compounds of the invention are advantageous in that they may be expected to have one or more of the following properties: tight binding to Hsp90, fast on-rate of binding to Hsp90, good solubility, good stability, good formulation ability, good oral bioavailability, good pharmacokinetic properties including but not limited to low glucuronidation, good cell up-take, good brain pharmacokinetics, low binding to erythrocytes, good toxicology profile, good hepatotoxicity profile, good nephrotoxicity, low side effects and low cardiac side effects.
EXAMPLES
General Methods
Fermentation of Cultures
[0161]Conditions used for growing the bacterial strains Actinosynnema pretiosum subsp. pretiosum ATCC 31280 (U.S. Pat. No. 4,315,989) and Actinosynnema mirum DSM 43827 (KCC A-0225, Watanabe et al., 1982) were described in the U.S. Pat. No. 4,315,989 and U.S. Pat. No. 4,187,292. Methods used herein were adapted from these patents and are as follows for culturing of broths in tubes or flasks in shaking incubators, variations to the published protocols are indicated in the examples. Both strains were grown on ISP2 agar (Medium 3, Shirling, E. B. and Gottlieb, D., 1966) at 28° C. for 2-3 days and used to inoculate seed medium (Medium 1, see below adapted from U.S. Pat. No. 4,315,989 and U.S. Pat. No. 4,187,292). The inoculated seed medium was then incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw at 28° C. for 48 h. For production of macbecin, 18,21-dihydromacbecin and macbecin analogues such as 21-deoxymacbecins the fermentation medium (Medium 2, see below and U.S. Pat. No. 4,315,989 and U.S. Pat. No. 4,187,292) was inoculated with 2.5%-10% of the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw initially at 28° C. for 24 h followed by 26° C. for four to six days. The culture was then harvested for extraction.
Media
Medium 1--Seed Medium
[0162]In 1 L of distilled water
TABLE-US-00001 Glucose 20 g Soluble potato starch (Sigma) 30 g Spray dried corn steep liquor (Roquette Freres) 10 g `Nutrisoy` toasted soy flour (Archer Daniels Midland) 10 g Peptone from milk solids (Sigma) 5 g NaCl 3 g CaCO3 5 g Adjust pH with NaOH 7.0
Sterilisation by autoclaving at 121° C. for 20 minutes.Apramycin was added when appropriate after autoclaving to give a final concentration of 50 mg/L.
Medium 2--Fermentation Medium
[0163]In 1 L of distilled water
TABLE-US-00002 Glycerol 50 g Spray dried corn steep liquor (Roquette Freres) 10 g `Bacto` yeast extract (Difco) 20 g KH2PO4 20 g MgCl2•6H2O 5 g CaCO3 1 g Adjust pH with NaOH 6.5
Sterilisation by autoclaving at 121° C. for 20 minutes.
Medium 3--ISP2 Medium
[0164]In 1 L of distilled water
TABLE-US-00003 Malt extract 10 g Yeast extract 4 g Dextrose 4 g Agar 15 g Adjust pH with NaOH 7.3
Sterilisation by autoclaving at 121° C. for 20 minutes.
Medium 4--MAM
[0165]In 1 L of distilled water
TABLE-US-00004 Wheat starch 10 g Corn steep solids 2.5 g Yeast extract 3 g CaCO3 3 g Iron sulphate 0.3 g Agar 20 g
Sterilisation by autoclaving at 121° C. for 20 minutes.
Extraction of Culture Broths for LCMS Analysis
[0166]Culture broth (1 mL) and ethyl acetate (1 mL) was added and mixed for 15-30 min followed by centrifugation for 10 min. 0.5 mL of the organic layer was collected, evaporated to dryness and then re-dissolved in 0.25 mL of methanol.
[0167]LCMS analysis procedure for fermentation broth analysis and in vivo transformation studies LCMS was performed using an integrated Agilent HP1100 HPLC system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive and/or negative ion mode. Chromatography was achieved over a Phenomenex Hyperclone column (C18 BDS, 3u, 150×4.6 mm) eluting over 11 min at a flow rate of 1 mL/min with a linear gradient from acetonitrile+0.1% formic acid/water+0.1% formic acid (40/60) to acetonitrile+0.1% formic acid/water+0.1% formic acid (80/20). UV spectra were recorded between 190 and 400 nm, with extracted chromatograms taken at 210, 254 and 276 nm. Mass spectra were recorded between 100 and 1500 amu.
In Vitro Bioassay for Anticancer Activity
[0168]In vitro evaluation of compounds for anticancer activity in a panel of 38 human tumour cell lines in a monolayer proliferation assay was carried out at the Oncotest Testing Facility, Institute for Experimental Oncology, Oncotest GmbH, Freiburg. The characteristics of 9 of the selected cell lines are summarised in Table 1.
TABLE-US-00005 TABLE 1 Test cell lines # Cell line Characteristics 1 MCF-7 Breast, NCI standard 2 NCI-H460 Lung, NCI standard 3 SF-268 CNS, NCI standard 4 OVCAR-3 Ovarian - p85 mutated. AKT amplified. 5 GXF 251L Gastric 6 MEXF 394NL Melanoma 7 UXF 1138L Uterus 8 LNCAP Prostate - PTEN negative 9 DU145 Prostate - PTEN positive
[0169]The Oncotest cell lines are established from human tumor xenografts as described by Roth et al., (1999). The origin of the donor xenografts was described by Fiebig et al., (1999). Other cell lines are either obtained from the NCl (H460, SF-268, OVCAR-3, DU145, MDA-MB-231, MDA-MB-468) or purchased from DSMZ, Braunschweig, Germany (LNCAP).
[0170]All cell lines, unless otherwise specified, were grown at 37° C. in a humidified atmosphere (95% air, 5% CO2) in a `ready-mix` medium containing RPMI 1640 medium, 10% fetal calf serum, and 0.1 mg/mL gentamicin (PAA, Colbe, Germany).
[0171]A modified propidium iodide assay was used to assess the effects of the test compound(s) on the growth of human tumour cell lines (Dengler et al., (1995)).
[0172]Briefly, cells were harvested from exponential phase cultures by trypsinization, counted and plated in 96 well flat-bottomed microtitre plates at a cell density dependent on the cell line (5-10.000 viable cells/well). After 24 h recovery to allow the cells to resume exponential growth, 0.010 mL of culture medium (6 control wells per plate) or culture medium containing a 21-deoxymacbecin analogue was added to the wells. Each concentration was plated in triplicate. Compounds were applied in five concentrations (100; 10; 1; 0.1 and 0.01 μM). Following 4 days of continuous exposure, cell culture medium with or without test compound was replaced by 0.2 mL of an aqueous propidium iodide (PI) solution (7 mg/L). To measure the proportion of living cells, cells were permeabilized by freezing the plates. After thawing the plates, fluorescence was measured using the Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm), giving a direct relationship to the total number of viable cells.
[0173]Growth inhibition was expressed as treated/control×100 (% T/C). For active compounds, IC70 values were estimated by plotting compound concentration versus cell viability.
In Vitro Bioassay for Anticancer Activity in Combination
[0174]A modified propidium iodide assay was also used to assess the effects of combined applications of the compounds on the growth of the DU145 cells as described above, except that for each standard agent 4 plates were prepared: one for the standard agent alone and three plates for the combinations of the standard agent with 3 different fixed concentrations of compound 14, respectively. Standard agents were applied at 10 concentrations in sextuplicates in half-log increments. Untreated cells, as well as cells incubated with the compound 14 alone were covered with 6 wells/plate, respectively. Growth inhibition is expressed as treated/control×100 (% T/C), and IC70 values for each combination were determined by plotting compound concentration versus cell viability.
Example 1
Sequencing of the Macbecin Biosynthetic Gene Cluster
[0175]Genomic DNA was isolated from Actinosynnema pretiosum (ATCC 31280) and Actinosynnema mirum (DSM 43827, ATCC 29888) using standard protocols described in Kieser et al., (2000) DNA sequencing was carried out by the sequencing facility of the Biochemistry Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW using standard procedures.
[0176]Primers BIOSG104 5'-GGTCTAGAGGTCAGTGCCCCCGCGTACCGTCGT-3' (SEQ ID NO: 7) AND BIOSG105 5'-GGCATATGCTTGTGCTCGGGCTCAAC-3' (SEQ ID NO: 8) were employed to amplify the carbamoyltransferase-encoding gene gdmN from the geldanamycin biosynthetic gene cluster of Streptomyces hygroscopicus NRRL 3602 (Accession number of sequence: AY179507) using standard techniques. Southern blot experiments were carried out using the DIG Reagents and Kits for Non-Radioactive Nucleic Acid Labelling and Detection according to the manufacturers' instructions (Roche). The DIG-labeled gdmN DNA fragment was used as a heterologous probe. Using the gdmN generated probe and genomic DNA isolated from A. pretiosum 2112 an approximately 8 kb EcoRI fragment was identified in Southern Blot analysis. The fragment was cloned into Litmus 28 applying standard procedures and transformants were identified by colony hybridization. The clone p3 was isolated and the approximately 7.7 kb insert was sequenced. DNA isolated from clone p3 was digested with EcoRI and EcoRI/SacI and the bands at around 7.7 kb and at about 1.2 kb were isolated, respectively. Labelling reactions were carried out according to the manufacturers' protocols. Cosmid libraries of the two strains named above were created using the vector SuperCos 1 and the Gigapack III XL packaging kit (Stratagene) according to the manufacturers' instructions. These two libraries were screened using standard protocols and as a probe, the DIG-labelled fragments of the 7.7 kb EcoRI fragment derived from clone p3 were used. Cosmid 52 was identified from the cosmid library of A. pretiosum and submitted for sequencing to the sequencing facility of the Biochemistry Department of the University of Cambridge. Similarly, cosmid 43 and cosmid 46 were identified from the cosmid library of A. mirum. All three cosmids contain the 7.7 kb EcoRI fragment as shown by Southern Blot analysis.
[0177]An around 0.7 kbp fragment of the PKS region of cosmid 43 was amplified using primers BIOSG124 5'-CCCGCCCGCGCGAGCGGCGCGTGGCCGCCCGAGGGC-3' (SEQ ID NO: 9) and BIOSG125 5'-GCGTCCTCGCGCAGCCACGCCACCAGCAGCTCCAGC-3' (SEQ ID NO:10) applying standard protocols, cloned and used as a probe for screening the A. pretiosum cosmid library for overlapping clones. The sequence information of cosmid 52 was also used to create probes derived from DNA fragments amplified by primers BIOSG130 5'-CCAACCCCGCCGCGTCCCCGGCCGCGCCGAACACG-3' (SEQ ID NO: 11) and BIOSG131 5'-GTCGTCGGCTACGGGCCGGTGGGGCAGCTGCTGT-5' (SEQ ID NO: 12) as well as BIOSG132 5'-GTCGGTGGACTGCCCTGCGCCTGATCGCCCTGCGC-3' (SEQ ID NO: 13) and BIOSG133 5'-GGCCGGTGGTGCTGCCCGAGGACGGGGAGCTGCGG-3' (SEQ ID NO: 14) which were used for screening the cosmid library of A. pretiosum. Cosmids 311 and 352 were isolated and cosmid 352 was sent for sequencing. Cosmid 352 contains an overlap of approximately 2.7 kb with cosmid 52. To screen for further cosmids, an approximately 0.6 kb PCR fragment was amplified using primers BIOSG136 5'-CACCGCTCGCGGGGGTGGCGCGGCGCACGACGTGGCTGC-3' (SEQ ID NO: 15) and BIOSG 137 5'-CCTCCTCGGACAGCGCGATCAGCGCCGCGCACAGCGAG-3' (SEQ ID NO: 16) and cosmid 311 as template applying standard protocols. The cosmid library of A. pretiosum was screened and cosmid 410 was isolated. It overlaps approximately 17 kb with cosmid 352 and was sent for sequencing. The sequence of the three overlapping cosmids (cosmid 52, cosmid 352 and cosmid 410) was assembled. The sequenced region spans about 100 kbp and 23 open reading frames were identified potentially constituting the macbecin biosynthetic gene cluster, (SEQ ID NO: 17). The location of each of the open reading frames within SEQ ID NO: 17 is shown in Table 3
TABLE-US-00006 TABLE 2 Summary of the cosmids Cosmid Strain Cosmid 43 Actinosynnema mirum ATCC 29888 Cosmid 46 Actinosynnema mirum ATCC 29888 Cosmid 52 Actinosynnema pretiosum ATCC 31280 Cosmid 311 Actinosynnema pretiosum ATCC 31280 Cosmid 352 Actinosynnema pretiosum ATCC 31280 Cosmid 410 Actinosynnema pretiosum ATCC 31280
TABLE-US-00007 TABLE 3 location of each of the open reading frames within SEQ ID NO: 17 Nucleotide position in Function of the encoded SEQ ID NO: 17 Gene Name protein 14925-17909* mbcRII transcriptional regulator 18025-19074c mbcO aminohydroquinate synthase 19263-20066c* mbc? unknown, AHBA biosynthesis 20330-40657 mbcAI PKS 40654-50859 mbcAII PKS 50867-62491* mbcAIII PKS 62500-63276* mbcF amide synthase 63281-64852* mbcM C21 monooxygenase 64899-65696c* PH phosphatase 65693-66853c* OX oxidoreductase 66891-68057c* Ahs AHBA synthase 68301-68732* Adh ADHQ dehydratase 68690-69661c* AHk AHBA kinase 70185-72194c* mbcN carbamoyltransferase 72248-73339c mbcH methoxymalonyl ACP pathway 73336-74493c mbcI methoxymalonyl ACP pathway 74490-74765c mbcJ methoxymalonyl ACP pathway 74762-75628c* mbcK methoxymalonyl ACP pathway 75881-76537 mbcG methoxymalonyl ACP pathway 76534-77802* mbcP C4,5 monooxygenase 77831-79054* mbcP450 P450 79119-79934* mbcMT1 O-methyltransferase 79931-80716* mbcMT2 O-methyltransferase [Note 1: c indicates that the gene is encoded by the complement DNA strand; Note 2: it is sometimes the case that more than one potential candidate start codon can been identified. One skilled in the art will recognise this and be able to identify alternative possible start codons. We have indicated those genes which have more than one possible start codon with a `*` symbol. Throughout we have indicated what we believe to be the start codon, however, a person of skill in the art will appreciate that it may be possible to generate active protein using an alternative start codon.]
Example 2
Generation of Strain BIOT-3806: an Actinosynnema Pretiosum Strain in which the gdmM Homologue mcbM has been Interrupted by Insertion of a Plasmid
[0178]A summary of the construction of pLSS308 is shown in FIG. 3.
2.1. Construction of Plasmid pLSS308
[0179]The DNA sequences of the gdmM gene from the geldanamycin biosynthetic gene cluster of Streptomyces hygroscopicus strain NRRL 3602 (AY179507) and orf19 from the rifamycin biosynthetic gene cluster of Amycolatopsis mediteffanei (AF040570 AF040571) were aligned using VectorNTI sequence alignment program (FIG. 4). This alignment identified regions of homology that were suitable for the design of degenerate oligos that were used to amplify a fragment of the homologous gene from Actinosynnema mirum (BIOT-3134; DSM43827; ATCC29888). The degenerate oligos are:
TABLE-US-00008 (SEQ ID NO: 18) FPLS1: 5' : ccscgggcgnycngsttcgacngygag 3'; (SEQ ID NO: 19) FPLS3: 5' : cgtcncggannccggagcacatgccctg 3';
where n=G, A, T or C; y=C or T; s=G or C
[0180]The template for PCR amplification was Actinosynnema mirum cosmid 43. The generation of cosmid 43 is described in Example 1 above.
[0181]Oligos FPLS1 and FPLS3 were used to amplify the internal fragment of a gdmM homologue from Actinosynnema mirum in a standard PCR reaction using cosmid 43 as the template and Taq DNA polymerase. The resultant 793 bp PCR product was cloned into pUC19 that had been linearised with SmaI, resulting in plasmid pLSS301. The cloned region was sequenced and was shown to have significant homology to gdmM, (FIG. 5). An alignment of the gene fragment amplified from cosmid 43 (A. mirum) with the sequence of the mbcM gene of the macbecin biosynthetic gene cluster of Actinosynnema pretiosum subsp. pretiosum shows only 1 bp difference between these sequences (excluding the region dictated by the sequence of the degenerate oligos), see FIG. 5. It was postulated that the amplified sequence is from the mcbM gene of the macbecin cluster of A. mirum. Plasmid pLSS301 was digested with EcoRI/HindIII and the fragment cloned into plasmid pKC1132 (Bierman et al., 1992) that had been digested with EcoRI/Hind III. The resultant plasmid, designated pLSS308, is apramycin resistant and contains an internal fragment of the A. mirum mbcM gene.
2.2 Transformation of Actinosynnema pretiosum subsp. pretiosum
[0182]Escherichia coli ET12567, harbouring the plasmid pUZ8002 was transformed with pLSS308 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Actinosynnema pretiosum subsp. pretiosum by vegetative conjugation (Matsushima et al., 1994). Exconjugants were plated on Medium 4 and incubated at 28° C. Plates were overlayed after 24 h with 50 mg/L apramycin and 25 mg/L nalidixic acid. As pLSS308 is unable to replicate in Actinosynnema pretiosum subsp. pretiosum, any apramycin resistant colonies were anticipated to be transformants that contained plasmid integrated into the mbcM gene of the chromosome by homologous recombination via the plasmid borne mcbM internal fragment (FIG. 3). This results in two truncated copies of the mbcM gene on the chromosome. Transformants were confirmed by PCR analysis and the amplified fragment was sequenced.
[0183]Colonies were patched onto Medium 4 (with 50 mg/L apramycin and 25 mg/L nalidixic acid). A 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL seed medium (variant of Medium 1-2% glucose, 3% soluble starch, 0.5% corn steep solids, 1% soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate) plus 50 mg/L apramycin. These seed cultures were incubated for 2 days at 28° C., 200 rpm with a 5 cm throw. These were then used to inoculate (5% v/v) fermentation medium (Medium 2) and were grown at 28° C. for 24 hours and then at 26° C. for a further 5 days. Metabolites were extracted from these according to the standard protocol described above. Samples were assessed for production of macbecin analogues by HPLC using the standard protocol described above.
[0184]The productive isolate selected was designated BIOT-3806.
2.3 Identification of Compounds from BIOT-3806
[0185]LCMS was performed using an Agilent HP1100 HPLC system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive and/or negative ion mode. Chromatography was achieved over a Phenomenex Hyperclone column (C18 BDS, 3u, 150×4.6 mm) eluting at a flow rate of 1 mL/min using the following gradient elution process; T=0, 10% B; T=2, 10% B; T=20, 100% B; T=22, 100% B; T=22.05, 10% B; T=25, 10% B. Mobile phase A=water+0.1% formic acid; mobile phase B=acetonitrile+0.1% formic acid. UV spectra were recorded between 190 and 400 nm, with extracted chromatograms taken at 210, 254 and 276 nm. Mass spectra were recorded between 100 and 1500 amu.
TABLE-US-00009 TABLE 4 compounds identified by LCMS Compound Retention time (min) [M + Na].sup.+ [M - H].sup.- Mass 14 11.4 525.2 501.2 502 15 9.7 541.1 517.1 518 A 8.6 506.1 482.1 483 B 9.3 539.2 515.1 516 C 10.9 543.1 519.2 520
Example 3
Production and Isolation of Novel Compounds
3.1 Fermentation and Isolation of 7-O-carbamoylpre-macbecin
[0186]Vegetative stocks of BIOT-3806 were prepared after growth in Medium 1 with 50 mg/L apramycin, and preserved in 20% w/v glycerol:10% w/v lactose in distilled water and stored at -80° C. Vegetative stocks were recovered onto plates of ISP2 medium (Medium 3) supplemented with 50 mg/L apramycin and incubated for 48 hours at 28° C. Vegetative cultures were prepared by removing two agar plugs, 5 mm in diameter from the ISP2 plate and inoculating them into 30 mL Medium 1 in 250 mL shake flasks containing 50 mg/L apramycin. The flasks were incubated at 28° C., 200 rpm (5 cm throw) for 48 h.
[0187]Vegetative cultures were inoculated at 5% v/v into 200 ml production medium (Medium 2) in 2 L shake flasks. Cultivation was carried out for 1 day at 28° C. followed by 5 days at 26° C., 300 rpm (2.5 cm throw).
[0188]The fermentation broth of BIOT-3806 (1 L, pink colour) was extracted three times with an equal volume of ethyl acetate (EtOAc). The solvent was removed from the combined EtOAc extract in vacuo to yield 2.34 g of brown oil. The extract was then chromatographed over Silica Gel 60 eluting initially with a CHCl3 and MeOH mixture (95:5) followed by an increase in MeOH concentration up to 10% and collection of several fractions (approx. 250 mL per fraction). The fractions were assayed for the presence of metabolites using HPLC. A particular fraction containing a new compound (fraction 5; 334 mg crude mass after removal of solvent) was further purified by chromatography over a Phenomenex Luna C18-BDS column (21.2×250 mm; 5 um particle size) eluting with a gradient of water:acetonitrile (80:20) to (50:50) over a period of 25 min, with a flow rate of 21 mL/min. Fractions were assayed by analytical HPLC and those containing the new compound were combined and the solvents removed to yield an off white solid (86 mg). Analysis by LCMS/MS, and by 1D and 2D NMR experiments carried out in acetone-d6 identified the compound as 7-O-carbamoylpre-macbecin (14)
##STR00005##
3.2 Fermentation and Isolation of 7-O-carbamoyl-15-hydroxypre-macbecin
[0189]Vegetative stocks of BIOT-3806 were prepared after growth in medium 1 containing 50 mg/L apramycin and preserved in 20% w/v glycerol:10% w/v lactose in distilled water and stored at -80° C. Vegetative stocks were recovered onto plates of ISP2 medium (Medium 3) supplemented with 50 mg/L apramycin and incubated for 48 hours at 28° C. Vegetative cultures were prepared by removing two agar plugs, 5 mm in diameter, from the ISP2 plate and inoculating them into 30 mL Medium 1 in 250 mL shake flasks containing 50 mg/L apramycin. The flasks were incubated at 28° C., 200 rpm (5 cm throw) for 48 h.
[0190]Vegetative cultures were inoculated at 5% v/v into 200 mL production medium (medium 2) in 2 L shake flasks. Cultivation was carried out for 1 day at 28° C. followed by 5 days at 26° C., 200 rpm (5 cm throw).
[0191]The fermentation broth of BIOT-3806 (1.3 L, cream colour) was extracted three times with an equal volume of ethyl acetate (EtOAc). The solvent was removed from the combined extract in vacuo to yield 2.87 g of a brown oil. The extract was then chromatographed over Silica Gel 60 eluting initially with a CHCl3 and MeOH mixture (95:5) followed by an increase in MeOH concentration up to 10% and collection of several fractions (about 250 mL per fraction). The fractions were assayed for the presence of metabolites using HPLC. A particular fraction containing a new compound (fraction 7; 752 mg crude mass after removal of solvent) was further purified by chromatography over a Phenomenex Luna C18-BDS column (21.2×250 mm; 5 um particle size) eluting with a gradient of water; acetonitrile (85:15) to (55:45) over 25 min, with a flow rate of 21 mL/min. Fractions were assayed by analytical HPLC and those containing the new compound were combined and the solvents removed to yield an off white solid (245.5 mg). For identification and characterisation MS, and 1 and 2D NMR experiments were carried out in Acetone-d6. Analysis by LCMS/MS, and by 1D and 2D NMR carried out in acetone-d6 identified the compound as 7-O-carbamoyl-15-hydroxypre-macbecin (15).
##STR00006##
3.3 Identification and Characterisation
[0192]A range of MS and NMR experiments were performed, viz LCMS, MSMS, 1H, 13C, APT, COSY-45, HMQC, HMBC. A thorough and exhaustive review of these data enabled the assignment of the majority of the protons and carbons of two analogues of pre-macbecin. The NMR assignments are described in Table 5.
TABLE-US-00010 TABLE 5 14 ##STR00007## 15 ##STR00008## 1H-NMR 13C-NMR Position 14 15 14 15 1 -- -- 171.8 172.5 2 -- -- 135.5 135.5 2-CH3 1.82 s 1.81 s 14.0 14.3 3 6.17 bs 6.02 s 133.8 133.7* 4 2.40 m 2.34 m 28.5* 27.7* 2.19 m 2.12*** 5 1.46 m 1.32 m 33.6* 36.1* 1.32 m 1.21 m 6 1.91 m 1.84 m 36.3 35.7 6-CH3 0.87 d, 7 0.86 d, 7 16.4 16.4 7 5.17 br.s 5.01 br.s 81.9 82.1 7-CONH2 -- -- 159.0 159.5 8 -- -- 134.0 134.5 8-CH3 1.50 s 1.44 s 14.4 13.9 9 5.35 d, 9.5 5.29 d, 9.5 131.4 132.7 10 2.45 m 2.42 m 36.0 35.7 10-CH3 1.01 d, 7 1.00 d, 7 18.6 18.8 11 3.60 dd, 8.5, 2.5 3.62 dd, 8.5, 2.5 76.3 75.9 12 3.18 ddd, 6, 3, 3 3.15 ddd, 6, 3, 3 84.1 83.4 12-OCH3 3.30 s 3.30 s 57.6 57.5 13 1.55 m 1.84** m CA 32.9 1.34 m 14 1.63 m 1.84** m 36.7 40.5 14-CH3 0.85 d, 7 0.75 d, 6.5 21.3 15.9 15 2.66 dd, 12, 1.5 4.62 d, 1.5 43.9 76.5 2.13 m 15-OH -- -- -- -- 16 -- -- 144.9 141.9 17 6.36 s 6.32 s 113.5 111.8 18 -- -- 159.3 158.5 18-OH 8.22 br.s 8.38 br.s -- -- 19 7.34 bs 7.16 s 106.1 107.2 20 -- -- 142.6 148.1 21 6.41 s 6.76 s 114.6 110.6 *connectivities for these carbons could not be made and assignments given are based on similarity to related molecules; CA, this carbon could not be assigned; **COSY correlations clearly distinguish these different signals; ***only observed as COSY cross peak.
Example 4
Generation of an Actinosynnema pretiosum Strain in which the gdmM Homologue mbcM has an In-Frame Deletion
[0193]4.1 Cloning of DNA Homologous to the Downstream Flanking Region of mbcM.
[0194]Oligos BV145 (SEQ ID NO: 22) and BV146 (SEQ ID NO: 23) were used to amplify a 1421 bp region of DNA from Actinosynnema pretiosum (ATCC 31280) in a standard PCR reaction using cosmid 52 (from example 1) as the template and Pfu DNA polymerase. A 5' extension was designed in each oligo to introduce restriction sites to aid cloning of the amplified fragment (FIG. 7). The amplified PCR product (PCRwv308, SEQ ID NO: 20, FIG. 8A) encoded 33 bp of the 3' end of mbcM and a further 1368 bp of downstream homology. This 1421 bp fragment was cloned into pUC19 that had been linearised with SmaI, resulting in plasmid pWV308.
4.2 Cloning of DNA Homologous to the Upstream Flanking Region of mbcM.
[0195]Oligos BV147 (SEQ ID NO: 24) and BV148 (SEQ ID NO: 25) were used to amplify a 1423 bp region of DNA from Actinosynnema pretiosum (ATCC 31280) in a standard PCR reaction using cosmid 52 (from example 1) as the template and Pfu DNA polymerase. A 5' extension was designed in each oligo to introduce restriction sites to aid cloning of the amplified fragment (FIG. 7). The amplified PCR product (PCRwv309, SEQ ID NO: 21, FIG. 8B) encoded 30 bp of the 5' end of mbcM and a further 1373 bp of upstream homology. This 1423 bp fragment was cloned into pUC19 that had been linearised with SmaI, resulting in plasmid pWV309.
TABLE-US-00011 BV145 (SEQ ID NO: 22) ATATACTAGTCACGTCACCGGCGCGGTGTCCGCGGACTTCGTCAACG SpeI BV146 (SEQ ID NO: 23) ATATCCTAGGCTGGTGGCGGACCTGCGCGCGCGGTTGGGGTG AvrII BV147 (SEQ ID NO: 24) ATATCCTAGGCACCACGTCGTGCTCGACCTCGCCCGCCACGC AvrII BV148 (SEQ ID NO: 25) ATATTCTAGACGCTGTTCGACGCGGGCGCGGTCACCACGGGC XbaI
[0196]The products PCRwv308 and PCRwv309 were cloned into pUC19 in the same orientation to utilise the PstI site in the pUC19 polylinker for the next cloning step.
[0197]The 1443 bp AvrII/PstI fragment from pWV309 was cloned into the 4073 bp AvrII/PstI fragment of pWV308 to make pWV310. pWV310 therefore contained a SpeII/XbaI fragment encoding DNA homologous to the flanking regions of mbcM fused at an AvrII site. This 2816 bp SpeII/XbaI fragment was cloned into pKC1132 (Bierman et al., 1992) that had been linearised with SpeI to create pWV320.
4.3 Transformation of Actinosynnema pretiosum subsp. pretiosum
[0198]Escherichia coli ET12567, harbouring the plasmid pUZ8002 was transformed with pWV320 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform Actinosynnema pretiosum subsp. pretiosum by vegetative conjugation (Matsushima et al, 1994). Exconjugants were plated on Medium 4 and incubated at 28° C. Plates were overlayed after 24 h with 50 mg/L apramycin and 25 mg/L nalidixic acid. As pWV320 is unable to replicate in Actinosynnema pretiosum subsp. pretiosum, apramycin resistant colonies were anticipated to be transformants that contained plasmid pWV320 integrated into the chromosome by homologous recombination via one of the plasmid borne mbcM flanking regions of homology.
[0199]Genomic DNA was isolated from six exconjugants and was digested and analysed by Southern blot. The blot showed that in four out of the six isolates integration had occurred in the upstream region of homology and in two of the six isolates homologous integration had occurred in the downstream region. One strain resulting from homologous integration in the upstream region (designated BIOT-3831) was chosen for screening for secondary crosses. One strain resulting from homologous integration in the downstream region (BIOT-3832) was also chosen for screening for secondary crosses.
4.4 Screening for Secondary Crosses
[0200]Strains were patched onto medium 4 (supplemented with 50 mg/L apramycin) and grown at 28° C. for four days. A 1 cm2 section of each patch was used to inoculate 7 mL modified ISP2 (0.4% yeast extract, 1% malt extract, 0.4% dextrose in 1 L distilled water) without antibiotic in a 50 mL falcon tube. Cultures were grown for 2-3 days then subcultured on (5% inoculum) into another 7 mL modified ISP2 (see above) in a 50 mL falcon tube. After 4-5 generations of subculturing the cultures were sonicated, serially diluted, plated on Medium 4 and incubated at 28° C. for four days. Single colonies were then patched in duplicate onto Medium 4 containing apramycin and onto Medium 4 containing no antibiotic and the plates were incubated at 28° C. for four days. Patches that grew on the no antibiotic plate but did not grow on the apramycin plate were re-patched onto +/-apramycin plates to confirm that they had lost the antibiotic marker. Genomic DNA was isolated from all apramycin sensitive strains and analysed by Southern blot. At this stage, half the secondary crossover strains had reverted to wild-type but half had produced the desired mbcM deletion mutants. The mutant strain encodes an mbcM protein with an in-frame deletion of 502 amino acids (FIG. 9).
[0201]mbcM deletion mutants were patched onto Medium 4 and grown at 28° C. for four days. A 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL seed medium (adapted from medium 1-2% glucose, 3% soluble starch, 0.5% corn steep solids, 1% soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate). These seed cultures were incubated for 2 days at 28° C., 200 rpm with a 2 inch throw. These were then used to inoculate (0.5 mL into 10 mL) production medium (medium 2-5% glycerol, 1% corn steep solids, 2% yeast extract, 2% potassium dihydrogen phosphate, 0.5% magnesium chloride, 0.1% calcium carbonate) and were grown at 28° C. for 24 hours and then at 26° C. for a further 5 days. Secondary metabolites were extracted from these cultures by the addition of an equal volume of ethyl acetate. Cell debris was removed by centrifugation. The supernatant was transferred to a clean tube and solvent was removed in vacuo. Samples were reconstituted in 0.23 mL methanol followed by the addition of 0.02 mL of 1% (w/v) FeCl3 solution. Samples were assessed for production of macbecin analogues
[0202]Chemical analysis by LCMS using the methods described in example 2.3 above unambiguously identified the presence of compounds 14 and 15 based on them having identical retention times and mass spectra.
4.5 Selection of Individual Colonies by Generating Protoplasts of BIOT-3872
[0203]Protoplasts were generated from BIOT-3872 using a method adapted from Weber and Losick 1988 with the following media alterations; Actinosynnema pretiosum cultures were grown on ISP2 plates (medium 3) for 3 days at 28° C. and a 5 mm2 scraping used to inoculate 40 mL of ISP2 broth supplemented with 2 mL of sterile 10% (w/v) glycine in water. Protoplasts were generated as described in Weber and Losick 1988 and then regenerated on R2 plates (R2 recipe--Sucrose 103 g, K2SO4 0.25 g, MgCl2.6H2O 10.12 g, Glucose 10 g, Difco Casaminoacids 0.1 g, Difco Bacto agar 22 g, distilled water to 800 mL, the mixture was sterilised by autoclaving at 121° C. for 20 minutes. After autoclaving the following autoclaved solutions were added; 0.5% KH2PO4 10 mL, 3.68% CaCl2.2H2O 80 mL, 20% L-proline 15 mL, 5.73% TES buffer (pH7.2) 100 mL, Trace element solution (ZnCl2 40 mg, FeCl3.6H2O 200 mg, CuCl2.2H2O 10 mg, MnCl2.4H2O 10 mg, Na2B4O7.10H2O 10 mg, (NH4)6Mo7O24.4H2O 10 mg, distilled water to 1 litre) 2 mL, NaOH (1N) (unsterilised) 5 mL).
[0204]80 individual colonies were patched onto MAM plates (Medium 4) and analysed for production of macbecin analogues as described above in example 2.3. The majority of protoplast generated patches produced at similar levels to the parental strain. 15 out of the 80 samples tested produced significantly more 14 and 15 than the parental strain. The best producing strain, WV4a-33 (BIOT-3870) was observed to produce 14 and 15 at significantly higher levels than the parent strain.
Example 5
Biological Data--In Vitro Evaluation of Anticancer Activity of Macbecin Analogues
[0205]In vitro evaluation of the test compounds for anticancer activity in a panel of human tumour cell lines in a monolayer proliferation assay was carried out as described in the general methods using a modified propidium iodide assay.
[0206]The results for 9 cell lines are displayed in Table 6 below; each result represents the median of duplicate experiments. Table 7 shows the mean IC70 for the compounds across the 38 cell line panel tested, with macbecin shown as a reference.
TABLE-US-00012 TABLE 6 in vitro cell line data Test/Control (%) at drug concentration Macbecin 14 15 1 10 1 10 1 10 Cell line μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL SF268 40 6 18 16 95 27 251L 58 44 18 16 86 29 H460 62 25 3 4 87 23 MCF7 42 19 11 9 99 19 394NL 16 13 14 13 75 18 OVCAR3 54 11 27 28 100 41 DU145 13 5 8 6 99 18 LNCAP 34 32 13 12 58 16 1138L 71 31 13 14 101 25
TABLE-US-00013 TABLE 7 average IC70 value across the 38 cell-line panel IC70 (μg/mL) macbecin 3.2 14 0.2 15 22.0
Example 6
Solubility Assay
[0207]Solutions (25 mM) of the test compounds were prepared by dissolving 3-5 mg aliquots in the appropriate amount of DMSO.
[0208]Aliquots (0.01 mL) were added to 0.490 mL of pH 7.3 PBS in glass vials. For each time point, 3 PBS vials were prepared in amber glass vials. For the six hour time point triplicate aliquots in DMSO were also prepared.
[0209]The resulting 0.5 mM solutions were shaken for up to six hours, with vials removed for analysis at 1, 3 and 6 hours. Samples were analysed by HPLC (0.025 mL injections). Compounds were quantified by peak area measurement at 274 nm.
[0210]Solubility in 2% DMSO in PBS at each time point was determined by comparing total peak areas for each chromatogram with mean total peak area for chromatograms produced from the corresponding 6 hour DMSO solutions. (Mean total peak area in DMSO solutions was assumed to be equivalent to a 0.5 mM solution). The results are shown below in Table 8.
TABLE-US-00014 TABLE 8 Solubility Results Solubility (microM) macbecin 81 18,21-dihydromacbecin 136 14 ≧500 * 15 ≧500 * Geldanamycin 1.7 17-AAG 171 * the solubility of these compounds was at or above the maximum measurable limit of this assay.
Example 7
Hsp90 Binding
Isothermal Titration Carorimetry and Kd Determinations.
[0211]Yeast Hsp90 was dialysed against 20 mM Tris pH 7.5 containing 1 mM EDTA and 5 mM NaCl and then diluted to 0.008 mM in the same buffer, but containing 2% DMSO. The test compounds were dissolved in 100% DMSO at a concentration of 50 mM and subsequently diluted to 0.1 mM in the same buffer as for Hsp90 with 2% DMSO. Heats of interaction were measured at 30° C. on a MSC system (Microcal), with a cell volume of 1.458 mL. 10 aliquots of 0.027 mL of 0.100 mM of each test compound were injected into 0.008 mM yeast Hsp90. Heats of dilution were determined in a separate experiment by injecting the test compound into buffer containing 2% DMSO, and the corrected data fitted using a nonlinear least square curve-fitting algorithm (Microcal Origin) with three floating variables: stoichiometry, binding constant and change in enthalpy of interaction. The results are shown below in Table 9.
TABLE-US-00015 TABLE 9 Kd values for Hsp90 binding Kd (nM) macbecin 240 14 3.2 15 6 Geldanamycin 1200
Example 8
Generation of an Actinosynnema pretiosum Strain in which mbcM has an In-Frame Deletion and mbcMT1, mbcMT2, mbcP and mbcP450 have Additionally Been Deleted
[0212]8.1 Cloning of DNA Homologous to the Downstream Flanking Region of mbcMT2
[0213]Oligos Is4del1 (SEQ ID NO: 29) and Is4del2a (SEQ ID NO: 30) were used to amplify a 1595 bp region of DNA from Actinosynnema pretiosum (ATCC 31280) in a standard PCR reaction using cosmid 52 (from example 1) as the template and Pfu DNA polymerase. A 5' extension was designed in oligo Is4del2a to introduce an AvrII site to aid cloning of the amplified fragment (FIG. 10). The amplified PCR product (1+2a, FIG. 11 SEQ ID NO: 31) encoded 196 bp of the 3' end of mbcMT2 and a further 1393 bp of downstream homology. This 1595 bp fragment was cloned into pUC19 that had been linearised with SmaI, resulting in plasmid pLSS1+2a.
TABLE-US-00016 Is4del1 (SEQ ID NO: 29) 5'-GGTCACTGGCCGAAGCGCACGGTGTCATGG-3' Is4del2a (SEQ ID NO: 30) 5'-CCTAGGCGACTACCCCGCACTACTACACCGAGCAGG-3'
8.2 Cloning of DNA Homologous to the Upstream Flanking Region of mbcM.
[0214]Oligos Is4del3b (SEQ ID NO: 32) and Is4del4 (SEQ ID NO: 33) were used to amplify a 1541 bp region of DNA from Actinosynnema pretiosum (ATCC 31280) in a standard PCR reaction using cosmid 52 (from example 1) as the template and Pfu DNA polymerase. A 5' extension was designed in oligo Is4del3b to introduce an AvrII site to aid cloning of the amplified fragment (FIG. 10). The amplified PCR product (3b+4, FIG. 12, SEQ ID NO: 34) encoded ˜100 bp of the 5' end of mbcP and a further ˜1450 bp of upstream homology. This ˜1550 bp fragment was cloned into pUC19 that had been linearised with SmaI, resulting in plasmid pLSS3b+4
TABLE-US-00017 Is4del3b (SEQ ID NO: 32) 5'-CCTAGGAACGGGTAGGCGGGCAGGTCGGTG-3' Is4del4 (SEQ ID NO: 33) 5'-GTGTGCGGGCCAGCTCGCCCAGCACGCCCAC-3'
[0215]The products 1+2a and 3b+4 were cloned into pUC19 to utilise the HindIII and BamHI sites in the pUC19 polylinker for the next cloning step.
[0216]The 1621 bp AvrII/HindIII fragment from pLSS1+2a and the 1543 bp AvrII/BamHI fragment from pLSS3b+4 were cloned into the 3556 bp HindIII/BamHI fragment of pKCl 132 to make pLSS315. pLSS315 therefore contained a HindIII/BamHI fragment encoding DNA homologous to the flanking regions of the desired four ORF deletion region fused at an AvrII site (FIG. 7).
8.3 Transformation of BIOT-3870 with pLSS315
[0217]Escherichia coli ET12567, harbouring the plasmid pUZ8002 was transformed with pLSS315 by electroporation to generate the E. coli donor strain for conjugation. This strain was used to transform BIOT-3870 by vegetative conjugation (Matsushima et al, 1994). Exconjugants were plated on MAM medium (1% wheat starch, 0.25% corn steep solids, 0.3% yeast extract, 0.3% calcium carbonate, 0.03% iron sulphate, 2% agar) and incubated at 28° C. Plates were overlayed after 24 h with 50 mg/L apramycin and 25 mg/L nalidixic acid. As pLSS315 is unable to replicate in BIOT-3870, apramycin resistant colonies were anticipated to be transformants that contained plasmid integrated into the chromosome by homologous recombination via the plasmid borne regions of homology.
8.4 Screening for Secondary Crosses
[0218]Three primary transformants of BIOT-3870:pLSS315 were selected for subculturing to screen for secondary crosses.
[0219]Strains were patched onto MAM media (supplemented with 50 mg/L apramycin) and grown at 28° C. for four days. Two 6 mm circular plugs were used to inoculate 30 mL of ISP2 (0.4% yeast extract, 1% malt extract, 0.4% dextrose, not supplemented with antibiotic) in a 250 ml conical flask. Cultures were grown for 2-3 days then subcultured (5% inoculum) into 30 mL of ISP2 in a 250 ml conical flask. After 4-5 rounds of subculturing the cultures were protoplasted as described in Example 3.6, the protoplasts were serially diluted, plated on regeneration media (see Example 3.6) and incubated at 28° C. for four days. Single colonies were then patched in duplicate onto MAM media containing apramycin and onto MAM media containing no antibiotic and the plates were incubated at 28° C. for four days. Seven patches derived from clone no 1 (no 32-37) and four patches derived from clone no 3 (no 38-41) that grew on the no antibiotic plate but did not grow on the apramycin plate were re-patched onto +/-apramycin plates to confirm that they had lost the antibiotic marker.
[0220]Production of macbecin analogues was carried out as described in the General Methods. Analysis was performed as described in General Methods and example 2. Compound 14 was produced in yields comparable to the parent strain BIOT-3870 and no production of compound 15 was observed for patches 33, 34, 35, 37, 39 and 41. This result shows that the desired mutant strains have a deletion of 3892 bp of the macbecin cluster containing the genes mbcP, mbcP450, mbcMT1 and mbcMT2 in addition to the original deletion of mbcM.
Example 9
Biological Data--In Vitro Evaluation of Anticancer Combination of Compound 14 with Standard Cytotoxic Agents Mitomycin C, Ifosfamid, Cyclohexylchloroethylnitrosurea (CCNU), Mitoxantrone and Vindesine
[0221]In vitro evaluation of 14 for anticancer activity when combined with standard cytotoxic agents against the tumour cell line DU145 in a monolayer proliferation assay was carried out as described in the general methods using a modified propidium iodide assay. Four separate concentrations of 14 were used from 0-80 nM for mitomycin C, and O-160 nM for ifosfamid, CCNU, mitoxantrone and vindesine along with 10 concentrations of the standard agent. The relative cell growth percentage treated/control (% T/C) values were plotted and used to draw the graphs shown in FIGS. 13-17. These graphs were then used to calculate the IC70 values shown in Tables 10-11.
TABLE-US-00018 TABLE 10 Concentration of 14 IC70 of mitomycin (nM) (μg/ml) 0 0.165 30 0.112 50 0.087 80 0.056
TABLE-US-00019 TABLE 11 IC70 of IC70 of IC70 of IC70 of Concentration ifosfamid mitoxantrone vindesine CCNU of 14 (nM) (μg/ml) (μg/ml) (μg/ml) (μg/ml) 0 90.14 2.08 0.0009 19.74 80 80.57 1.00 0.0008 11.30 120 52.09 1.25 0.0006 9.34 160 17.32 0.11 0.0001 1.63
The data shows that the effect of standard cytotoxic agents, such as the alkylating agents mitomycin C, ifosfamid and CCNU, the topoisomerase II inhibitor mitoxantrone and the mitotic inhibitor vindesine may be improved by the addition of Compound 14. The usefulness of cytotoxic agents such as mitomycin C, ifosfamid, CCNU, mitoxantrone and vindesine is known to be limited by their toxicity and they are conventionally employed in therapy at high levels close to their maximum tolerated dose. It may therefore be deduced that Compound 14 and other compounds of the invention should have useful effect in sparing the amount of cytotoxic agent (such as mitomycin C, ifosfamid, CCNU, mitoxantrone, vindesine) to be used in therapy. Thus it may be possible to achieve greater therapeutic efficacy or efficacy with fewer side effects than can be achieved with the cytotoxic agent alone or combinations of a cytotoxic agent with another cytotoxic agent.
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[0302]All references including patent and patent applications referred to in this application are incorporated herein by reference to the fullest extent possible.
[0303]Throughout the specification and the claims which follow, unless the context requires otherwise, the word `comprise`, and variations such as `comprises` and `comprising`, will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.
Sequence CWU
1
3411638DNAStreptomyces hygroscopicus NRRL 3602 1atgaccggga aggaggcggc
agtggacacc gcgcgggaaa cggacagcct cgaggccgag 60gtgctgatcg tcggctacgg
accggtgggc cagctactgt cggtgctact ggcccagcgc 120gggcggcgcg tgacggtcgt
ggagcgctgg ccggagccgt accggcaccc ccgggcggtc 180gggttcgaca gtgaggccgc
gcgccttctg gcctcggccg ggatcggcga ctcgctcgac 240aagttcaccg aacccgcgcg
ggaccacgcc tggcagaaca cgaagggcga gacgctgatc 300gaccacgagg tggccgaccg
ggggcactgc acctggccgg aggctttgtc ggcgtatcag 360cccgccctgg agtccgcgct
gatcgagcac ggggagacgc tgccgccgct gcggatcctg 420cgcggatacg aggcggtggg
actcgcggac gacggcgacc atgtgacctt gaccgtggtc 480ggcccggacg gggagaagac
ggacctcacc gcgctgtggg tggtcggctg cgacggcgcg 540aacagcctgg taaggacggg
cgtcggcacc accatgacgg acctcgactt ctcgtacgac 600tggctgatct gcgatgtgcg
gttgcacgag caccgcgagt tccggccgaa caacctggag 660atctgcgatc cggcgcgccc
ccggacggcg gtgtccgcgg gtcctggcca ccggcggtac 720gagttcatgc gggtgcccgc
ggacgacccc gaacacttcg gcaccgtgga gagcgcctgg 780gagctgctgc ggctgttcga
tgtgacgccc gagaacggcg ttctggaccg gcacgcggtc 840tacaccttcc aggcccgctg
ggcggagcgc tggcggaccg gacggatggt gctggccggg 900gactcggcac acctcatgcc
gccgttcgcg gggcagggca tgtgctccgg attccgtgac 960gcggccaatc tggcctggaa
actggacctg gtcctgggcg gacacgcggc gccgacgctg 1020ctggacacct acaccaccga
gcggcgggca cacgtgcggc acgcggtgga gatgtcggtg 1080ggcctgggcc gggtggtgtg
catggcggac ccggccgcgg cggcggaccg tgacgcggcg 1140atgctggccg cgcgcaaacg
caacatcggc ccgagtgccg cccgccgttc cgtggtgagg 1200ccgctcgtgg acgggctgct
acggcaggac ggtcagggcc gcccggcacc gtacgccggc 1260caggcgggcc cccagtggcg
agtgtgccgc gcgggaacca ccggcctgtt cgacgacgtg 1320gtgggcaccg gtttcgtcct
cctctacgcc gaggacgtgt tccccgcgct ggacgcgcgg 1380cggctgacat tcctcgacag
catcggcacc cgactggtgc gcatggtccc cgcggacacg 1440cccccggccg ccctggggcc
acgggacgcg ctggacgtgg aggaccggta cctgtcctat 1500ctgtcggaga tggacgcgct
ggcggtactg gtacgcccgg acttctacct gttcggcatc 1560gcggaggacg agggcgaact
cctctctctc gtagacgact tggccaccca gctgagcccg 1620tcacccactc cttcgtaa
163822455DNAAmycolatopsis
mediterranei 2atgaccggga aggaggcggc agtggacacc gcgcgggaaa cggacagcct
cgaggccgag 60gtgctgatcg tcggctacgg accggtgggc cagctactgt cggtgctact
ggcccagcgc 120gggcggcgcg tgacggtcgt ggagcgctgg ccggagccgt accggcaccc
ccgggcggtc 180gggttcgaca gtgaggccgc gcgccttctg gcctcggccg ggatcggcga
ctcgctcgac 240aagttcaccg aacccgcgcg ggaccacgcc tggcagaaca cgaagggcga
gacgctgatc 300gaccacgagg tggccgaccg ggggcactgc acctggccgg aggctttgtc
ggcgtatcag 360cccgccctgg agtccgcgct gatcgagcac ggggagacgc tgccgccgct
gcggatcctg 420cgcggatacg aggcggtggg actcgcggac gacggcgacc atgtgacctt
gaccgtggtc 480ggcccggacg gggagaagac ggacctcacc gcgctgtggg tggtcggctg
cgacggcgcg 540aacagcctgg taaggacggg cgtcggcacc accatgacgg acctcgactt
ctcgtacgac 600tggctgatct gcgatgtgcg gttgcacgag caccgcgagt tccggccgaa
caacctggag 660atctgcgatc cggcgcgccc ccggacggcg gtgtccgcgg gtcctggcca
ccggcggtac 720gagttcatgc gggtgcccgc ggacgacccc gaacacttcg gcaccgtgga
gagcgcctgg 780gagctgctgc ggctgttcga tgtgacgccc gagaacggcg ttctggaccg
gcacgcggtc 840tacaccttcc aggcccgctg ggcggagcgc tggcggaccg gacggatggt
gctggccggg 900gactcggcac acctcatgcc gccgttcgcg gggcagggca tgtgctccgt
tgacggacac 960ggacgtcgtc atcgtcggaa acggcccgat cggcgccacg ctgtcggtgc
tgctcgcgca 1020acgcggttgg cgggtgacgg tgctcgaacg ccggccccgg ccgtacaagc
tgccgcgggc 1080gaccagcttc gacggcgaga cggcccggct gctggccgcc accggcatcg
gcccggacct 1140cggccggatc accgcgccgg ccaacggcta ccagtggcag accgccgacg
ggcagacgct 1200gctcgacatc gccttcacca cagacggccc gtacggctgg ccggacgcca
acaccatgca 1260ccagcccgcg ctcgaggagc tcctcgcggc ccgggcggcg gacctgcccg
ggatcaccgt 1320gctgcgcggg cacgaagtcg tggccatcac cgacggcgac tcgttcgtcc
aggtgaccgc 1380caccggcgaa gacgacgttc cgcggacgat ctcgggccgg tgggtggtcg
gctgcgacgg 1440tgccaacagc ttcgtgcgcg agcacctcga cgtgccggtg accgacctcg
gcttctccta 1500cgagtggcta ctctgcgacg tccggctgaa cgagccgcgc gagttcgtgc
cgacgaacgt 1560gcagatctgc gacccggccc ggcccaccac gctggtgggc agcgggcccg
gccgccgtcg 1620ctgggagttc atgcgcctgc cgggcgaaaa cgcggcggag ctcaacaagg
acgagacggc 1680gtggcggctg atggccccct tcggcgtcac ccccgaaacc gccaccctgc
tgcgcagcac 1740cacctacatc ttccaggccc gctgggccga ccggtggcgg gtggggcacg
tcctgctcgc 1800cggcgacgcg gcgcacctga tgccgccgtt cgccgggcag ggcatgtgct
ccggcatccg 1860cgacgtcacc aacctggcgt ggaagctcga cctcacgctc cgcggcttgg
ctccggagtc 1920ccttttggac agctacgggg acgagcggcg cgagcaggcg cgcgaggcca
tcctcgcgtc 1980ggtccagctg ggccgggtga tctgcgtgac ggacccggcg gccgccgccg
agcgggactc 2040gaccgtgctg gccaaccgcc gcggcaagcc cgcgggccgg ccggaaccgg
cgaagccgct 2100caccggcggg ctgcgccacg aaagcccggg cgcgggcgtg gtggtgccgc
agggccgggt 2160gcaggtcggc gacgccaccg ggctcttcga cgacgtcatc ggccggggtt
tcgtcctgct 2220gaccaccgag gagacccact cggacttcct caccgaactc ggagcccacg
tcgtccggct 2280cgacgagggg gtggacgtcg acgacgtcta ccggccgttc ctggcccggt
tcggggcggc 2340ttcggtgctc gtgcggccgg actaccacgt cttcggcacg gccgggccgg
gcgggctcga 2400agccctggtc ggcgccctgc gcgaccggct gcgcgccgcg gtgcccaccg
gctag 24553793DNAActinosynnema mirum 3ccgcgggcgg tcgggttcga
cggtgaggcg acccgcgtgc tggcctcggc cgggctcggg 60cccgcgctgg ccgagttcgg
ggagcccgcg ggcgactacg agtggcgcac cgcgtccggg 120gagacgctga tcgcgttcac
cgtgcgggag gaggggcact gcggctggcc cgaggcgacc 180tcggcctacc agcccgcgct
ggaggacgcg ctgatcgcgc gcggcgaggc gctgccgggg 240gttcaggtgc ggcgcggctg
ggaggtgacc gggctgaccg accggggcga ccacgtgcgg 300gtggtggcca ccgaccccgg
cggggcgcgc gtgaggctga cggcgcggtt cgcggtcggc 360tgcgacgggg cgaacagcgt
ggtgcgggcc cgcaccggca ccgacgtgac cgacctggac 420ttctcgcacg actggctggt
gtgcgacgtg cggctgcacg accggcgccc ggtgacgccg 480aacaacctcc aggtgtgcga
cccggccagg ccgcgcaccg cggtgtcggc ggggccaggg 540caccggcggt acgagttcat
gcgggtgccc ggcgacgacc cggagcggtt cggcacgccg 600gagagggcgt gggagctgct
ggcgctgttc ggcgtcgggc gcggcgacgg ggtgctggac 660cggctggccg tgtacacgtt
ccaggcgcgg tgggcgcggc ggtggcgggc gggccggatg 720ctgctggccg gggacgccgc
gcacctgatg ccgccgttcg ccgggcaggg catgtgctcc 780ggcatccgcg acg
79341572DNAActinosynnema
pretiosum 4gtggcgggcg aggtcgagca cgacgtggtg gtcgtcggct acgggccggt
ggggcagctg 60ctgtcggtgc tgctggcgca gcgcggctgg cgggtgctgg tgctggagcg
ctggccgacg 120ccgttccggc tgccgcgcgc ggtcgggttc gacagcgagg cgacccgcgt
gctggcctcg 180gccgggctcg ggcccgcgct ggccgagttc ggggagcccg cgggcgacta
cgagtggcgc 240accgcgtccg gggagacgct gatcgcgttc accgtgcggg aggaggggca
ctgcggctgg 300cccgaggcga cctcggccta ccagcccgcg ctggaggacg cgctgatcgc
gcgcggcgag 360gcgctgccgg gggttcaggt gcggcgcggc tgggaggtga ccgggctgac
cgaccggggc 420gaccacgtgc gggtggtggc caccgacccc ggcggggcgc gcgtgaggct
gacggcgcgg 480ttcgcggtcg gctgcgacgg ggcgaacagc gtggtgcggg cccgcaccgg
caccgacgtg 540accgacctgg acttctcgca cgactggctg gtgtgcgacg tgcggctgca
cgaccggcgc 600ccggtgacgc cgaacaacct ccaggtgtgc gacccggcca ggccacgcac
cgcggtgtcg 660gcggggccag ggcaccggcg gtacgagttc atgcgggtgc ccggcgacga
cccggagcgg 720ttcggcacgc cggagagggc gtgggagctg ctggcgctgt tcggcgtcgg
gcgcggcgac 780ggggtgctgg accggctggc cgtgtacacg ttccaggcgc ggtgggcgcg
gcggtggcgg 840gcgggccgga tgctgctggc cggggacgcc gcgcacctga tgccgccgtt
cgccgggcag 900ggcatgacct ccgggttccg ggacgcggcg aacctggcgt ggaagctgga
cctggtgctg 960cgcggcgagg ccgggtcggc gctgctggac agctacacgc tggagcgcgc
cgagcacgtg 1020cggcacgccg tgacgatctc ggtgggcctg gggcgggtgg tgtgcgtggc
cgacccggcg 1080gtggctgcgg accgggacgc ggcgatgctg gcggcgcgcg agcgcgagct
gacaccgggc 1140gcgtcggccc ggtcggtgct caagcccctg gaggacgggg tgctgcaccg
ggacggcgac 1200ggcgccctcg cgccgcacgc gggggccgtg ggcccgcagt ggcgggtggg
gcgcggcggg 1260cgggtcgggc tgttcgacga cgtggtgggg accgggttcg cgctgctcac
caggggcggg 1320ctggtggcgg ggccggaggt gcgggcgcgg ctggacgggc tgggcgcgcg
ctacgcgcac 1380ctggtgcccg ccggggcggc ggcggacggg ccggacgacg tggtcgacgt
gagcgggaac 1440tacctgacgt ggctggagga gctggacgcg gcggcggtgc tgctgcgacc
ggacttctac 1500gtgttcggcg cggccgggga cgcggcgggg ttggccgggc tggtggcgga
cctgcgcgcg 1560cggttggggt ga
15725545PRTStreptomyces hygroscopicus NRRL 3602 5Met Thr Gly
Lys Glu Ala Ala Val Asp Thr Ala Arg Glu Thr Asp Ser1 5
10 15Leu Glu Ala Glu Val Leu Ile Val Gly
Tyr Gly Pro Val Gly Gln Leu20 25 30Leu
Ser Val Leu Leu Ala Gln Arg Gly Arg Arg Val Thr Val Val Glu35
40 45Arg Trp Pro Glu Pro Tyr Arg His Pro Arg Ala
Val Gly Phe Asp Ser50 55 60Glu Ala Ala
Arg Leu Leu Ala Ser Ala Gly Ile Gly Asp Ser Leu Asp65 70
75 80Lys Phe Thr Glu Pro Ala Arg Asp
His Ala Trp Gln Asn Thr Lys Gly85 90
95Glu Thr Leu Ile Asp His Glu Val Ala Asp Arg Gly His Cys Thr Trp100
105 110Pro Glu Ala Leu Ser Ala Tyr Gln Pro Ala
Leu Glu Ser Ala Leu Ile115 120 125Glu His
Gly Glu Thr Leu Pro Pro Leu Arg Ile Leu Arg Gly Tyr Glu130
135 140Ala Val Gly Leu Ala Asp Asp Gly Asp His Val Thr
Leu Thr Val Val145 150 155
160Gly Pro Asp Gly Glu Lys Thr Asp Leu Thr Ala Leu Trp Val Val Gly165
170 175Cys Asp Gly Ala Asn Ser Leu Val Arg
Thr Gly Val Gly Thr Thr Met180 185 190Thr
Asp Leu Asp Phe Ser Tyr Asp Trp Leu Ile Cys Asp Val Arg Leu195
200 205His Glu His Arg Glu Phe Arg Pro Asn Asn Leu
Glu Ile Cys Asp Pro210 215 220Ala Arg Pro
Arg Thr Ala Val Ser Ala Gly Pro Gly His Arg Arg Tyr225
230 235 240Glu Phe Met Arg Val Pro Ala
Asp Asp Pro Glu His Phe Gly Thr Val245 250
255Glu Ser Ala Trp Glu Leu Leu Arg Leu Phe Asp Val Thr Pro Glu Asn260
265 270Gly Val Leu Asp Arg His Ala Val Tyr
Thr Phe Gln Ala Arg Trp Ala275 280 285Glu
Arg Trp Arg Thr Gly Arg Met Val Leu Ala Gly Asp Ser Ala His290
295 300Leu Met Pro Pro Phe Ala Gly Gln Gly Met Cys
Ser Gly Phe Arg Asp305 310 315
320Ala Ala Asn Leu Ala Trp Lys Leu Asp Leu Val Leu Gly Gly His
Ala325 330 335Ala Pro Thr Leu Leu Asp Thr
Tyr Thr Thr Glu Arg Arg Ala His Val340 345
350Arg His Ala Val Glu Met Ser Val Gly Leu Gly Arg Val Val Cys Met355
360 365Ala Asp Pro Ala Ala Ala Ala Asp Arg
Asp Ala Ala Met Leu Ala Ala370 375 380Arg
Lys Arg Asn Ile Gly Pro Ser Ala Ala Arg Arg Ser Val Val Arg385
390 395 400Pro Leu Val Asp Gly Leu
Leu Arg Gln Asp Gly Gln Gly Arg Pro Ala405 410
415Pro Tyr Ala Gly Gln Ala Gly Pro Gln Trp Arg Val Cys Arg Ala
Gly420 425 430Thr Thr Gly Leu Phe Asp Asp
Val Val Gly Thr Gly Phe Val Leu Leu435 440
445Tyr Ala Glu Asp Val Phe Pro Ala Leu Asp Ala Arg Arg Leu Thr Phe450
455 460Leu Asp Ser Ile Gly Thr Arg Leu Val
Arg Met Val Pro Ala Asp Thr465 470 475
480Pro Pro Ala Ala Leu Gly Pro Arg Asp Ala Leu Asp Val Glu
Asp Arg485 490 495Tyr Leu Ser Tyr Leu Ser
Glu Met Asp Ala Leu Ala Val Leu Val Arg500 505
510Pro Asp Phe Tyr Leu Phe Gly Ile Ala Glu Asp Glu Gly Glu Leu
Leu515 520 525Ser Leu Val Asp Asp Leu Ala
Thr Gln Leu Ser Pro Ser Pro Thr Pro530 535
540Ser5456501PRTAmycolatopsis mediterranei 6Met Thr Asp Thr Asp Val Val
Ile Val Gly Asn Gly Pro Ile Gly Ala1 5 10
15Thr Leu Ser Val Leu Leu Ala Gln Arg Gly Trp Arg Val
Thr Val Leu20 25 30Glu Arg Arg Pro Arg
Pro Tyr Lys Leu Pro Arg Ala Thr Ser Phe Asp35 40
45Gly Glu Thr Ala Arg Leu Leu Ala Ala Thr Gly Ile Gly Pro Asp
Leu50 55 60Gly Arg Ile Thr Ala Pro Ala
Asn Gly Tyr Gln Trp Gln Thr Ala Asp65 70
75 80Gly Gln Thr Leu Leu Asp Ile Ala Phe Thr Thr Asp
Gly Pro Tyr Gly85 90 95Trp Pro Asp Ala
Asn Thr Met His Gln Pro Ala Leu Glu Glu Leu Leu100 105
110Ala Ala Arg Ala Ala Asp Leu Pro Gly Ile Thr Val Leu Arg
Gly His115 120 125Glu Val Val Ala Ile Thr
Asp Gly Asp Ser Phe Val Gln Val Thr Ala130 135
140Thr Gly Glu Asp Asp Val Pro Arg Thr Ile Ser Gly Arg Trp Val
Val145 150 155 160Gly Cys
Asp Gly Ala Asn Ser Phe Val Arg Glu His Leu Asp Val Pro165
170 175Val Thr Asp Leu Gly Phe Ser Tyr Glu Trp Leu Leu
Cys Asp Val Arg180 185 190Leu Asn Glu Pro
Arg Glu Phe Val Pro Thr Asn Val Gln Ile Cys Asp195 200
205Pro Ala Arg Pro Thr Thr Leu Val Gly Ser Gly Pro Gly Arg
Arg Arg210 215 220Trp Glu Phe Met Arg Leu
Pro Gly Glu Asn Ala Ala Glu Leu Asn Lys225 230
235 240Asp Glu Thr Ala Trp Arg Leu Met Ala Pro Phe
Gly Val Thr Pro Glu245 250 255Thr Ala Thr
Leu Leu Arg Ser Thr Thr Tyr Ile Phe Gln Ala Arg Trp260
265 270Ala Asp Arg Trp Arg Val Gly His Val Leu Leu Ala
Gly Asp Ala Ala275 280 285His Leu Met Pro
Pro Phe Ala Gly Gln Gly Met Cys Ser Gly Ile Arg290 295
300Asp Val Thr Asn Leu Ala Trp Lys Leu Asp Leu Thr Leu Arg
Gly Leu305 310 315 320Ala
Pro Glu Ser Leu Leu Asp Ser Tyr Gly Asp Glu Arg Arg Glu Gln325
330 335Ala Arg Glu Ala Ile Leu Ala Ser Val Gln Leu
Gly Arg Val Ile Cys340 345 350Val Thr Asp
Pro Ala Ala Ala Ala Glu Arg Asp Ser Thr Val Leu Ala355
360 365Asn Arg Arg Gly Lys Pro Ala Gly Arg Pro Glu Pro
Ala Lys Pro Leu370 375 380Thr Gly Gly Leu
Arg His Glu Ser Pro Gly Ala Gly Val Val Val Pro385 390
395 400Gln Gly Arg Val Gln Val Gly Asp Ala
Thr Gly Leu Phe Asp Asp Val405 410 415Ile
Gly Arg Gly Phe Val Leu Leu Thr Thr Glu Glu Thr His Ser Asp420
425 430Phe Leu Thr Glu Leu Gly Ala His Val Val Arg
Leu Asp Glu Gly Val435 440 445Asp Val Asp
Asp Val Tyr Arg Pro Phe Leu Ala Arg Phe Gly Ala Ala450
455 460Ser Val Leu Val Arg Pro Asp Tyr His Val Phe Gly
Thr Ala Gly Pro465 470 475
480Gly Gly Leu Glu Ala Leu Val Gly Ala Leu Arg Asp Arg Leu Arg Ala485
490 495Ala Val Pro Thr
Gly500733DNAArtificialPrimer 7ggtctagagg tcagtgcccc cgcgtaccgt cgt
33826DNAArtificialPrimer 8ggcatatgct tgtgctcggg
ctcaac 26936DNAArtificialprimer
9cccgcccgcg cgagcggcgc gtggccgccc gagggc
361036DNAArtificialPrimer 10gcgtcctcgc gcagccacgc caccagcagc tccagc
361135DNAArtificialPrimer 11ccaaccccgc cgcgtccccg
gccgcgccga acacg 351234DNAArtificialPrimer
12gtcgtcggct acgggccggt ggggcagctg ctgt
341335DNAArtificialPrimer 13gtcggtggac tgccctgcgc ctgatcgccc tgcgc
351435DNAArtificialPrimer 14ggccggtggt gctgcccgag
gacggggagc tgcgg 351539DNAArtificialPrimer
15caccgctcgc gggggtggcg cggcgcacga cgtggctgc
391638DNAArtificialPrimer 16cctcctcgga cagcgcgatc agcgccgcgc acagcgag
3817100588DNAActinosynnema pretiosum 17gatctggggc
gacgagccgc ccgccgggcc ggggccggcg ttgcaggcgc tcgtctcccg 60gctgcggcgg
gcgctcggcg cgccgggcgc ggtcgcgctg ggggtgggcg ggtaccggct 120cgtggcggac
gtggacgcgg cgcggttcga ggagctggcc gcgcggggcg gggaggacgc 180gctgcgggag
gccgccgcgc tgtggggcgg gcgggtcggg ggcgagccgc cggtggtcgc 240ggccgtcgcg
ccgcgggtgg cgacccggct ggcgcggctg tcggtggagg tggtgctgga 300cctggcggag
gtcgagctgg cgctcgggcg caccggggcg gccatcggtg gggcgagcgg 360ggtgctggcc
gagcacccgg cgcacgagcg ggccgccggg gtgctggtgg acgcgctcgc 420gggcgcggga
cggcaggccg aggcgctggc ggcctacgag cgggtccgcg cggcgctggc 480cgacgagctg
ggcgccgacc ccggcacggc cctgcgcgag cgccacctgc ggctgctgcg 540cgccaccccg
ccaccgctcc cccggccgaa cgcgctgccc gcgccggtga cgggcttcct 600cggccgggac
gccgacctcg cccgcgtcgc cgacctgctg gccgccgggc ggctggtcac 660cgtcgtcggg
cccggcgggg tgggcaagac ccggctggcc gtggaggcgc tgcgccggga 720ccgggacgcg
ctgctggtgg acctcgcgcc ggtcgccgag ccctcggagg tcgtcgccgc 780cgtgctcgcc
gggatcgggc tgcgcggcga ccgcgaccgg ccgggcgggg acgcgacggc 840gctgctggcc
gccgagctgg cggcgcgcag gtcggtgctg ctgctggaca actgcgagca 900cctggtcgac
gccgtggccc acctggtcgc gctcctgctc ccccgctgcc ccgagctgcg 960cgtgctcgcc
accagccggg aacccctggc ggtcgacggg gaggcgctgg tcccgctggg 1020gccgctcgcg
ctgcccggaa tcggggacgg gcttgacgcc gcggtcggca cggcctcggt 1080gcggttgttc
gcccaacggg cgtcggcggt gcgccccggt ttcgccgtcg acgccacgac 1140gctgccggac
gtggtgcgcc tggtgcgggc gctggacggg ctgccgctgg cgctggagct 1200ggccgccgcc
cggttgcgcg ccctgccgct gcccgacctg gtggccgggt tgtcggcgcg 1260gttccgcctg
ctggcgggcg ggaaccgggc cgcgccgccc cggcaccgca cgctgcgcgc 1320ggtgatcgcg
tggagctggg acctgctgga cgggcccgag cgggccgtgg ccgagcggat 1380ctccgtgctg
cccggcgggg tcaccccgga gtcggccgcc gccgtctgcg cgggcgccgt 1440gcccgccgac
gaggtgcccg aactgctggc cgcgctggtc gaccggtcgc tgctgagcct 1500ggtcgggggt
cggcggcgga tgctggagac ggtgcgcgcg tacggggtcg agcgcctggc 1560cgccgccggg
gacttgagcg cggtccgcga cctggccgcc gcgcacgtgg cgggggtgct 1620ggcggggcag
gacgcggtgc tgcgcgggcc ggggcagcgc gcggcggtgg cggcgatcgg 1680cgcggagcac
gacaacgcgg tggccgcgct gcaccaccgg tgcgccaccg gggacgcgga 1740cggggcgctc
gcgctggcgc tgtcgctggt ctggtactgg caggtgttcg gccgccagtc 1800cgagggcgcg
cactggctcg ggcgggcgct ggcggtgccc ggcgggccgt ccccggagcg 1860ggactgcgcg
cgggccgccc acctgctcgg cctggccgac ggcgggcacg gggtgggtga 1920tcgcggggag
gtgggggcgc tcgcggaccg ggtgctggcg caccgggggc tccccggtca 1980cctgcgggtg
ctcggggcgg tcctgctgtt cctgctgggg cgcggcgagg gggtgttccg 2040ggagctgggc
gcgggcggcg ggtggttgtc cgggctggcg cacctgttcc tggccgagct 2100ggcggagaac
gcgggcgagc tggaccgggc gcgcgggcac gcggaggtgt ccctggaccg 2160gttccgggcg
gccggggacg ggtggggcgt ggcgggggtg ctgccggtgc gggcgcgggc 2220gcggcggtac
gacgacctgg acgggacgtg ggcggacctt cgggaggcgc gggcgctgga 2280gggggagttc
ggggcgctga gccccggtga ccgggtgcgg gcggacctgc ggtgggtcga 2340cctgcacgag
cggcgcggtg acagcggggc ggcgctggag gtgctggccg cggcccgtgc 2400tcggggggag
caggtcgcgg tggtggacgc gcgggaggcc gcgctgcggg tgcggctcgg 2460ggacctgggg
cgggcgggtg agctgctggc cggggtgggt ggggcggtgg gcgacctggc 2520gcgggccgcg
tatcgggtgg cctcggggga cctggcgggt gcggagcggg cgttgcggcg 2580ggcgcgggtg
gtggcggctg cgagcgggga gctgcccgcg ctggccccgg tggcggtggg 2640ggcggcggcg
ctggagcagg cgcgggggcg gtgggcgggg tcgggggtgc tgctcgggac 2700ggccgcgcgg
gtgcggggcg cgcacgaccg caccgacccc ctggtgcgcg agctggtcga 2760ccgggggcgg
gcggcggtgg gcgggagcgc gttcgcggcg gcgtacgcgc gggggtggga 2820ggcggagcgg
gacgtggcgg cggcgttcgt gctctgagcg ccgggatcgg gcgggcgggg 2880tcaggcgggc
ggggtcatgt gggcggggtc aggcgggcca ggtcacacgt ccagggaccc 2940cgcccagtcc
gcgatcgtcc ggacttcggc ctgcgtcggg aagaccttct cggtgagcac 3000gcggtgcacc
tcggggtcgc cgtccaggca gccgtcggcc aggacggtga gctggaagtc 3060caggtcggcg
gcctggcgga gggtggacag gaccacgccg ctggtcgcga tgccggtgag 3120caccaggtgg
tcgacgccct gggcgcgcag gacgaggtcc aggtcgctgc ccgcgaacgc 3180gctgacgcgg
cgcttggtca ccaccacctc gtcgtcgagc ggcgcggtct cggggtggaa 3240gtcggtggcg
ccggagcccc tgggggccgc ggccaggcgg ccgaacatct tgttgcgcgg 3300gtggatctcc
gcgtagtcgg ggcggaagcc gacgccgacg tggatcaccg gcacggacgc 3360ggcgcgggcc
gcctcgagcg cggtggcgag cctggggagg taggccgggt cggggtagcg 3420ggcgaccacg
gcgggctgga cgtccatcac cagcagggcg ggggtgggga tctcgggcct 3480cgtttcggtg
gtggcggcgc gggggccgcc ggtgggggtc aggggtgcgg gggtgccggg 3540gtgagcaggc
tggtgacggt gagcaggcgg tcggcgagtt cctcggggcg cagcgggtcc 3600tcggcgcgca
cgacccagtc gtggacgatc gcgccggtgc cgtgggagat cagcacggcc 3660aggtcgcggg
cggcccgctc gtccacctcg cccaggccgg cgcgcagggc ctcggtggtg 3720atgagggtgc
ggaagagctc ggccagggcc tccgccagcc gccacgcgca cgggccggtg 3780agcacggcgc
ggtagaaggg gcggtggtcg gcgaagtggc gggccacggc caggaggcgg 3840gcgtggcgcg
gggcccgcgg gtcggccagg tgcggcagga gctcgcgccg caccaggtcc 3900gccgcagcgg
cgacgaggag cgtgtcgcgg tcgccgaagt gctggtagag cagctgcctg 3960ctgacgtcgg
cggcctcggc caggtcggtc accgggaccg ccgccccgcg ctcggcgacc 4020aggtcgacgg
cggcggccat gagggcggcc ctggagcggg cgacccggcg gtcggggcgg 4080gtggtcacgg
gggtgaaact agacagttgt caataaatga gcaagtgtcg tcgaacgcgc 4140gcgcgggaat
ctccggtgcg cggggcccgt ccctggcagc atgatcacgc gatgaccgag 4200gtgaggacgc
gcccgtacgc cgggcccgcg gacctgcgcg cgatgcaggg gttggcgcgg 4260cggatctgga
cgccgtcgag ccggtggcac gtcggcgacc tggcctggca gcgcaaccag 4320cacaccgggc
gcgaggccga gtggccgacc gcgctgtggg aggcgggcgg cgaggtggtg 4380gcgtgggggt
gggccgagct gccgggtgag ctggcgctgc tggtcgaccc cgcccggccg 4440gagcttgcgg
gggcggtgct cgactggttc gcgggcgtgg ccaccgcgcc ccggcggtcg 4500gtcaccgtgc
tggacgccga accgcacctg gtcgccgcgc tggaggctcg cgggtacgag 4560cggctgggcg
ggccgcactt ccggcactcg gtgcgcgcgc tggacgacct gccgacgccc 4620gaactgcccg
ccgggtaccg ggtccgcgcc gtgcggggcg aggaggacgt ggcggcgcgg 4680gtcgcggcgc
accgggcggc ctggtggccg tcgcgggtca ccgaggagag ctaccgggcg 4740gtgatggggg
cgtggccgta ccggccgggg ctggactggg tggtggaggg gccggacggg 4800cggttcgcgg
ccacctgcct gatctggttc gacgagcgca acggcgtggg cgagctggaa 4860ccggtcgggg
tcgaccccgg tctgcggcgg cgcgggctgg ggcgggcggt gtgcctggcg 4920gcgctgggcg
cgctgcgcga ggcgggcggg cgggcggcgg tggtgtaccc gctgcacggg 4980caccccgacc
accccgcgcc cgcgccgctg taccgggggc tggggttccg cgagcacgcc 5040cgcacgatca
ccttcaccgc gctggaggcg cgcgggtagc agcggccggg cggggcgagc 5100ggacccggtc
gacgagcggc tccgctgtcg gagcggtcgt cccagcgcgt ggacaccagt 5160gccacgacca
gaccgcgccc cgcttcgcgt ggtcggctcg ggggtcgacc gcggtgaggc 5220tctcgccggg
gtgggtgaac cacgtcctgg cgatggcctg caccgcgagc accgggtgcc 5280gcccgtggcg
ctggacgtca ccgacgcagc cgccgtcgac cgggccgggc cggccgcgtt 5340cggccgttgc
gccgcgccgg ccgagtccga cgccaggtgg cggccggtcc ccgggtccgc 5400ctggaactga
ccccgccggc ctccccgccc gcccgtccgg ccgggcgccg aacccgcctc 5460aggcgtgctc
gaccgcgcgc accgatcccc ccaccaccac cggcatcggg acgtggtgca 5520cggtcgtcgg
gctgcggtcg cggcgggggc gggacaggag gagttccacg gccatcgcgc 5580ccaggcggtg
gtgcggcagg gcgacggtgg tcaggcgcgg gcgcatccag gcggccacgg 5640ggtggtcgtc
gaagccgacc acggagacgt cgtccggcac ggacaggccc gcctccgcga 5700gcgcctggca
cgcgccgaac gccaggcggt cgttgaagca cagcagcgcg cgagggcggt 5760ggtgggacag
gaggtccagg gtggcgcggt agccgttctc cggcatccac tccacgcacg 5820ggcgcacgct
ctccacctcc acccccgccg ccgcgaaggt ctccagcgcg ccggagaggc 5880gggccacggc
ggcgatgtgg cgcgggtcga tcgcctcggc cgtgggcccg gtgccgatca 5940ggtgcacgcc
ctcgcggtgc ccggcgtcga gcagcacgcg cgccgccgaa cggccgccgc 6000cgcggtcgtc
ggggagcacg gcgtgcgcgg ggaagtcgtt ggcgggcagc acgttcagca 6060gcacggacgg
cccgtcgcgc agcccgtccg ggacctccag cagccggggg aacctggccg 6120cgaagaccac
gccctccacc tggcgggcgc gcagcgaggc caccagcgcc gcctccacct 6180cgcggtcgcc
gccgctctca ccggcgaaca gggtgaaccc gtgccggtgg gcggcgccga 6240ccgcgccctc
gatcagctca ccggacagct tggccgaggc cacggcgtcc gagacgaaac 6300cgagggtctt
ggtgcgggag gcggacagca gcgtgtcgcg gcggtagccg agctgctcgg 6360ccgtcgcccg
caccttgcgc tccaccgccg ccgagatgcg cagctcccga gcgcggccgg 6420agagcaccag
ggaggcggtg ggcaccgaca cggcgcaggc ggacgcgacg tcggccagcg 6480tgacgcgcgt
ccgcccgctc tcgcggacac ctgctcgcgg gggtgtgccc gtcacccgtg 6540cctcccgtca
ccggtcgcgc gacagccccg cgcgaggtcc taccccatcg tgcaggccgc 6600gccgttcaag
gagaaccccg aaggtggggc cgcgtccccg ccgtgggtga cctggtagcc 6660gatgctgact
ttgccaccgg gtgggatcgc cgcgttgtag cccgcgtcgc gggcggtcac 6720ccggcccgag
ctgggcgcgt acgaggcgtt ccagccggag gtgatcacct ggcccgcggg 6780cagcgcgaac
tccagcgacc agccctgcac ctgcgtggtc ccggtgttgg tgatggcgag 6840ctccgccgtc
aggccgttgc cccaggcgtt gacggtggcc gacacccggc aggcccccgg 6900ctgcggttcg
ccgggcgtgg tggtggtggt ggtggtggtg gtcgtggtcg tggtggtgct 6960ggtcgggtcg
gggccggttc cggcgaactg ggtgaagaac cgccaggtct cctcgggcgc 7020ccacgtcctg
gtgccgctgt cgccgggcgc gttgtcctgc ggtgcggcga tgtggccctc 7080gtcgaacgcg
acccagcgca ccgggtagcc gtcgcggcag ccggtgtagg tggtgccccg 7140gtgggtcagg
ctgccctggg acggttccgg cgggttctgc gcggcgcagc cgttgttgcg 7200cacgaaccgg
tcgcgcatcg agcgcccgcc ggagatgttc aggacgctgt cgcgcaggcc 7260gtggatgccg
aggtaggcga tgggctgcgt gccgccggcg cagccgctga gcacgccgcc 7320cgcgatgacc
gcgaccgcgc ggaacaccgt cggccgcgag caggccaccg agtaggacat 7380cgcgccgccg
tagctgaagc cggtggcgaa ccgctgggtg gtgtccacgc acagcccggc 7440gtcgagctgg
cggacgatgt cgtcgacgag ggtgatgtcc tcgccgccgt tgttggccca 7500gccgttgttg
aagccctgcg gcgccacgaa gatcgtgctg ctgcccgcca ggcgcttgag 7560gccgtagtag
gaccagacgt cccgctgcac ggtctggccg gtggcgacgt cgttcgcggt 7620gccgctgagc
cagtggaagc cgaagacgac gcggtggggg cggttccggt cgtagccgtc 7680cgggatcgac
aggatgtagg tgcgggactt gccgctgctg gtgatcgtgc gcgtgccgct 7740ggtgagcgcg
ggcgccttgc cgcagccctc cgtcgtggcg gacgcgccgg gggcgccgga 7800cgcgccgggc
gctccggtcg cgctggtggt gatcagcccc gcggcgaggg tgagcagcgc 7860gatgcccgct
gccgcgagga ccctgttgcg cgccaaggga ttcgcccttc ctgtggtggt 7920tccggtgggt
gtggtcacgg ggtggtgagg tcgaagcggc gggcggtgac ggagccgccg 7980agcgcggcgg
tggcgtggtt gaagacggcg aagcggtagc ccatgaagaa ccgccagtcg 8040ttcttgagcg
tgaacgccgg gccgaaggcg gtgaagttga cgccgtcggt gctgtaggag 8100aaccgggcct
gcctgccgtt gccggggcgg atgtcggcgt tggcgcgcaa ccagatccgg 8160gagccgccca
ggtcggcgct cgcgacctcg tagccggttc cggtggtgcg ccaggagccg 8220tccatggtca
ggccggtgac ggagacgatc cggttgcggc cgttgtcgcg cttgacgccg 8280atccacgccg
aggagtcgcg cagcacggcc agcccggtgc ggtcgccgtc gcgcatcccc 8340gacaggtcca
gttccacggt gccggtggag gtggggccct ggatgcggtg ggtgagggtg 8400ttgcgggcgg
agtacaggtc gttggtgacg gtcgcggtgg acaggcgaag gccgttgttc 8460acgctgtact
tggcggtgtc cgggttgtgg ttccactccc actgcgggcc gagcgcggcg 8520ccggagaagg
tgtcggcgcc gatcatgggt ttgacctggc gcgggggcgc gggcaggttc 8580ggcttcgggt
aggtcgcgcc ccagccgccg ttgacggtgg tgacgcgcgg ccagccgtcc 8640gaggtccagg
tgatcggggc gagcaccggc acgcgcccgc cggggtaggc gtcgacgaac 8700gccaggtagt
gccagtcgcc gttctgggtc tgcaccaggc cgccctggtg cggcactccc 8760ccgccctgga
tcggcgaggg caggtcgagc agcacctgct ggatcgagta cgggccgaac 8820gggctggacg
acttgagcac gtactggccg ttcgcgggcc tggtgagcca gatgtagtag 8880ttgccgccgc
gcttgtagaa gcgcgcccct tcgagggtgc cgatgttcga gggggtctgg 8940aacacctgct
gggagcggac ctccgacttc ccgtcggcgg agagctgggc gacgctgatg 9000ctggtgttgc
cgtaggcgac gtacagggtg tcgtcgtcgt ccacgagcat cccggcgtcg 9060tagtagcact
tgttgatggt ggtgtgcttg gaccactggc cgtcgacggc ggtcgcggtg 9120tacaggtgcg
tctgggcgaa gtcgacgcag ccgccccagt agaaggtgcg gttgctcttg 9180cggtgcgcca
ggaacgacgc ccagatgccg ttgacgtacg cgcgggagcc gttgcccatg 9240tcgtacttgg
ccccgaagtc caggcgtggc acggagtgcc cggcgaactc ccagttgacc 9300aggtcgtagg
agcgcagcac gggcgcgccg ggcgagtagt gcatggtgga ggccgagtag 9360tagtaggtgt
cgtccacgcg caggacgtcg atgtcggcga agtcctgcca cagcacgggg 9420ttggtgtagg
tcccggccgc gcgggccggg tgggtggtgg tggcggtcag gctcgccgcc 9480acgaggccga
gcacggccag ggcgatgggc gcccatcggc gttgacgggg catcggtgtg 9540cctctcctgg
tgtccgggag ttggctctgg gcgcggcggc ggtggacttg tcgggcgcgg 9600cggtggtcgt
gggggtcagc agggggagtt ggtctgggtc agcaggccca gccgccaggg 9660caggcggttg
tagtcgccgg acgcgttggg gtccaggccc tggtacaggt agctcagctt 9720gcaggggttg
atctccatgg tctggtcggt cccgctgcgc accagctcgc cgtggctgat 9780gtcgcgcgtc
cactggccac cggggaacgt ggtgttgttg gccctggcga acgggttcga 9840ctcgctgtcg
gccagcgcgg tccacggtcc ggcgatcgcc ggggcggtcc aggagcggaa 9900ccagcggcgg
ccgtccgagc cgatcgcctc gtggagcatc agccactggt tcttgccggc 9960gaccttgtag
atgttggacg cctcgaacaa ccggttgcgg ttgctgtcct gcatggcgat 10020cacggtgttg
gtgaagccgt tggggaactg ggcgaggctg gtctccgagc ggtacaggtg 10080gccgttgtcg
tccgaggaga acaggtggca cttggccgtg tcgcagacgg tccagaagtc 10140gacccagtag
ccgttgccga tgttgtcccg gatgatctgc ggcatcccgt tggcgtagaa 10200gttcctcggc
gcggaccagg acgcggggtt ctcgatgtcg gcggtcgtcg agtacgaggc 10260gttggacccg
gtctggtaca ccaggtacca caggcgttgc ggggcgaagt agaacacctg 10320cggcgcggcc
cggtagcccg tgccgatccc ggagcggtcc aggtagtggt gcggggcgga 10380cgcggcctgg
gaccagtcgg tgaagctggt gtgcacgagg ttgtagccgt tggtgtagac 10440cgaggcgaac
acgtggtagc ggccgttgtg gcgcaccacg ctggggtcct tgacggagac 10500cgtggcgtgc
gaggagtcgg gcttgggtcc gatcagcgcg ccgctggagg accaccggaa 10560gctgctcggc
agcgagccgc ccggttgctg cggggtcgtg gtggtgggcg gcgtggtcgg 10620gggcgtggtg
gtcgtgggcc cgactcctcc cgtgcacgtg gtcccgttga ggctgaacga 10680ggtcgggtcg
gggttggacc cggtggaggt cgcggtgaac ccgaactcga cgcggccccc 10740ggtggggatg
gcggcgttgt aggaggcgtt gcgggcgctc acctggccgc cggactggga 10800cacctcggcg
ttccaggcct gcgcgacctg ctggcccgag ccgtaggtcc aggtgagcgt 10860ccagccgtcg
acggcgtcac cgaggttggt gatggcgacg ctcgcggtga aaccgccctg 10920ccactgggag
gtcgccgcgt aggcgatcga gcaccccggc gccgcggcgg cctggggtgg 10980gagggcggcg
agcgcggcca ccatggcgag cgaggtggtg gccatggcgc cgatccgggc 11040ccggcgcggg
gtgaacagcc ttgcgaggag catggtcgcc cttcgtcgtc gtcgacgggt 11100ggtcggcgcg
gccgaccggg agcggcggga gcgggctggt actcccccac ttcctgcaat 11160ctagccaggt
ggcacagggt ggtcaaagct aaaaagggcg gacgcggttt agcttccagc 11220gcaaaggttt
cgcgcgttct ttcggccggg gggcaggtgg atcggggcgg gctcggggcg 11280aggacggggc
tgggaatggg gcgggggatg gggcgggctc ggggcggggg tcgcccggag 11340cccgccacgg
gtcagaggcg cacgcggacg acggtgaacg ggaggttcgg ctcggcgatc 11400tggtactgga
agttgaccac cagcaggtcg tcgccgtcga aggtcgcggt ggacgggacg 11460tccatgcccc
tgccgttgac ccgccgcagc accgtggcgc gggagtggtc ctcgctcagc 11520cgcaggacgc
tgatctcgcc ctccgggtgg aacaggctgg tgacgctgta gaggtcgttg 11580ccgcgcagga
gcagcccgtc cgagccgatg tcgcccacgc cgcccaggtc gatcggggtg 11640acggcgccgg
tgcgggtgct gatgcggtgg aacgcctggg agttggtgtc ggcgagcagc 11700acgtggcggc
cgtccggggt gaccacgagg ccgttgccgt tgatgccctc ctcgtagcgc 11760accggggagt
cggcgaggtc cacgaacgtc ctcagtggct ggtcgacctc ggggctcgcc 11820agctgggcgg
cggtgatccg gtagaggacg gggcggaacg agtcgctgac gtaggcgtcg 11880ccgttcgggg
cgatggcgac gtcgttgacc aggccgtcgc gggcgccgga gtcgaacacg 11940tgcaggagcg
cgccggtgcg ggtgctgtgg acgaagacct tgccggtggc gccgcccgcg 12000atgaccagcc
tgcctcgggt gatcttcatg ccgacggcgg tggtgcggcc gtgctgaccg 12060gcgggcagga
acggctccag ggcggggcgg tcgacgtggc cgcgccagat cgtgccgtcg 12120gtcgtgccgc
cgacgtagaa gtgcggcgtg cccggctcgc ggacgatgcc ctccgggtag 12180gcgcggtcgc
cggggaccac gtagcgggtg acggggtggt gcgcggcggc ggcgggtggc 12240acggcggcgg
cgggtggcgc ggcggcggcg ggtggcgcgg cggcggggag ggctggggcg 12300agcgcggtga
ggagcagggt cgcggtgagg agggctcggg tggtggtcac ggaagggctc 12360cgggggtcga
aggggtgtct ggcgccagac aagcgcttcg tggcgcgggg tggcagtggg 12420cgcttgtcgg
gggtagttct tcacccccct tccgggcggg gcggccgact agggtgagcg 12480gtgtgggcga
tcttgggcgg cacccggtgg cgaaccgctc cgacgtgcgg gacttcctgg 12540tcagcaggcg
cgcgagggtg agtccggggc gggccgggct gcccgtgctc gggcggcggc 12600gggtgccggg
gttgcggcgg gaggaggtcg cgctgctcgc cggggtcagc gtggactggt 12660acacccgctt
ggagaagggg cacatcggcg gtgtctcgcg ggaggtgctc gacgccgtgg 12720ccggggtgct
gcggctcgac gccgaggagc gggtctacct gttcgacctg gcgcgcgcgg 12780cccggcgtcc
ccgcgccgcc gaggtggcgg cggaggccgc gctgcccgcg acggcgcagt 12840ggctgctgga
cagcatgacg ctgtcgtcgg cgatggtgac cgggcggcgg caggacgtgc 12900tggcggtcaa
cccgctggcc cgcgcgctct acgcgccgct gttcgccagc gccaccacgc 12960gggacggcgg
ccgggcgaac ctcgcccgct accacttcct cgacgcgggc gcccgcgagt 13020tctacgggga
ctgggcgggc accgccgacg tgctcgtcgc cgcgctgcgc gccgaggccg 13080ggcgcgaccc
gcgcgacggg gccacccgcg agctggtggg cgagctgacg gccgcgagca 13140ccgagttccg
ggcgcggtgg agcgcgcacg acgtgctgct gcacccgcgc ggcgccaaga 13200ccttccggca
ccccgaggcg ggtgagctga gcctgagcta ccactcggtg gacctgccga 13260tctccgccac
cgagacccgg cacgtgtgcg cgtgcaccgc cgaacccggc tcgaccgacg 13320aggcgaggct
gcgcgcgctc gtcgggtgag ccgggggtgg ccggccaccg ccgtcgcgct 13380cgcggcggcg
gggcggggcc gccggtcaga gcgtgagcgc catcccgatc gagccgggcg 13440cggtctcggt
gaagccgtgc ttgaggtaca gcgggcggcc gggcgcgtcg gccaggaggg 13500tcacgaacgc
gccgggcggg gcggcctcgc ggatgcgccg gagcagcgcg tccatgatcg 13560cgccgccgac
gccccttccc tggtggtcgg gcagcacggc catgtcgacg acgtggaagt 13620accagccgcc
gtcgccgagg acccggccca tgccgacggt cccgccgtcc gcgtgcgtga 13680cgtggaagga
ggcccaggcg ccgggcaggg cggcggcggc ctgctcggcg gtcttgggcg 13740acaggccgga
ctcggcgcgc aggcggaggt agtcggcgac ggacggcggg gtcgggtgga 13800gctcgtagtc
ccggtgcacg cggtcaggct cccacgggcg cgggcgggcc cccgcccgac 13860ctgacgattt
ccccgtcggc ggggatgcgg gcgggcgctc gcggattttc gacatccccc 13920ggcccggcga
gacgcggcgg cgccgctgaa aagagcgccg tcgcggccct tcgcgccgcc 13980cccgacatcc
cccgcgcggc gaccggtcaa tgcggtccac gcctgggggt ttccctccca 14040cgtcgaacac
cgccaccacg cgcccacgcg ccgcgtcgac caccccgacg ccgaggaaca 14100cctgttcacg
ggcacgggaa gccgcagcgg agggggaacc gggaatggcc gcaggcgatc 14160gcggcacgac
gtccgcacat caccgcgagc agaatcgcga ggcgttcacc ggggcggcgg 14220aggaagattc
cagcgcccct ctcgaagaac ctgcgggaag ccctggaaga aaacccggac 14280ccgaaacgcg
acaaaattgc ggacacccac ccgtgaaaca ccgggcgccc ccaccaggtc 14340acccgctgac
atcacgctca gtcagtatcg gcacgctccc ccgccgaggc ggagcgcgac 14400accccgccca
accgggcacc gagcgggcac ctccactcgg cccagcaccg ccccaagatc 14460gcacgtagca
cgggttgaaa ccgctcaagc gcatctcaac ccgttcggag cagagtggcg 14520cccgtcacgt
cccgaccggt cacggttggc aacgggtcca gtccacgcga ggtggcatca 14580agcgcacttg
ccccgatcac acccgcccgt gcaaccgaat gcagcaggga tatctttccc 14640gagaactcgg
ccgttaaccg ggagtggagc caggcccacc cctaagacgc ttgcccacat 14700gcccaacaat
ggtgaagatg gaacggcgga ccgcaccgcg aacgcgaacc gaactccgcg 14760agagggcacg
gtgaacgatc ctggaacagc tactgccgcg tagctcaagg gtggaacgcc 14820cggctcgcgc
gcggcggagg gaataacggc ttttacgccc tcgacaacag cttgtcaacg 14880aaacccgtgc
acccgagcgg tccccgcgcg caccgctcgc gggggtggcg cggcgcacga 14940cgtggctgcc
cggcgtcgac gacgacgcga gttccccgac cgcccgcgaa ggcggtcgcg 15000gatcgccacg
acggggcacc cggaccacgc ctcccccgga acagcgcgcc cacgcgcggt 15060tccggcgcgc
gccgggaccg ccgcacccgc cggagcgccg ccaccggccg gggccggtcc 15120ccgcggaccg
ggtcgccttc cggaccacca ctccacggac cacggaaagg accactcccc 15180cagtggagct
tctgcgcgca cccgagatcc agtcggccgt cgagcacctc gcggtggacc 15240tgccggaccg
ggcgggacgg gcgttcctgg tggacggacc gcccgcctgc ggcaagacga 15300cggccctgcg
gcggctcgtc gaccggatcg cccacgagga ccacctcgtg ctcaccgcca 15360cctgcacccc
gccggagacg gagctgccgt tcggggtgct caagcagctc ctcgcctccc 15420ccggcatggc
cagggtcgac ccgcgcctgg tcgccgacct cggcgagctg ctcgccccgg 15480ccccgccgcc
cgccgacgac tcggcgctcc tgcagctgta ccactcgctg tgcgcggcgc 15540tgatcgcgct
gtccgaggag gtgccgctgg tcatcgcggt ggacgacgtc cgccacggcg 15600acaccgcctc
gctgcacgtg ctgctgcagc tggtgcaccg gctggacacg gcgcgggtgc 15660ggctgctgct
caccgacgac ctgctgctgc cggtgagctt cccgccgctg cgctacgagc 15720tgctgcgcct
gcgcgggctc ggcctggtcc gggtcgcgcc gctgcctgcg gccagggtgc 15780gggaggaggc
ggtgcgggcg gtcggcgcgg acgtcgcgaa gcgggtcgac ttcgccgcgc 15840tgaccggcgg
caacccgctg ctgctgcacg cgctggcggt ggacgtgctg gaggcgggcg 15900agccgcgcga
gatcggctac ggcaactcgt tcctgtcctg cctgcaccgc aacgaacccc 15960tgttcctgga
caccgtgcgg gcgctggccg tgctgggcgg cggctcggcg tcggacctgg 16020gcaggctgtc
cgggcacgag ccggagcagg tcgcccaggt gctgaacgcc ctgcgggagt 16080cggggctgct
ggccgaggac gggttccggc acgacgcggc gcgccgcgcg gtcgtcgcgg 16140acaccccggt
cgccgagcac gaggtgctgc accgccgcgc cgcgcggctg ctgcgcgacc 16200agggcggcgc
ggtcaccgac atcgccgacc acctgctgcg ggcgggccgc atcaccgacc 16260cgtgggcggc
ggacctgctg gtggacgcgg cggagctggt ggtgcagcgc ggcgagccga 16320cggcggcggt
ggcgctgctc cagcgcgcgc tcgactgcag cccggaccgg gagcgcagga 16380cggccgtgca
ggcgcggctg gccacggccg agtggctggt gaacccgtcg acctcggcaa 16440ggcaccacac
cgcgctgctg gcggcgttcc acgcgggcag gttgtcggtg cgcgacagcg 16500cgacgctgat
gaagcacctg cgctgggccg ggaacaccgc cgactcggac gcggtgctcg 16560cccggctgcg
gaccgacccg cgcgccgccg aggacgtgcc ggtgctggag cactggctga 16620ccagcaccta
ccccggcgcg gcccggccca ggaccgtgct ggggcgggac gtggactcgg 16680cgcgcagcag
ggcggacctg gtgccgaggg cgaacgcggt gctgctggac gtgctggtgg 16740ccggggacag
cgacgacgtg gccgaccggg cggaggcggt gctgcgggag ctgcggctgg 16800cgcgggagtc
cgggtgctac ggcggtgcgg ccgtgctggc gctgtccgcg ctgctctact 16860cggaccgcgc
ggacgtggcc gcctcgtggt gcgagcagct gctgtcggcg cgggccgtgc 16920cgctgctgcc
gatgccgcgc gcgcaggtgc tggcgctggc ggccgagtcg gcgctgcgcc 16980ggggcgacca
cccgagcgcg gacgagctgg cgcgggaggc gctgaccgtg gtgtccccga 17040ccgcgtgggg
ggtgtcggtg gggctgccgc tgagcaccag ggtgctggcg ctgaccagga 17100tgggccgcta
cgacgaggcg gcggccgtgg tggcgcagcc ggtgccgaac gggatgttcg 17160ggcaccgcaa
cagcgtggac tacctgtacg cgcgcgggca cttcttcctg gcgcgggaac 17220ggccgcgcgc
ggcgctgggc gacttcctgc tgtgcgggga gcagctgacc cggtggggcc 17280tgggcagcgg
gtgcgcgccg gtgccgtggc ggaccgcggc ggcggaggcg tggctggcgc 17340agggcaaccg
ggaccaggcg cgggtgctga tccacgagca gctcggcagg ccgggcacgg 17400acagccgccg
ggcgcgcggg caggcgctgc ggctgctcgc ggcgaccagc tcggtgaagc 17460ggcacccgca
gctgctgcgg gaggcggtgg cggtgttcga ggccgtcgac gacaagtacg 17520agctggcgcg
gaccctgcgc gacctgggga gggcgcagcg ggcgctgggc gagaacaagc 17580tggcgcgccg
ggtgatccgg cgggggtggc acgtcgcccg gatgtgcgag gcggcgccgc 17640tgtgcgagga
gctgatgccc accgccgacg ggctggtgcc cgcgcagccc gcgtcggcgg 17700cccgcaggtc
ggacctggac cggttgacca gctcggagca ccgggtggcc gcgctcgcgg 17760cgtcggggct
gacgaaccgg gagatcgcgg tgaagctgta cgtcacgcac agcacggtgg 17820agcagcacct
gacgcgggtg ttccgcaagc tcgggatcaa gcagcgggag cagctgccgc 17880cggagctgag
cgtcgaccgg tcgaagtgac gcggacgggg cggtcccgtg gatctggggc 17940cgccccgtcc
ggtcccggtc cgtccggtcc cgccctgtcc ggtcccgccc tgtccggtcc 18000cgccccgtcc
ggtcccggtc cgcctcaggc tcggggcatc gcggccaggg tggtggcgac 18060gacgtgctcg
tcgacgtcgc gcaccagctc ggcgccgcgc gggccgtcga gcacgaacgc 18120caggccgccg
gtggacttct tgtcgcggcg catgaaccgc agcagctcgt cgtccggcac 18180gcccggcggc
agcgcgacgg gcagcccgta gcccgcgacc acggagtggt gctcggccac 18240ccggtccggg
ccgatccggc cgagcgcgcc cgcgaggcgg ccggcgaaga ccgtgccgat 18300cgcgacgccc
tcgccgtgcc gcaccgcgaa accggtggcg agctccaggg cgtggccgag 18360ggtgtggccg
tagttgaggg tgtgccgcag gccggagtcg cgctcgtcgg cggccacgac 18420gcgggccttg
agggcgacgc tcgccgccac ctggtccagc agcggcagcc ggtccaggcc 18480cggcgcgccg
atgaagtggc agcgggcgat ctcgccgagg ccgttgcgca gctcgcgctc 18540gggcagggtg
gcgagcaggt cgaggtcgca cagcacggcg gcgggctgcc agtaggcgcc 18600gacgaggttc
ttgccctcgg ggaggttgac cgccgtcttg ccgccgacgc tcgcgtcgac 18660ctgggccagc
agcgaggtcg gcacgtgcac caccggggtg ccccggtggt agagcgaggc 18720ggcgaggccg
accgcgtcgg tggtggtgcc gccgccgcag gagacgacga cgtcggcgcg 18780ggtcaggccg
aactcggcga accggctgca caggtgggcg acggtggcga gggtcttgtc 18840gtgctcgccg
tcgcgggccg ggaggacgag ggaggggacg ccggggtcgg gcgtctggtc 18900cgccgggcgg
gcggtgacca cgacggcgcg gcgcgcgccg agggcccgca cgacgtccgg 18960gagggcggcg
cgcacgccgt gtccgatgtg gacggtgtag gcgcgctcgc ccagctcgac 19020ccggacctcg
cgggtggtgg cggcggtggg ggcggggtgg gtggagctgg gcactgcttc 19080ctcctcgggt
gggcgggacg gggggcgatc gggggacgcg gaggggtgac gggaaagcaa 19140tcgggcagga
atgggaacgg gtccgggggc gaacgggcag gaattcgaat gggggcaagc 19200gaccgggagc
gatcccagtg gtggggcgga agtgcgggcg gcggaaaggc ggtcgtgctg 19260cctcagccgc
cgcccgcggc gcccgtcacg agcgtggtgc gcagggtgag cgccgcccgc 19320gccgccacgg
ccgcgccgac gccgaggcgg ttgtgggtca gggtcgcctg ctcgaacagc 19380accggcaggg
ccgggcggcg gcggtccgcg gcggcgcgca gctgctccag gtagcgccgc 19440cacgcgcccg
ccgagccgac cacgcgcccg ccgggcggcg ccccctgcgc ggcgacggcg 19500gcctcggtgc
gctcgaccgg gcggagcggc ctgctccagc tctgccagca gtggaacagg 19560aacgcgggcc
tggccggttc cggggccagc gcgaccagtt cggccaggtg gtgcagcacc 19620gtggcctgcg
cgcccgactc gccagggtcc tcgccccaca ccgggctcac caccgacgcg 19680acgtccaggg
ccagttcgct ggacgcggtc gcgaccagcg gctcgaccgg cccgcccggc 19740aggcccccac
ccggcaggcc ctcgcccgcc gggtcgacgc gcaggtcgcg ggccgccgcc 19800tcggcgcaca
gcacggcgag caccgggccc cgcgcggcct cggtcgtgcc cagccacagc 19860gccaccaccc
ccgccccgcg caggaacgac cagtgcgcgc cccggtcccg cgcgcgcagc 19920tcccgcacga
cggggggcag cacctcggcc accgagcggg ctccaccgcc tgccagcgac 19980cagcccgacc
aggacggggc gcccgccgcc gggggtccgc cgaggggcgc tctcgcggaa 20040ccggtccgcg
tcatgtccac caccacttcc gccttggcga gaacgggtcc tgcgggatca 20100ccgcgctgtt
ccgacgccgc cgacaatagc gacgcgcaat acgccgaatt caccgccaaa 20160tcaggtcagg
ggggttgagg gggatgcctt agggggcgag tgcccgcaaa gcggaagaag 20220aatcggaagc
acatgcagga gcgacttcca agctcaggcc gcaggaccgg gtccgcgtcg 20280tcgcggacac
cccggtcctg cgcgtgcgcg caccgaagga cgtggtgaca tgcttcggac 20340cgacctgatc
cgcccggttc ccgaactgct cggggccaac gcggatcgct tcggcgacag 20400gaccgcctac
tcggacggtc gccgttcggt cgggcacgcc gggctggaac ggcgcacgcg 20460ccgcctcgcc
ggtcacctcg ggcagttgcg gctgcacccc ggcgaccgcg cgatgatctg 20520cctgggaaat
cgcgtcgaaa tgatcgagag ctatttcggc gtgctccgcg cggacgccgt 20580ggcggtcccg
gtgaacccgc gttccaccga cgcggagctg acccacctgc tcgccgacag 20640cggggcccgg
ctggtgatca ccgacgcggc gcgcgccgag cggttcgacc ggttgcgcgc 20700cgagcggttc
ggcgacctga ccgtgatcgc cacccaggac ggcccgctgc ccgacggcgt 20760catcgcgttc
gagccgctgg ccgccgagga gccggagctg cccgcgcgcg acgggctcgg 20820gctcgacgac
gtggcctgga tgctctacac ctccggcacg accgggcgcc ccaagggcgt 20880gctgtccacg
cagcgcagct gcctgtggtc ggtggccgcc tgctacgtgc cggtgccgga 20940cctgcgcgcc
gaggaccgcg tgctgtggcc gctgccgctg ttccacagcc tgtcgcacat 21000cacctgcctg
ctggccgcca cggccgtggg cgcgaccacg cgcatcgtgg acggcacgtc 21060cgcgcaggac
gtgctcgcgg cgctggagca ggagcggtcg acgttcctgg cgggcgtgcc 21120gacgctgtac
cggtacctgg tcgacgccgc ccgcgagcgc gggttcaccg ccccggacct 21180gcgggtgggc
ctggtcggcg gggcggtgac gacggcggag ctgctgcgcg cgttcgagga 21240cacgttcggc
gtgccgctga tcgacgccta cggcagcacc gagacgtgcg gggcgatcgc 21300ggtgaactgg
ccgaccgggg cgcgcgtggc gggctcgtgc gggctgccgg tgccggggct 21360gacggtgcgg
ctggtggacc cggagacgct gctggacgtg cccgccgggc gggagggcga 21420gttctgggtg
tcggggccga gcgtgatgct gggctaccac aaccagcccg aggcgacggc 21480cgaggtgctg
cgggacggct ggtaccgcac gggcgacctg gggcggcgcg acgaggccgg 21540gttctgcacg
gtcaccgggc ggatcaagga gatgatcatc cggggtgggg agaacgtgca 21600ccccggcgag
gtcgaggccg tggtgcgggc ggtgccgggg gtggcggacg tcgccgtcgt 21660gggcaagccg
cacgacgtgc tgggcgaggt gccggtggtg ttcgtggtgc cgggcgcggg 21720cgggttcgac
ccggcggcgg tgctggcggc gtgccgggag gagctgtcgt acttcaaggt 21780gcccgaggag
gtctacgaga tcgagcgggt gccgcgcacg gcgtcgggca agaccacccg 21840gcacgtgctg
ctggacctgc ccgcccggtt gcgggcggcg tcgagcgggc agttccagtc 21900gctgctgcgg
ctggactggg tgccgaggac ggcgctgccg ggtgaggagg tcccggcgag 21960ctgggtgctg
gtggacggcg acccgctggg gctcgcggac gggttgcggg ccacgggcgc 22020gcgggtgcgg
gtgggcgagc cgggcgcgga tgcgctgggc gacggcggat cggacgccga 22080cgagccgggc
gcgagcagcg cgggcgaacc gggctcgggt ggctcgggtg agccgggctc 22140gggtggctcg
ggcgaaccgg gctcgggtga accgggcgcg agcagcgcgg gtgagccggg 22200tgcgggtgag
ccgggcgcgg ccgaaccccc gcaggtcgtg ctggtcgccg cggtccccgg 22260tgagcgtggt
gaggtcgcgc gggacgtgga ggcgctcgcg gacgggctcg cgcggcggct 22320cgtcgggtgg
ctggccgacg agcggttcgc gggggcgcgg ttcgtcgtgg ccacctcggg 22380cgcggtgtcg
acctcccccg gcgaggacct gcgggagctg cgggcggccc cgctgtgggg 22440tgtggtgcgg
tcggtgcagg ccgcgttccc cggtcgggtg gtggcggccg acctggacgc 22500gtccggcgac
gggcgggcgg cggcgctggc tcgcgtcgtc gcgggcgggc acgaccaggt 22560ggccgtgcgc
ggcgacgtgc cgctggcgcc ccggctggcc agggtgtccg tgccgtccga 22620cccggccccc
gccccggcgc tggacccgga cgggctggtc gtggtcaccg gtggcgactc 22680ggcgcgcggc
gcggccctcg cgcggcacct ggtggccgcg cacggcgcgc ggcgcctgct 22740gctggtctcc
cccgacgggc tgcccgacca ggccgccgcc gacctggagg ccgggttcgc 22800ggcggcgggc
gcgcgggcgg agtcggtggt gtgcgacccg gccgacccgg tcgcgctgcg 22860cgccctgctc
gacgcgcagg accgcccggt cacggccgtg gtgcacgtgc agggcgggcg 22920ggcgctgctg
gactcggcgc gcgccctcgt cgccctgcac gagctgaccc gccaggcgcg 22980accggcgctg
ttcgtcgtgg tcacctcggt ggccgggctg ctgggctcgg cgggcgaccc 23040ggcgcgcgcg
gcggccgacc agttcgccga ggcgctggtg cgcaggcgcg ccgaccgggg 23100cctgccgggg
ctggccgtgg cctggggtcc gctgccgggc gagcccgcgc aggcgggcgc 23160gggcgcgctg
ccgatggccg aggcgctgac cctggtcgac gccgcgctcg ccgccgacca 23220gggcccgctg
gtggtgctcg ggctcgacgc ggtcgggtcg cggcgcgcgg tgggcgcggt 23280gccgccggtg
ctgcacgacc tggtcgacgg cggtcgcgcc gcgcgggtcg cgccgggcgc 23340ggtggccgag
ttcacgcgca ggctcgcgga ggcgggtggg cagcgggccc gacgcgtcgc 23400gctggacctg
gtgcgcgagc acgtcgcggc ggcgctcggc ctgcccgagg acaccccggt 23460gcgcgccgac
caggcgttcc gcgacttcgg cgtcacctcg ctgaccgccg tggcgctgcg 23520cgaccggatc
aacgccgcga ccggcgcgtc cctgcccgcg acggcggtgt tcgaccaccc 23580gaccccggcc
gcgctcgccg accacctggt gcgcgaggtc accggcgacc ggccgcacgt 23640cgagcgggcg
cgggacgagc gggcgcgcgg gacctcgcgc gcggacgagc cggtggcgat 23700cgtcgccatg
gggtgcaggc tgcccggcgg cgtggcctcg ccggaggacc tgtggcggct 23760ggtggacgag
ggcgtcgacg cgatcggccc gttcccgacc gaccggggct gggacctggc 23820caccctgctc
gacggctcgg actcgccggg gaggtcctcc gtggaccgcg gtggtttcct 23880gccgggcgcg
ggcgacttcg acgccgggtt cttcggcatc tccccgcgcg aggccctggc 23940catggacccg
cagcagcggt tgctgctgga ggtggtgtgg gagaccgtgg aacgcgccgg 24000gatcgacccg
cgctcgctgc acggcgaaga cgtcggcgtg ttcagcggcc tgatgtacca 24060cgactacggg
accgaacccg gttccgcgcc ggagggcctg gaggggttcg tcagcaccgg 24120cagcgcgggc
agcgtggtct ccggccgcgt cgcctacgcg ctcggcctga ccggcccggc 24180gctgaccgtg
gacacggcgt gctcgtcgtc gctggtggcg atccacctgg cggcgcaggc 24240gctgcgctcg
ggcgagtgct cgatggcgct cgcgggcggg gtcgcggtga tggggcagcc 24300gacgtcgttc
gtggagttct cccggcagcg cgggctcgcc gccgacgggc gctgcaagtc 24360gttctccgac
gacgccgacg gcacgaactg ggccgagggc gtgggcgtgc tgctgctgga 24420gcggctctcg
gacgcgcgcc gcgacgggca cccggtgctg gcggtgctgc gcggcagcgc 24480ggtgaaccag
gacggggcca gcaacgggct gaccgcgccc agcggcccgg cgcagcagcg 24540ggtcatcagg
caggcgctgg cgaacgccgg gctgcgaccg tccgaagtgg acgccgtgga 24600ggcgcacggc
accggcacca ccctgggcga cccgatcgag gcgcaggcgc tgctcgccac 24660ctacgggcag
gaccgcgagc agccgctgtg gctgggctcg ctcaagtcca acctcgggca 24720cgcgcaggcg
gcggcgggcg tcgcgggcgt gatcaagatg gtgatggcgc tgcggcacgg 24780cgtcctgccc
cgcaccctgc acgtcggcac gccctcgtcc aaggtcgact ggtcggcggg 24840cgcggtcgag
ctgctgaccg aggccaggcc gtggcgcgcg aacgggcggc cacgccgggc 24900gggcgtgtcc
tcgttcgggg tcagcggcac caacgcgcac gtcgtggtgg aggagcaccg 24960ggaaccggcc
gccgcgccgg tcgacccggt ctcccccggc ctggcggtca gcggcggcgt 25020cgcgccgctg
gtgctgtccg ggcgcacccg ctccgcgctc gccgcgcagg ccgcggccct 25080gctggggcac
ctggccgacg ggaccgaccc ggcggcgctg ggccgcgcgc tcgccaccac 25140ccgcaccgcg
ttcgagcacc gggccgcggt cctcgcgccg gacgtcgacg ccgcgcgcgc 25200cggggtgcgc
gcgctcgccg aggaccggcc cgcgccgaac ctggtcaccg ggcaggccga 25260cgtggacggc
ccggtcgtgt tcgtcttccc cggccagggc gcgcagtgga ccggcatggg 25320ccgggagctg
ctggagacct cgccggtgtt cgccgcgcgg ctgcgcgagt gctcggaggc 25380gctggagcgg
tggaccggct ggtccctgct cgacctgctc gccgacgggg cggagctgga 25440ccgggtcgac
gtgctccagc ccgcctcgtg ggcggtgatg gtggcgctgg ccgcgctgtg 25500ggagtcgtgc
ggggtgcgcc cggacgccgt ggtcgggcac tcgcagggcg aggtggccgc 25560cgcgtgcgcc
gccgggtggc tgtcgctgga cgacgcggcc agggtggtgg cgctgcgcag 25620ccgcgcgatc
gccgagcacc tggccgggcg cggcggcatg atgtccgtcg ccgccggggc 25680ggagcgggtg
gccgggctga tcgccgaccg gcagggccgg gtgtcggtgg ccgccgtgaa 25740cgggccgtcc
gcgaccgtgg tggccggggc cgccgacgcg ctgcccgagc tggccgcgcg 25800ctgcgagcgg
gagggcgtgc gggcccggat catcccggtg gactacgcca gccacaccga 25860gcacgtggac
gcgctcgacg gggtgctgca ggaggtgctg gcgggcgtca ccgcgcaggc 25920cgggcacgtg
ccgtggctgt ccaccgtgga cggcgagtgg gtcgacggct cggggctgga 25980cgcggactac
tggttccgga acctgcgcgg gaccgtgcgg ttcgccgacg cggtggcggc 26040gctggcgggc
tccgggcacc gggtgttcgt ggaggtgtcc agccacccgg tgctcaccgc 26100cgcgaccggc
gaggtgctgg aggccgccgg ggtgcgcgac gcgctggtgg tcggctcgct 26160gcggcgcgac
gacggtggcc ccgagcggtt cctcaccggg ctcgccgagc tgcacgcgcg 26220cggcgtcccg
gtggggctgg aggcggtgtt cgcgggcgcg gacgggcggg tggagctgcc 26280gacgtacgcg
ttccagcacg agcggtactg gctggcgcgc ggcccggtgg ccggggacgt 26340gtccgggtcg
gggctggtgg acgcggcgca cccgctgctc ggggcggtcg tgccgctgcc 26400gggcacgggc
ggggtgctgc tgtccgggcg gctctcgcac cggcggcagc cgtggctggc 26460cgagcacgcg
gtggccggga cggtgctgct gccgggcgcg gcgatcgtgg agctggccgt 26520gcgcgcgggc
gacgagaccg ggtgcggggt gctgcgggag ctggtgatcg ggcagccgct 26580ggtggtgccg
ccggacgccg aggtggacct gcaggtgctc gtcggcggcc cggacgacgg 26640gggcgtgcgg
gacctgcggc tgtactcgcg gaccggggcg gcggcggagt gggtcgagca 26700cgcggcaggc
gcgctcgccc ccggcggcgc ggtcggcggg gcgcgaccgg ccggggcgcg 26760gacggccggg
gcgcgactgg acggggcgcg actggacgga cagtggccac ccgcgggcgc 26820ggaacccgtt
gcgctggaag gcttctacga gaacctggcg gagctgggct acgagtacgg 26880gccgctgttc
cgggggctcg cggcggcgtg gacgcgcgac ggcgaggtgt tcgccgaggc 26940cgtgctgccc
gaggaggcgt tgtccgggca ggcgttgtcc gggcaggcgg ggtccgggca 27000ggcggggtcc
gggaacgggt ccgggaacgg gttcggcatc cacccggccc tgctggacgg 27060ggcgctgcac
gcgggcaacc tgtgcgtgcc gcccgcgccg ggccggacgc tgctgccgtt 27120cgcgtggaac
gaggtgcggc tgcacgccac cggggcgacg gcggtgcggg tgcgcgtgcg 27180ggcgaccggc
gaggactccc tggagctgga gctgttcgac gccgacggcg cgcccgtggc 27240gagcgtcggc
gggctgaccc tgcgaccggc ggtcacgggc gcgcgcccgg ccgagtcgct 27300gcacgaggtg
gagtggaccg aggtcgcggc gggcggttcg tggccggagg tcgccgacac 27360ccgcgactgg
gaggccgccg ccgacctgcc gacccggtcg cgcgagctgg ccgcccgcgc 27420gctggaactg
gtgcaggacc ggctggcggg cgtggacggc gcaccgctgc tggtgatcac 27480cacgggcgcg
gtggcggtgg ccgacgacgc cgaggtcacc gacccggccg ccgccgccgt 27540ctgggggctg
ctgcgctcgg cgcagtccga gcaccccggc cggttcgcgc tggtcgacgt 27600cgacggcggc
gcggcggccg aggtcgccgc gctcgtgccc ggcgacgagc cgcagaccgc 27660gctgcgcggc
gggctcgtgc gggctccgcg cctgcgccgc ctgccccccg gtctcgtgcc 27720gcccgccggg
gcgcactggc acctggacgc agtcaccacc ggcacgctcg acgggctcgc 27780gctcgtggcc
tcggaaccgg tcccgctgcg ggccggggag gtgcggatcg aggtcagggc 27840ggccgggcag
aacttccggg acgtgctggt ggcgctggac ggcgtcgcgg gccaggaggg 27900catcggcggc
gagggctccg ggatcgtgac cgaggtcggc cccgaggtga ccggattcgc 27960cgcgggcgac
cgggtgatgg ggctgttccc gcgctcgttc gggccgctgg ccgtggccga 28020cgcccgcacg
gtggtgcggg tgccgcgcgg ctggtcgttc accgacgcgg cggccgtgcc 28080ggtcgcgttc
ctgaccgcgc tgcacggact ccaggacgtc gccgggctgc gggccgggga 28140gacggtgctg
gtgcacgcgg cggcgggcgg cgtcgggcag gccgccgtgc agctcgccca 28200ccacttcggc
gcgcgcgtgc tggccaccgc gcacccggcc aagcacagcg tgctgaccgc 28260gctgggcgtg
cccgccgagc ggctcgcctc cagccgcgac ctcggctacg cgcggcggtt 28320cggcgacgtc
gacgtggtgc tgaactccct ggtcggcgag cacgtcgacg cctcgctgcg 28380gctgctgcgc
gcgggcggcc ggttcgtgga gatcggcaag aacgacgtcc gggacgccga 28440ctcggtcggg
gacgtccgct accgggtgtt cgacctgggc gcggacgccg ggccggaccg 28500gatcggcgag
ctgctggagc agctggtggg cctgttcgag tcgggcgcgc tgcggccact 28560gccggtgcgc
acgtgggacg tcacccgcgc ggcctcggcg ttccgcgaga tgagccgggg 28620cgggcacacc
ggcaagatcg tcctgacgat cccgcgccgc ctcgaccccg agggcacggt 28680gctgatcacc
ggcggcgccg gcacgctcgg ggccaccgcc gcccgccacc tggtcaccgc 28740gcacggcgcg
cggaacctgc tgctggtcgg caggcggggc cccgacgcgc ccggcgcgag 28800cgagctggcg
gaggagctgc gcgggctggg cgcggacgtg cgggtggcgg cgtgcgacgt 28860cgccgaccgg
gccgcgctcg acgccctgct cgcctcggtc ccggccgggc gcccgctgac 28920ggcggtcgtg
cacgcggcgg gcgcgctcga cgacggcacg gtcaccgcgc tcaccccgga 28980gcggttcgac
gcggtgttcc gccccaaggt ggacgcgatc gcgcacctgg acgaggcgac 29040ccgcgacgcc
gacctggccg cgttcgtcgt ctactcctcg gcggcgggcg tgctcggcaa 29100cgcggggcag
ggcaactacg cggcggcgaa cgccgtgctg gacgcggtgg cccgcacccg 29160gcacgcccgc
gccctcccgg cgacctcgct ggcctggggg ttgtggagcg acacgagcgc 29220gctgaccgcg
acgatggacg ggcgcgcggt ggaccgcacg cggcgcgcgg gcgtgctggg 29280catgggcaac
gacgaggcgc tggcggcgct ggacgcgggc ctggcgtccg ggctgcccgc 29340gctggtggcc
gcccggatcg acccggccgc gctgcgcgac cccgcgtcgg ggtcgccgct 29400gctgcgcggg
ctggtgcgcg ccacccgccg cacggccgcc acccgcgacc gggacgccgt 29460gggcgggctg
gccggacggt tggccgggtt gtcggccgcg gagcaggacg agctgctgct 29520gggcctggtg
cgcagcgagg ccgccgccgt gctcgggcac gcgagcgccg agcgggtcga 29580gccgcaggtg
gcgttccggg acatggggtt cgactcgctc accgccgtgg agctgcgcaa 29640ccggctcgcg
gcggcgaccg ggctgcggct gcccgcgacg gcgacgttcg accacccgac 29700gccggtgcgg
ttcgccgcgc tgctgcgggg cgagctgctg ggcgccgtcg tggctcccgg 29760agccgtgacc
gccgccgcgg ctcccgtgac cgccgccgcg cccgccgacg agccgatcgc 29820gatcgtgtcg
atggcgtgcc ggctgcccgg cggggtggtc gacccggccg ggctgtggga 29880gctgctcacc
ggggagcggg acgggatcgt ggacttcccc gacgaccggg gctgggacct 29940ggagtcgctc
taccacccgg acgccgactc ccccggcacc tcctacgtgc tgcgcggcgg 30000gttcctggac
gacgcgggcg ggttcgacgc cgggttcttc ggcatctccc cgcgcgaggc 30060cctggcgatg
gacccgcagc agcgggtgtt cctggagacc tgctgggagg cgttcgagcg 30120cgccgggatc
gacccggtct cggtgcgcgg cagcgacacc ggggtgttcg ccgggatcat 30180cgaccaggac
tacggggtgc gcgcgggcac ggcccccgag gagctggagg gctacctgct 30240caccggcacc
gccacgtcgg tggcgtccgg gcgggtggcc tacctgttcg ggctggaggg 30300cccggcggtc
accgtggaca cggcgtgctc gtcgtcgctg gtggccacgc actgggcggt 30360gcaggcgctg
cgccggggcg agtgctcgat ggcgctggcg ggcggcgcga ccgtgatggg 30420gcggccgtcg
gcgttcgtgg agttctcccg gcagcgcggg ctggcgcggg acgggaactg 30480caaggcgttc
ggcgcggacg cggacggcac cgcgttcagc gagggcgcgg gcgtgctgct 30540gctggagcgg
ctctcggacg cgcggcggcg cgggcacccg gtgctcgcgg tgatccgggg 30600gtcggcgctg
aaccaggacg gggcgtcgaa cgggctgacc gcgcccagcg gaccggcgca 30660gcagcgggtg
atccgggcgg cgctggccga cgcgggcctg cggccgtcgg acgtggacgc 30720ggtggaggcg
cacggcaccg gcaccgcgct cggcgacccg atcgaggcgg gcgcgctgct 30780ggcgacctac
ggcgcggacc gggagggcgc ggaaccggtg tggctggggt cgctcaagtc 30840caacaccggg
cacacgctgg cggcggcggg cgtgtcgagc gtgatcaaga tggtgctggc 30900gctgaaccac
ggcctgctgc cccggtcgct gcacgtgcgg gagccgagcg cggcggtgga 30960ctgggagtcg
ggcggcgtgc gcctgctgac gagcgcccgg ccgtggccgg agagcggcag 31020gccccggcgg
gcgggggtgt cgtcgttcgg gatcagcggc acgaacgccc acctggtgct 31080ggaagccgcg
cctgcggagg agggcgcggg ggcgcggagt ggggcggcgg cgccgggacc 31140ggacacccgg
tcggcgccca ccccggacgc cccagcgggc cccgtccaga cctccggcgt 31200gatcccctgg
ccgttgtcgg cccgctccgc cgacgcactg cccgcgcagg ccgcgaagct 31260ggccgcccac
gtgcgggcgc acgacgacct ctcgccgctc gacgtcggct ggtccctcgc 31320gaccacccgc
accgcgcacc cgcaccgcgc cgtgctcgtc ggcggcaccc gcgaggcgct 31380gctgtcggcc
gccgacgcgc tcgcgggcgg cgaggccagc caggccgtgc tcaccggctc 31440cgccgtcggg
tcgggttcgg cgaagaccgt gttcgtgttc cccggccagg gcgcgcagtg 31500ggcgggcatg
ggccgtgagc tgctggggtc ctcgccggtg ttcgccgcgc ggctgcgcga 31560gtgcgccgac
gcgctggccc cgcacaccga ctgggacctc ctggacgtgg tgcgcggcgc 31620ggagggcgcg
ccggggttcg agcgggtcga cgtgctccag cccacctcgt gggcggtgat 31680ggtggcgctg
gccgcgctgt ggcgctcgtg cggggtggag ccgtccgccg tcgtcgggca 31740ctcgcagggc
gaggtggccg ccgccgtggt cggcgggtac ctggcgctgg gcgacgcggc 31800gcggctgatc
gcgcggcgca gcagggccat cgcgcaggag ctgaccgggc gcggcgggat 31860gctgtccgtg
ctcacctcgc ccgagcgggt cgccgaactg ctggagccgt gggccgggaa 31920gctgtggatc
gcggcggtca acagccccgc gtccgtctcg gtgtccggtg acgccgaggc 31980gctgggcgag
ttcgtgcggg tgctggccaa ggcccggatc aaccggtggc ggctgcccgg 32040cgtggacttc
gccgggcact ccgggcacgt cgacggcatc gaggcgcggc tgcgcgagga 32100gctggccgac
gtcaccgccg cggcgggcga agtgccctgg ctgtccaccg tggacgggcg 32160gtgggtggag
cgcaccaggc tggacgccga ctactggtac cgcaacctgc gcgacgtggt 32220ccgcttcgac
gaggccgtcc gcgcgctggt ggacgccggg caccgggcgt tcgtggaggt 32280ctccacgcac
ccggtgctga ccaccgcgat cggcgaggtc gccgacgagc ggcaggacgt 32340gcgggtcgcc
gtggcgggca cgctgcgccg cgacgacggc ggcgcggacc gggtcgtggg 32400cgcgctcggc
gaggtggccg cctcgggcgt ggcggtggac tgggcggcgg tgttcggcgg 32460gaccggggcc
gcggtggtgg agctgccgac gtacgcgttc cggcacgagc ggttctggct 32520caccccgtcc
ggcggcgacg tgcgcgcggt ggggctgcgg caggccgggc acccgctgct 32580gggcgcggtg
gtcagcgtcc cggacaccgg cggcgtgctg ctgaccgggc ggctgtcgct 32640gtccgcgcag
ccgtggctgg ccgaccacgc gctgtccggc gtgccgctgc tgccggggac 32700ggcgctggtg
gagctggcgg tgcgcgcggg tgacgagacc ggcacgccgg tggtggcgga 32760gctggtgctg
ggcaggccgc tcgtgctgcc gcgcaccggg tcggcgcagg tgcaggtgct 32820ggtgggcgag
gaggcgcggg acgggcggcg gccggtcgcg gtgtactcgc gggcgggcga 32880cgaccggccg
tggaccgagc acgcctcggg ctcgctcgcg ccggacgagg acgccgcgcc 32940gggagcggag
ggcgacgagt ggccgcccgc cggggccgag ccggtggacc tcggcggctt 33000ctacgacggc
ctcgccgaac ggggctacga ctacggcccg gccttccggg gcctggtgcg 33060cggctgggtc
aggggcgacg aggcgttcgc cgaggtcggg ctgcccgacg accagcacgg 33120cgcggcggcc
cggttcgggc tgcacccggc gctgctggac gcggccctgc acgcggcctc 33180gctgtgcgcg
ggccacggcc ggggcacggc gctgccgttc acctggaccg gcgtgcggct 33240gcacgcggcc
ggggcgacgg cgctgcgcgt gcggctggag gcggacgggc cggagcggtt 33300gtcgctgcgg
gcgagcgatc cggcgggcac gcccgtggtg accgtcgggt cgctgctgct 33360gcgcgccgcc
gacgcggacc ggctgcgggc gacagcggcg gcgacggcgg cagcggcggc 33420ggacgacggg
ctgcacgcgc tggagtggac cccgcacccg ctgcccgagg agacgaccgg 33480ttcccccgcc
gtcctggaca ccagggcgtg ggagctgccc gagggcgtcg ggcgggccga 33540ggcgatcacc
acgcgggtgc tcgccgagct ccaggccgag ctcgacggga cggcgaccct 33600ggtcgtggtg
acgcggggcg cggtggccgt gcatgacgac gccgaggtca ccgacccggc 33660cgccgccgcg
gtgtgggggc tggtgcgcgc cgcgcaggcc gaggaacccg gacgcgtcgc 33720cgtggtcgac
gtcgacgacg cctccgaggc cgcgctggac gccgccgcgc acgccgcggg 33780cgcagaaccg
cagctcgccc tgcgcggcgg ggcggcgttc gcgccgaggc tggtcgaggc 33840gtccggggcg
ctggccgtgc cggacgggcc gtggcggctc gacagcaccg gccggggcac 33900cctggagaac
ctggcgctcg tgcccaaccc cgccgccggg gcgccgctcg cgcccggtca 33960ggtgcggatc
gtggtgcggg cgggcggcct gaacttccgg gacgtgctga tcgcgctcga 34020cgcctacgag
tcggagatcg gcaccgaggg cgcgggcgtg gtcgtggagg tcgcgccgga 34080cgtcacccgc
gtggccgtgg gcgaccgcgt gatgggcatg atccccggct cgttcgggcc 34140gctggccgtg
gccgacgccc gcacggtggt gcggatgccg cgcggctggt cgttcaccga 34200cgcggcgggc
gtgccggtcg cgttcctgac cgccctgtac gggctgcgcg acctcggcgg 34260cctggcggag
ggcgagaccg tgctggtgca cgcggcggcg ggcggcgtcg gcatggccgc 34320cgtgcagctc
gcccggcact tcggcgcgcg cgtgctgggc accgcgcacc cggccaagca 34380cgccgcgctg
gacctgcccg ccgaccacct ggcctccagc cgggacctcg cctacgcgca 34440gcggttcggc
gacgtcgacg tggtgctgaa ctccctggtc ggcgagcacg tcgacgcctc 34500gctgcggctg
ctgcgcgcgg gcggccggtt cgtggagatg ggccgggcgg acctgcgcga 34560cgccgacgag
gtggcgcgcg agcaccccgg ccgcgcctac ctcccgttcg acctcggcgg 34620cgacgccggg
ccggaccgga tcgccgagct gctggtggag ctggtggccc tgttcgagtc 34680gggcgcgctc
cgcccgctgc cgacccggcg caccgacctg gtgcgcgccc ccgaggcgtt 34740ccgggccatg
agccagggcc gccacgtcgg caagctcgtg ctcaccccgc cccgcgcgct 34800cgaccgcgac
ggcacggtcc tgatcaccgg cggcacggga accctcggcg cggctctggc 34860ccgccacctg
gtggacgcgc acggcgtccg gaacctgctg ctggtcagcc gcagcggccc 34920caacgcgccg
ggtgcggccg acctggtcgc ggagctggcc gagcggggcg cgagggtccg 34980ggtggccgcg
tgcgacgtgg ccgagaagga cgcgctcacc gcgctgctcg cctcgatccc 35040caccgggcgc
ccgctcaccg gcgtcgtgca cgcggcgggc gcgctggacg acggggtgct 35100caccgccctg
gacgccgacc gggtcgcggc ggtgctgcgc cccaaggccg acgccgccct 35160gctgctgcac
gaggccaccg aggacgccga cctcgcgctg ttcgccctgt gctcgtcggt 35220ggcgggcgtg
ctgggcaacg cgggccaggc gaactacgcc gccgccaaca cctacctgga 35280cgcgctggcc
cagcaccggt cggccgccgg tctggccgcg ctgtcgctgg cctggggccg 35340gtgggcgcag
accagcgccc tcaccgcaga cctgcccgcg cccggcggtc gccgcgacct 35400ggtgcgcccc
atggacaccg cgtccgcgct gcgcctgctc gacgccgcgc tccgcaccgg 35460acgctcgacg
gtcgtcgccg ccgagctgga cgtcacggcg gccaccgccg cgaacccggt 35520gctgcgcggc
ctggtccggc ccgcccggcg cgcgctggcc acgtccgcgc gggacgagcg 35580cggcgtggcg
gcggcgctgg ccgggctggg cgaggccgac cggcgccggt tcgtgctgga 35640cctggtgcgc
tcgcacgccg ccgtcgtgct gggcctggcg ggcaaggagg ccgtggacgc 35700cgagcgcgcg
ttcaccgaga ccggcttcga ctcgctcacc gccgtggagc tgcgcaaccg 35760gctcgccgcc
gcgaccgggc ttcggctgcc ctccacgctg gtgttcgacc acgccacccc 35820gaccgcgctg
gccgcgcacc tgcgcgccga gctgaccggc gacgacctgc cgcaggcgcg 35880ggccgtcgcc
gccacggcgg gggcgcggga cgacgacccg gtggtgatcg tgtcggcgag 35940ctgccgcctc
cccggcggcg cggactcgcc ggaggcgctg tgggagctgc tggagcgggg 36000cagggacgcc
atcaccccgt tcccgcgcga ccggggctgg gacctggagg cgctctacga 36060cgccgacccg
gaccggccgg gcaagagcta cgtgcgcgac ggcgggttcc tcgccgacgc 36120ggccgggttc
gacgccgagt tcttcggcat ctccccgcgc gaggcgctgg ccaccgaccc 36180gcagcagcgg
ctgctcgccg agacctcctg ggagctgttc gaacgcgcgg gcatcgcccc 36240gacctcggtg
cgcggcagcg acgtcggcgt gttcgcgggc gtgatcaacc aggagtacgg 36300cgtgcacagc
ggcacgaccc ccgccgagct ggaggggtac gtgatgaccg gctcgaccac 36360cagcatcgcc
tccggccggg tggcgtacct gctcgggctg accgggcccg ccgtcaccgt 36420ggacaccgcg
tgctcctcgt cgctggtggc gatccacctg gcggcgcagg cgctgcgctc 36480gggcgagtgc
tcgatggcga tcgcgggcgg cgcgacggtg atcgcgaggc cgggcgggtt 36540cgtctcgttc
tcccggcagc gcggcgcggc ccccgacggg cgctgcaagg cgttcggcga 36600cggcgcggac
ggcatggcgt tcgccgaggg cgtcggcctg gtgctgctgg agcggctctc 36660ggacgcgcgc
cgcaacgggc acccggtgct ggcggtcgtg cgcggcacgg ccctgaacca 36720ggacggcgcg
tccaacggcc tgaccgcgcc gaacgggccc gcgcagcagc gggtgatccg 36780gcaggcgctg
gccaacgccg ggctgtcccc cgacgaggtg gacgcggtcg acgcgcacgg 36840caccggcacc
gcactcggcg acccgatcga ggcgcaggcg ctgctcgcca cctacgggcg 36900ggaccgggac
ccgcggcggc cgctgtggct ggggtcggtg aagtcgaaca tcgggcacac 36960ccaggcggcg
gcgggcatcg cgagcgtgct caagatggtg ctggcgatgc agcggggcgt 37020gctgcccgcg
accctgcacg ccgacacccc gacgacgaag gtcgactggt cctcgggcgc 37080ggtggcgctg
ctgtcgcggg cgcggccgtg gccggagacc gggaggccgc gccgggcggg 37140cgtgtcctcg
ttcgggatct ccggcaccaa cgcgcacgtg ctgctggagc aggccccgca 37200ggacgcgccc
gccacgccgg tcgccccgcg gggcgccggg ctggtcgggg cggtggcctg 37260gccggtgtcc
gggcgcacgc ccgccgcgct gcgcgcccag gccgccaggc tcgggacgca 37320cctggcgggc
gcgcaggccg gacccgccga cgtgggctgg tcgttggcgg gcacgcggac 37380ggcgttcgcg
cagcgggcgg tcgtggtggc cgggacggcg gagcaggccc gtgacgggct 37440ggcggcgctg
gccgaaggcc gctcgtccgc gctcgtgacg accggtgagg ccggggtcga 37500cgggcgcgtg
gtgttcgtgt tccccggcca aggggcgcag tggatcggca tgggcgcgga 37560gctgatcgac
gcgtcgccgg tattcgccga gcggttgcgc gagtgcgcgg aggcgctgga 37620accgttcgtg
gacttcgacc tgatcgaggt gctgcgcgga cgcgggtcgc tggagcgggt 37680cgacgtggtg
cagcccgcgt cgtgggcggt gatggtgtcg ctggcagcgc tctggcggtc 37740gctgggcgtg
gaaccggacg ccgttgtcgg gcactcgcag ggcgagatcg cggcggcggc 37800ggtcagcggg
gcgctcagcc tgcccgacgc cgcagccgtg gtcgcgttgc gcagcaaggc 37860gatcgcccag
gacctggccg ggctcggcgg catgatgtcc gtcgccctgc ccgccgacga 37920cgtcgacctg
agcgggtatc ccggacgcct gtgggtcgcc gcgcacaacg gccccacctc 37980gaccgtggtg
gccggtgacg tggacgcgct gcgcgagctc cacgcccact acgagggcgc 38040cgaggtccgg
gcccggatca tccccgtcga ctacgccagc cacaccgggc acgtcgacac 38100catccgcgag
cggctcgccg aggcactggc gcacgtgcgg ccgagggcgg gcacgatccc 38160gtggctgtcg
accgcgaccg gcgagtggac caccggtgag gacgccgacg ccgactactg 38220gttccgcaac
ctgcgcggcg cggtgggctt ccacaccgcc atcaccaccc tcgccgagca 38280gggccaccgg
gtgttcgtgg aagtctccag ccaccccgtg ctcaccaccg ccatcgaggc 38340cacgctcgaa
ggaaccggac ccaccgccgt caccggaacc ctccgccgcg acgacggcgg 38400ccccgaccgc
ctcctcacca gcctcgccac cctgcacgtg cgcggcgtcc acgtcgactg 38460ggacgcggtc
tacgcgggca gcggcgcgca ccgcacgacg ctccccacct acgcgttcca 38520gcacgagcgc
tactggctca ccgagccgga cgcgccgcag gccgtcgcgg acgccccgtt 38580ctgggacgcc
gtggacagcg gcgacgtggc cgcgctcgcc gggtccctgg gcgtcgagcc 38640cgccgccctg
gagccggtgc tgccggggct gacgagctgg cgggcccgca accgggacgg 38700cgcggccgtg
gacgactggt cctaccggat cggctgggag cgggtggacg tgcccgccgc 38760cccgctgtcc
gggacgtggc tggtcgtggt gcccgaggca ctcgccgacg acacctcggt 38820cgccgaggtc
gcggcggcgc tggccgcgcg cggcgcgacg ccgaggatcg tggcggcggg 38880cccggacctg
ggcccggacc tgggtgacga gccggacggg gtgctgtcgc tgctggcgtg 38940ggacgaccgc
ccggccgggg gcggcacgct ctcgcgcggc gtcgtggacg cggtcgggct 39000ggtgcgggag
gcggtgcggc gcggctggtc ggccccgctg tggtgcgcca cgctcggcgc 39060ggtcgccgtc
gccgaccccg gcgaggtgac ggccgagttc gggccgcagc tgtggggcac 39120gggcgtcgtg
ctgggcctgg acctgccgga cacctggggt ggcctggtcg acctgcccgc 39180gcggccggac
ggggtcgcgc tggacctgct gtgcgcggtg gtcgcgggcg cgggcgacga 39240ggaccagctg
gcggtgcgcc cggccggggt gttcgcgcgg cgcatgaccc gacgcccggt 39300cgcgtcggcg
cccgcgtggc gaccgcgcgg gacggtgctg gtcaccggcg gcaccggcgg 39360cctcggcggc
tacgtcgccc ggtgggcggc ggagcggggc gcgcgggacg tggtgctgct 39420ctcgcgcggc
ggcccggacg cgccgggcgc ggacgccctg gtcgccgaca tcacggcggc 39480gggcgcccgc
tgcgcggtgc tggcctgcga cgtcaccgac cgggacgcgc tggccgaggt 39540ggtcgcgaac
ctgccggacg ggccgctgtc ggtggtgcac gccgcgggcg tggcgcgacc 39600gggacggccg
ctggtggaga ccacgccgga ggagttcgcg gccatcggcc ggggcaaggt 39660cgcgggcgcc
cgcctgctgg acgagctgct gggcgaccgg gagctggacg cgttcgtgct 39720gttctcctcc
ggcgcggcgg cctggggcag cggcgggcag gccgggtacg cggcgggcaa 39780cgccttcctg
gacgggctcg cgcagcgcag gcgcgcccga gggctcgcgg ccacctcggt 39840ggcctggggc
gcgtggggcg gcgtcggcac ggtcgacgag gtgctgggcg agcagtggcg 39900gcgcgccggg
ctgctcacca tggacccgcg cctggccacc ctcgccctcg cgcacgccgt 39960gggctcgggc
gaggcgcacc tgctcgtcgc ggacgtcgac tgggcccgct tcgcccccgc 40020ctacgcgctg
gccaggccgc gcccgctgct ggcggcgctg cccgaggtcg ccgacgcgct 40080ggcggtcgtg
gacgcgcccg ccgacgccgg ggggatcggg gcgcggctgg ccgggctgcc 40140gcccgccgag
caggagcggc tgctcaccga gctggtgcag gcggaggcgg cggccgtgct 40200gggcctgggc
ggcatcaccg gcgaccgggc gttccgggag gtcgggttcg actcgctcac 40260ggccgtggag
ctgcgcaacc ggctcggcgc ggccacgggt ctcaccctgc ccgcgacgct 40320ggtgttcgac
cacccgcgcc cgagcgccct ggccgcgcac ctgcggtccg cgctgggccc 40380ggccgccgcg
ccggtggact cggtggcggg cgtgctggcc gagctggacc ggctggaggc 40440ggccatcccg
gcgctgccgt cggccgagat cggccggtcc cggctggagc tgcggctgcg 40500gcggttgagc
gcccgcgtcg gcgagctggt cgccgcgaac ggcgagcggg cgaacggcgg 40560gcgcgcgaac
ggcgggcgcg cggcggccga cgagctggac gacgcggggg ccgaggacgt 40620gctcgcgttc
atcgaccggg agttcgggga cgcgtgagcg gccacacgag ccccgacccc 40680ggcccccacc
gcggccccca caacgacgac cctggcgagg aacagatggc gaacgacgag 40740aggctcctca
gctacctcaa gcgggtcacc gccgacctgc accgcacgcg ggagcggctg 40800cgcgaggcgg
agtccggggc ggacgagccg atcgcgatcg tcggcatggc ctgccgcttc 40860cccggcggcg
tgcgcacccc ggacgagctg tgggagctgg tggcgtccgg ccgcgacggc 40920atcggcccgt
tcccggacga ccggggctgg gacctgggcg cgctgttcga cccggacccc 40980gacgccaccg
gccgctccta cgtcaccgag ggcgggttcc tggacgacgc ggccctgttc 41040gacgcgggct
tcttcgggat ctccccgcgc gaggcgctgg ccaccgaccc gcagcagcgg 41100gtgctgctgg
agaccgcgtg ggagaccttc gagcaggcgg gcatcgaccc gacctcgctg 41160tccgggcagg
acgtgggcgt gttcaccggg gtcgccaacg gggactacgc gctgaccgtg 41220gaccgggtgc
cggagggctt cgagggctac ctgggcatcg gcggggcggg cagcatcgcc 41280tccgggcgca
tctcgtactc cctgggtctg gagggtccgg ccgtcacgct ggacaccggc 41340tgctcgtcgt
cgctggtcgc gatgcactgg gccgggcacg cgctgcgggc gcgggagtgc 41400tcgctggcgc
tcgcgggcgg cgtgatggtg atggcgacgc cgggtgggtt cgtcgggttc 41460tcccggcagc
gcgggctggc ccgcgacggg cggtgcaagt cgttcggcga cggcgcggac 41520ggcacgtcgt
ggtcggaggg cgtgggtctg ctgctgctgg agcggctgtc ggacgcgcgg 41580gccaacgggc
acgaggtgct tgcggtggtg cgcgggtcgg cgatcaacca ggacggggcg 41640tccaacgggc
tcaccgcgcc caacgggccg tcgcagcagc gggtgatccg cgcggcgctc 41700gacgcggcgg
ggctcgggca cgcggacgtc gacgcggtgg aggcgcacgg caccgccacg 41760gtgctcggcg
acccgatcga ggcgcaggcg ctgctgaaca cctacgggcg gcaccgggac 41820ggggcgcagc
cgctctacct ggggtcggtc aagtccaacc tcgggcacac ccaggcggcg 41880gcgggcgtgg
ccggggtgat caaggcggtg caggcgatgc gccacggcgt gctgccgccc 41940accctcaacg
tcggcacgcc caccaccaag gtcgactggt cctcgggcgc ggtggaggtg 42000ctggcggagg
cccggccgtg gccggagacc gggcgtccgc gccgggtggg cgtgtcgtcg 42060ttcggcgtga
gcggcaccaa cgcgcacgtg atcctggagc aggcacccga gcacgagcca 42120gcgccggagg
agccgggtgg gcgcgggccg gtggcggcgg gcggcgcgac gccgtggacg 42180ctgtccgggc
gcacgcccgc cgcgctcgcc gaccaggcgc ggcggctggc cgggcacgtg 42240acggccgacc
tgcgggcgga ggacgtcggg ttctcgctgg ccaccaccag ggcgcacctg 42300gagcaccggg
cggtggtggt cggctcggac gggctggcgg cgctggccga aggccgctcg 42360tccgcgctcg
tgacgaccgg tgaggccggg gtcgacgggc gcgtggtgtt cgtgttcccc 42420ggccaagggg
cgcagtggat cggcatgggc gcggagctga tcgacgcgtc gccggtattc 42480gccgagcggt
tgcgcgagtg cgcggaggcg ctggaaccgt tcgtggactt cgacctgatc 42540gaggtgctgc
gcggacgcgg gtcgctggag cgggtcgacg tggtgcagcc cgcgtcgtgg 42600gcggtgatgg
tgtcgctggc agcgctctgg cggtcgctgg gcgtggaacc ggacgccgtt 42660gtcgggcact
cgcagggcga gatcgcggcg gcggcggtca gcggggcgct cagcctgccc 42720gacgccgcag
ccgtggtcgc gttgcgcagc aaggcgatcg cccaggacct ggccgggctc 42780ggcggcatga
tgtccgtcgc cctgcccgcc gacgacgtcg acctgagcgg gtatcccgga 42840cgcctgtggg
tcgccgcgca caacggcccc acctcgaccg tggtggccgg tgacgtggac 42900gcgctgcgcg
agctccacgc ccactacgag ggcgccgagg tccgggcccg gatcatcccc 42960gtcgactacg
ccagccacac cgggcacgtc gacaccatcc gcgagcggct cgccgaggca 43020ctggcgcacg
tgcggccgag ggcgggcacg atcccgtggc tgtcgaccgc gaccggcgag 43080tggaccaccg
gtgaggacgc cgacgccgac tactggttcc gcaacctgcg cggcgcggtg 43140ggcttccaca
ccgccatcac caccctcgcc gagcagggcc accgggtgtt cgtggaagtc 43200tccagccacc
ccgtgctcac caccgccatc gaggccacgc tcgaaggaac cggacccacc 43260gccgtcaccg
gaaccctccg ccgcgacgac ggcggccccg accgcctcct caccagcctc 43320gccaccctgc
acgtgcgcgg cgtccacgtc gactggaagg ccgtgttcgc cggcacgggc 43380gcgcgccgcg
tcccgctgcc gacctacgcg ttccagcacg agcgctactg gctggaccgg 43440ggcgcggcgg
ccggtgacgt cacgggcgcg ggcctggccg acgcggcgca cccgctgctg 43500gccgccgtcg
cccagctgcc cggcaccggc ggggtgctgc tgagcgggcg gttgtcgcgg 43560gcgacgcacc
cgtggctggc cgagcacgtg gtgaacggga ccgcgctggt gcccggcacg 43620gccctggtgg
agctggcgct gcgcgcgggc gacgaggtgg acgcgcccgt gctgcgcgag 43680ctggtgatca
cccggccgat gccggtgccg gagcggggtt tcctgcacgt gcaggtggac 43740gtcgcgggtg
cggcggacga cgggtcgcgg gcggtgcgga tctggtcgcg cgccgaggac 43800gcggcgagcg
agacggcccg ctggaccgag cacgccaccg gctccctcgc ccccgacgac 43860gcggccccgc
ccgcccgcgc gagcggcgcg tggccgcccg agggcgcggc ggccgtggac 43920gtggacgact
tctacgaccg cctcgcgggc gcgggctacg agtacgggcc gctgttccag 43980ggcctcaccg
ccgcgtgggc cggggacggg caggcgtggg ccgaggtggt gctgcccggt 44040gaggcgggcg
ggttcggcgc gcacccggcg ctgctggacg cggcgctgca cgtgggcacg 44100ttctgcctgc
ccggcgggcc ggggtcgcgc acgctgctgc cgttcgcgtg gacgggcgtg 44160cggctgcacg
ccaccggcgc gacggcggtg cgggtgcacg cccgcgccac cggcgacgac 44220ggcctcgtcg
tggagctgcg cgacgcggac ggggaaccgg tcgtgacggt cgacgcgctc 44280gtgctgcgcg
acgccgaccc cgccgacgcg caggccccgg acgtcacggc ggacgcgttg 44340tggcgggtgc
ggtgggtcga gcagccgccc gcgcccgcgg cgcccggctg ggtgctgctg 44400ggcgggccgt
ccgggcacgc cgggttcgcc gccctgccgg tgttcgccga ccctgcggcc 44460gtggcggcgc
tgcccgaggg cgaccggccc gcggtggtcg tcgtggacac caccgcgtgt 44520cgggagccgg
ggcgggacgt gccgggggcg gcgcgggcgt tcgtggtgcg ggcgctggag 44580ctgctggtgg
cgtggctgcg cgaggacgcg ctggccggga cccgactggt gctagtcacc 44640agcggcgcgg
tgccggtgcg cgcggacgcc gaggtcaccg accccgctgc cgcggcggtg 44700tggggtctgc
cgcgctcggc gcagtcggag cacccggacc gggtgtggct gctggacgtc 44760gacgagccgg
gcgcggcgcc gggcgcgctg gcctcgccgg agccgcagct ggccgtccgg 44820gcgggcgcgg
ggttcgcgcc ccggctcgcc agggccgagg ccgcgcccgg cgcgctgccg 44880gtcgacgggc
cggtgctggt caccggcggc accggcacgc tcggcgcgct cgtggcccgg 44940cacctggtca
ccgcgcacgg cgcgcggaac ctgcacctgg tgagcaggcg cggcccggac 45000gcgtccggcg
ctcgagaact cctggacgag ctgcgcgggc tgggtgccga ggtcgagctg 45060tcggcgtgcg
acgtggccga ccgggtggcg ctcgccgccc tgctggggcg cgtgcgcccc 45120gccgccgtgg
tgcacgcggc gggcgcggtg gacgacggcc tgctcaccga cctgaccgcc 45180gaccggttcg
acgccgtgct gcggcccaag gtcgacgcgg tcgcccacct ggacgaccta 45240ctcggggacg
tgccgctggt ggtgttctcc tccgcgaccg gcaccctcgg cacccccggc 45300caggcgaact
acgccgcggc caacgcggtc gccgacgcgc tcgtgcagcg ccgccgcgcg 45360cggggcctgc
cgggcgtgtc gctggcgtgg ggcctgtggt cggacaccag cgagctgacc 45420gcgaccatgg
acgccgccga cgtggcccgc acccgccggg gcggggtgct cggcctggac 45480gcggcgcgcg
gcctggcgct gctcgacgcc gcgctcggcg cggacgacgc gctgctcgtg 45540ccgatccacc
tggacaccgc cgcgctgcgc cggggggccg acccggctcc gccgctgctg 45600cgcggcctgg
tccgccgcgc ccggcgcgcg gcgggcgcgg cccggcaggc cgcgctgccg 45660ctggtggcgc
gactggccgg ggtggacgcg gcggagcggc ggcgggcgct ggtggagctg 45720gtgcgcgccg
aggccgccgc cgtcctcggg cacggcggcc cggacggcat cgggcaggac 45780cagccgttcc
gggaggtcgg gttcgactcg ctcacggccg tggagctgcg caaccggctc 45840ggcgcggcca
cgggtctcgc gctgcccgcg acggtggtgt tcgaccaccc gacgtccgcg 45900cgggtcgccg
agcacctgcg ggagctgctg ttcggcgcgg agacggctca ggcccccgcg 45960cggcgggagg
tggtggccga cgacgacccg atcgccgtgg tgggcatggc ctgccggttc 46020cccggcgggg
tcgccgacgc ggacgggctg tggcggctgg tcgccgagga gcgcgacggc 46080atcggcccgt
tcccggacga ccggggctgg gacctggcgg cgctgttcga cccggacccc 46140gaccacgcgg
gcacctcgta cgtgcgggaa ggcgggttcc tcgacggggc gaccgggttc 46200gacgcgccgt
tcttcggcat ctccccgcgc gaggcgctgg ccatggaccc gcagcagcgg 46260ctgctgctgg
aggtggcgtg ggagacgttc gagcaggcgg gcatcaaccc gcgctcggcg 46320cacggcaccg
acaccggggt gttcgcgggc gtgatctacc acgactacgg cgaggcggcg 46380ggcgagctgc
ccgagggcgc ggagacctac cgcagcaccg gcacgtcggg cagcgtggtg 46440tccggccgcg
tcgcctacgc gctgggcctg accggcccgg cgctgaccat cgacacggcc 46500tgctcgtcgt
cgctggtggc gatccacctg ggcgcgcggg cgctgcgggc gggcgagtgc 46560tcgatggcgc
tggtcggcgg ggtgacggtg atgtcgacgc cgggcgggtt cgtgagcttc 46620tcgcggcagc
gcgggctggc cccggacggg cggtgcaagt cgttctcgga gggcgcggac 46680ggcaccgggt
tcagcgaggg cgtcgggctg gtgctgctgg agcggctgtc ggacgcgcgg 46740gccaacgggc
acgaggtgct tgcggtggtg cgcgggtcgg cggtgaacca ggacggggcg 46800tccaacgggc
tcaccgcgcc caacgggccg tcgcagcagc gggtgatccg cgcggcgctc 46860gacgcggcgg
ggttggggca cgcggacgtg gacgcggtgg aggcgcacgg cacgggcacc 46920accctcggtg
acccgatcga ggcgcaggcc gtgctcgcca cctacgggca ggaccgcgag 46980cagccgctgt
ggctgggcac gctcaagtcc aacctcgggc acacccaggc ggcggcgggc 47040atcggcagcg
tgatcaagat gatccaggcg atgcggcacg gcgtgctgcc gcgcaccctg 47100cacgtcaccg
agccgaccac ggccgtggac tggggcgcgg gcgcggtgga gctgctgacg 47160cgggcgcggg
agtggccgga gaccgggcgt ccgcgccggg cgggggtgtc gtcgttcggg 47220gtgagcggca
cgaacgcgca cgtgatcctg gagcaggccc ccgaaccggt ggcggtggag 47280gcggcgccgg
aggcgggggt gctgccgtgg gtgctgtcgg cccgcacgcc cgaggcgctg 47340cgggagcagg
ccgaccggct cgtggcgcac ctgggcggtg agtcgtcctc ggcggccgtg 47400gcccggtcgc
tggtgctggg tcgggcggcc ctggaggagc gggccgtggt cgtgggcgac 47460cgggcgcgcg
ccggggaggc gttgcgggcg ctggccgagg ggcggccctc ccccgcgctc 47520gtcaccgggc
ggaccggggt cgaggggcgc gtggtgttcg tgttccccgg tcagggcgcg 47580cagtgggtcg
gcatggggcg tgcgctgctg gacgcctcgc cggtgttcgc cgaacgcctg 47640cgcgagtgcg
cggcggccct gcgcccgtac accgactggg acctggtcga ggtgatcacc 47700tcgggtggcg
cgctggacga cgtggacgtc gtgcagcccg cgtcgtgggc ggtgatggtg 47760tccctcgcgg
cgctgtggcg ctcgctcggc gtcgaaccgg acgcggtgat cgggcactcg 47820cagggcgaga
tcgccgccgc gaccgtcgcg ggctggctca gcctccagga cggcgcgaag 47880atcgtcgcgc
tgcgcagcca gctgatcgac gaggagctga ccgggctggg cggcatgatg 47940tccgtcgccc
tgcccgccga ggacatcgac ctgagcggtt acgagggccg gttgtgggtc 48000gcgacggtca
acgggccgag cgcgaccgtg gtcgccgggg acaccggggc actggaggag 48060ctgcggcgcg
gctgcggcga ggcggtccgc acgcgggtga tccccgtgga ctacgccagc 48120cacaccgggc
acgtcgacgc catccgcgac cagctcgccc ggatgctcgc cgacgtcacc 48180ccgcggcccg
gcgagatccc gtggctgtcc acggtgaccg gcgagtggat cacccccggc 48240gacgacgacg
ccgactactg gttccacaac ctccgccgca ccgtccactt cgccgacggg 48300atcaccaccc
tgctcgacgc cgggcaccgg gtgttcgtgg aggtctcctc gcaccccgtg 48360ctggcggcgg
cggtgcagga gagcgccgag gcggccgggg acgcgcgggt cgccgtgacc 48420ggcacgctgc
gccgcgacga cggcggctgg gaccgggtcc tgaccggcct ggccgagctg 48480cacgtgcgcg
gcgtggacgt ggactggacg cgggtgctgc ccgaggcggg gcgggcgccg 48540ctgccgacgt
acgcgttcca gcacgagcgc tactggccgg aacccgcgcg cccggccgcc 48600gcgccgggcg
gtggtgacga cgcgctgtgg gcggtgatcg agggtggtga cgcggcggac 48660ctggccgggg
agctggccgt ggacgaggac gagctggcgc gggtgctgcc cgccctgacc 48720tcctggcggc
ggcgcagccg ggcaaggagc gcgctcgacg gctggcgcta ccgggtcgac 48780tgggtcccgg
tccccacgag cgggtctggg ctgcccggcg ggcaagcgct gtccggcggg 48840caggcgctgt
ccggcgggcc gaggtcgtcc ggcggggcag ggctctccgg cggtcagggg 48900acgccaggcg
ggtccgggtc gcccggcgga gcggcactgc caggcgggcc agggtcgccc 48960ggcggagcgg
cgctgcccgg ccgggtggcc gtggtggtgc ccgcgaacga cgagcgggcg 49020cgggcggtcg
cgggcgggct ggtcgcgcgg ggtgtggacg tgaccgtcgt ggcggcggtc 49080gacgccaccc
gcgacgggct ggcgaaggcg ctgcccgacc gccccgacgc cgtggtgtcg 49140ctgctgtcct
gggacgcggg ggccgacgag ccgggcgcgc ccggttcggc cacggccgcg 49200ctggtgcagg
ccctggccga ccggggtgcc accgggccgc tgtggtgcgc gaccgggggc 49260gcggtgagcg
tcgcgggcga ggacgccgac cccgaccagg ccgccgtgtg ggggttgggc 49320ggggtgctgg
ccctggacct gccggaggcg ttcggcggac tggtcgacct gccgcggcag 49380cccaccgacg
ccgacctcga cgcgttcgcc gccgcgctga ccgcccccgg cggcgaggac 49440cagctcgcgg
tgcgcgacgg ccgcctgctg gcccgccgcc tggtccgcga cggggccgac 49500gcgccggagt
ggacgccgcg cggcgcggtg ctggtcaccg gcggcaccgg cggcctcggc 49560acgcacgtgg
cccgctggct cgcccgctcc ggggccgggc acgtcgtgct cgccagccgc 49620tccggccccg
ccgcccccgg cgcggccggg ctggccgccg aggtggaggc gctgggcgcg 49680cggtgcagcg
tggtggccct ggacgtggcc gaccgggacg cggtggccgc cgtgctcgcc 49740gacgtcgagc
gggacgggcc gctgaccgcc gtggtgcacg cggcgggcgc gggactggcc 49800ccgacgccgg
tggtggagct gaccgccggg cggtacgcgg acgtcgcggc cggcaaggtc 49860gagggcgcgc
gggtgctgga cgaggtgctc gccgaccggg cgctggacgc gttcgtgctg 49920ttctcctccg
gcgcggccgt gtggggcagc ggcgggcagg ccccgtacgc ggcggccaac 49980gcgttcctgg
acgggctggc cgcccgcagg cgggcgcgcg ggctcgtggc cacctcggtg 50040gcgtggggcg
gctggggcgg cggcctcggc atgatcggcg acggggacgc ggagcggtgg 50100gcccggctgg
gcatccgcac gatggacccg gaggcggcgc tgcgcggcat ggcgctggcg 50160gtcggctccg
ggcgggccgc gagcgtggtg gccgacgtcg actgggcccg gttcgccccc 50220ggctacgccc
tggcgcggga gcgcccgctg ctgcgcgggc tgccggaggt ggtggcgctg 50280ctggccgaac
cggacgagcc cgccgcggcg gtggacgcgc ggggcgcgct ggcggcccgg 50340ctgaccgggc
tggacgcggc cgggcaggac gagctgctcg cggacctggt gcgggcgcag 50400gcggcggcgg
tgctggggtt cgccgaccct ggcgcggtcg cggcggaccg ggcgttcaag 50460gacgccgggt
tcgactcggt gaccgccgtg gagctgcgga accggctggg cgcggccacc 50520gggctgcggc
tgccgccgac cgtggtgttc gaccacccga aacccctggc tctggcgcgc 50580gtgctgcgcg
ccgagctggt cccccagcgg ggggacgggg tgacggcggc gcaggtggcg 50640caccgggagg
acgcgatccg gcgggtgctg gcgtcggtgc cgctggcccg gttcgaggag 50700ctgggcgtgc
tcggcgcgct cgtggacctc gtgcccgccg cgccaccggc gggcggcgcg 50760gcgacagcgg
agcgggacga cctggcggac ctggcggagc tggacctgga cggtctggtc 50820cgcagggcga
tgcgcggcac caccgccggg aacgactgag gctttgatgc ggagcggaga 50880gagcatgagc
gcgggcacct cgccggagag cgtcgtccag gccctgcgga ccacgctggt 50940ggacaacgag
cggctgcggc gggagaacga gcggctggtc gccgaggccg gtgagccggt 51000ggcgatcgtg
tcgatggcgt gccggctgcc cggcggcgtc accgacccgg agtcgctgtg 51060ggagctggtg
cgcgagggcc gggacgccat cgggccgttc ccgaccgacc ggggctggga 51120cctggggtcg
ctgttcgacg acgacccgga cgcggcgggc tcctcgtacg tgcgggaggg 51180cgggttcctg
gcgcgggcgg gcgggttcga cgcgccgttc ttcggcatct ccccgcgcga 51240ggccctggcc
atggacccgc agcagcggct gctgctggag gtggcgtggg aggccgtgga 51300gcgggccggg
ctcgacccgc gctcgctgga gggccgggac gtcgcggtgt tcgcgggcgg 51360caacccgcag
ggctacggcg gcggaccggg tgacgccccg gagggcctgg aggggttcct 51420gggcgtcaac
gcctcgtcgt cggtgatctc cgggcgggtc tcctacaccc tgggcctgac 51480cggcccggcc
gtcaccgtgg acacggcgtg ctcgtcgtcg ctggtggcga tccacctggc 51540ggtgcggtcg
ctgcgctcgc gggagtgctc gatggcgctc gcgggcgggg tgaccgtgat 51600ggggcagccg
accgcgttcg tggagttctc gcggcagcgc gggctcgccc cggacgggcg 51660gtgcaagtcg
ttcggcgacg gcgcggacgg cacgtcgtgg gccgagggcg tcggcgtgct 51720gctgctggag
cggctctcgg acgcgcggcg cgacgggcac gaggtgctgg cggtgatccc 51780cggctcggcg
gtgaaccagg acggggcgtc caatggcctg accgcgccga acggcccgtc 51840ccaggagcgg
gtgatcgcgg cggccctggc cgacgccggt ctcggcctgg ccgacgtgga 51900cctgctggag
gcgcacggca ccggcaccag gctgggcgac ccgatcgagg cgcgggcgct 51960gctcaacacc
tacggccggg gcaggccgca ggaccggccg ctgtggctgg ggtcggtgaa 52020gtcgaacctc
gggcacgccc agtcggcgtc gggggtggcg ggcgtgatca aggtggtgca 52080ggcgatccgg
cacggcctga tgccgcgcac gctgcacgcc gacgagccga gctcgaacgt 52140ggactgggcg
gcgggggcgg tggagctgct ggcgcgcgag cgggagtggc cggagaccgg 52200gcgggcgcgg
cggggcgcgg tgtcgtcgtt cggggtgagc ggcacgaacg cgcacgtgat 52260cgtggagcag
gcgcccgagg aggccgccgc cggggtcgcg gcggcggggc ggcccgcgcc 52320caggtcggcg
ggcgggcagg acgccgggat cgcggcggtg accgggcagg ccgcccccgc 52380cgctggcccc
gccaccgccg aacccgccgc gtcggccgtc gaggacggga ccggcgtcgc 52440ccccggcccg
gtcgcgaccg gcggggtcgt gccgtgggcg ctgtccgggc ggaccgccgc 52500cgcgctggcc
gcccaggcgg cccggttgcg cgcgcacctc gccgcgcacc cggcggcccg 52560cccggtggac
gtggcctggt cgctggccac gacccgctcg gtgctggagc accgggccgt 52620cgtgcccgcc
gcctcgctcg acgaggccct ggcggggttg gacgcgctcg cctcgggccg 52680cgcggaccgg
tcggtggtcg tcggcgaggc ggcgcccggc cgggtggcgg tgctgttcac 52740cgggcagggc
agtcagcggg ccggtgcggg gcgcgagctg cgggagcggt tcccggtgtt 52800cgcgcgggcg
ttcgacgccg cgtgcgccgc cgtgggcgag ctgcccaccg gcgacggcgg 52860cgcgatcggg
ctcgccgagg tggcgctggc cgaccccggc acgcccgccg ccgcgctgct 52920cgaccggacc
gcgttcaccc agcccgccct gttcgcgctg gaggtcgcgc tgttccggct 52980ggtccagtcg
tggggcgtgc gcccggcggc gctggccggg cactcggtcg gcgagatcgc 53040cgccgcgcac
gtggccgggg tgctctccct cgccgacgcc gccgcgctgg tgcgcgccag 53100gggcgggctg
atgcaggagc tgcccgaggg cggcgcgatg gtggcggtgg aggcggccga 53160ggacgaggtc
gtgccgctgc tcggggacgg ggtgtcgctg gccgccgtca acggcccgac 53220ctcggtggtg
ctctccggcg acgaggaggc cgtcaccgcc gtcgccgcga ggctggcgca 53280gcggggcagg
cgcaccaaga ggctcgccgt ctcgcacgcc ttccactcgc accgcgtgga 53340cccggcgctg
gccgccttcc gcgccgtggc cgaggagctc gcctacgccg cccccacgat 53400cccgatcgtc
tccaccctca ccggccgccc cgtcaccccc gacgagctgc gctcccccga 53460ctactgggtg
cggcacgcgc gcggcgccgt ccggttcctg gacgccgtgc gggcgctggg 53520ggacgcgggc
gcgcgcacgt tcctggagct gggcccggag ggcgtgctca cggcggcggg 53580cgcggactgc
ctgccggacg cggtgttcgc ggcgacgctg cgcgccgacg tgcccgaggc 53640gcgggccgtg
ctcgccgggg tcgcgggcct gcacgtgcgc ggcgcgacag tcgactgggg 53700ttcgctgttc
acgggcgcgg acgcgcggcg cgtcccgctg cccacctacg cgttccagca 53760cgaggaccac
tggctggtgc gccgctccac cgccgccgac gtgggcgcgg tcggcctgcg 53820cgaggccggg
cacccgctgc tgggcgcggt cgtcgcgctg ccggagagcg gcggggtgca 53880gctgagcggt
cggttgtcgg tggcggcgca gccgtggctg gccgagcacg tcgtctccgg 53940cacggcgctg
gtgccgggcg cggcgctggt ggagctggcg gtgcgggcgg gcgacgagac 54000cggcacgccc
gtgctggagg agctggtgat cggccgcccg atgccgctgc cggacggcgg 54060cgcgctgagc
gtgcaggtcg tcgtcggccc ggacgagggc gggcgccggt cggtgcgcgt 54120gtactcccgc
gcggacgggg cggtggactg ggtcgagcac gcggcggggg cgctgaccgc 54180gccggaggcc
gcgccgaccg ccgacgcggg cccgtggccg ccggagaacg ccgaacccgt 54240ggacacgcgg
ggcttctacg acaccctcgc ggagggcggc tacgcctacg gcccgctgtt 54300ccggggcctg
acctcggcgt ggcgcggcga gggcgaggcg tgggcggagg tggcgctgcc 54360cggtgacgcg
accgggttcg gcatccaccc ggccctgctc gacgccgcgc tgcacaccgc 54420gcacttctgc
ctgcccaccg ggaccgagcg gcgggccggg ctgctgccgt tcgcctggac 54480cggcgtgcgg
ctgcacgcgg gcggcgcgac gaccgcgcgg gtgcacgccc gcgccaccgg 54540cgacgacggc
gtgaccgtgc gcctgctcga cggtgccggt cagccggtcg cggacgtggc 54600cgccctgacc
ttccgcgccg cagccgacac cccgtccgcc gaggtcccgg acgcgctgtg 54660ggcggtggag
tggaccgagc acccgctgcc cgcggacggg accacccccg cgggcgggac 54720caccacggcc
gtggtggtcg tggacacccg gagcgtcgac gcccccgacg acggccccgc 54780ccgcgcccgc
gcgctgaccg cccacgtcct cgccgagctg cagcggcacg ccgacgacga 54840ccggccggtc
gtcgtggtca cctcaggcgc ggtcgccgtg cgcgtcgacg gcgaggtcac 54900cgaccccgcg
tccaccgccg tgtgggggct ggtgcgggcc gcgcaggtcg agcagcccga 54960ccgggtccgg
ctggtcgacg tcgagccggg ggccgacccg gtgctcacct cgcccgagcc 55020gcaggtggcg
ctgcgcggcg ggaccgcgca cgtgcccagg ctggtccgcg cccgccgcgc 55080cctcccggcg
ccgaccgcga cgtcgtggcg gctgggctcc gaccgccccg gcacgctgga 55140ctccctcgcc
ctgctcccgg acgactccgg cacggccccg ctcgcccccg gcgaggtgcg 55200gatcgcggtc
cgcgcggcgg gcctgaactt ccgcgacgtg ctggtcgcgc tggggatgta 55260ccccggtcgc
gcggtgatcg gcgcggaggg cgcgggtgtg gtcgtggagg tcggccccgg 55320ccccgacgac
accgacgccg gcgacaccgg ccccggcgac accggctcgg gcggcctggc 55380cgtgggcgac
cgggtgatgg gcctgttccc cggcgcgttc ggcccgctgg ccgtggccga 55440ccaccgaatg
gtgacccgga tgccggacgg ctggtcgttc accaccggcg ccggcgtgcc 55500catcgcgttc
ctgaccgccc tctacgggct gcgcgacctc ggcgggctca ccgcgggcga 55560gaccgtgctg
gtgcacgcgg cggcgggcgg ggtcggcatg gccgccgtgc agctcgcgcg 55620ggcgttcggc
gctcgggtgc tgggcaccgc gcacccggcc aagcacgcgg ccgtgacccg 55680cctgggcgtc
cccgagtccc acctgtcctc cagccgcgac accgcctacg ccgacctgtt 55740cggcccggtg
gacgtggtgc tgaactcgct caccggcgag cacgtggacg cctcgctggg 55800gctgctgcgc
gcgggcggcc ggttcctgga gatgggcaag accgacctgc gcgacgccga 55860cgaggtcgcg
aaggcgcacc ccggcgtcgc ctaccgcccg ttcgacctgg gcggcgaggc 55920gcccgccgag
cgcgtcgcgg agctgctggc cgagctggtc gcgctgttcg aggcgggccg 55980catccacccg
ctgcccaccg cggcctggga gatcacccgc gcgccggagg cgttcggctg 56040gatgagccgg
gccgggcacg tgggcaagat cgtgctgacc ctcccccgcc gccccgaccc 56100ggacggcacg
gtgctggtca ccggcggcac cggctcgctc ggcgcggtcg cggcccggca 56160cctggtcacc
gcgcacggag cccgccacct gctgctcgcc tcccgacgcg gcgagcaggc 56220ccccggcgcg
gcggagctga ccgacgggct gcgcgggctg ggcgcggacg tgcgggtcgc 56280ggcgtgcgac
gtcgccgacc gggacgcgct cgccgcgctg ctcgccacga tccccgccgc 56340gcacccgctc
accgccgtcg tgcacacggc gggcgtgctc gacgacggcg tgctcgccgc 56400gcagaccccc
gagcgcctgg acgcggtgtt ccgccccaag gtcgacgccg tcgcgaacct 56460gcacgagctg
accggcgacc cggccctgtt cgcggtgtac tcctcggcct ccggcgtgct 56520cggcggcgcg
ggccagacca actacgccgc cgcgaacgcc tggctcgacg gcctcgccca 56580cgtccggcgc
gcggcgggcc tgcccgcgac ctcgctggcc tggggcctgt gggcgcagga 56640cggcggcatg
acgggcggcc tggcgggcgg accggccggg ccgggcgggc gggcccgccg 56700gggagccgtc
gcgccgctgt ccaccaccga gggcatggcg ctgttcgacg cggccgtcgc 56760gtcgggccgc
ccgctcctgg ccccgatcag gctcgacccc gccgcgctca ccgccgacgg 56820cgcgcagccg
cccgcgctgc tgcgcggcct ggcccgcccc acccgccgca ccgccgtcgc 56880ggccaccacc
gacgacggcc tcgcgggcag gctcgccgcg ctcgacggcc ccggcaggca 56940gcggctgctc
gtggagctgg tgcgggagca ggccgccgcc gtgctgggct tcgcgacccc 57000ggacgccgtg
tcgccgggcc gggcgttccg ggacctgggc ttcgactcgc tgacggccgt 57060ggagctgcgc
aaccgcctct ccgccgccac cggcctgcca ctgcccgcca ccaccgtgtt 57120cgaccacccg
accccgctgg acgcggcggc ccacctgctc gacgcgctgg gcgtcgcccc 57180cgcgcccgcc
ccggccaccc cggtcgtgac ggccgcgcgg gacgacgacc cgatcgcggt 57240cgtcgccatg
ggctgccgcc tgcccggcgg cgtgtcctcc ccggaggacc tgtggcggct 57300gctcgacggc
ggcgtcgacg ccatcggccc gttcccggac gaccggggct gggacctggg 57360gtcgctgttc
gacgacgacc ccgacgcggt cggcaagtcc tacgtgcgcg agggcgggtt 57420cctggcgggc
gcgggcgggt tcgacgccgc gttcttcggc atctcccccc gcgaggcgct 57480cgccatggac
ccgcagcagc ggctgctgct ggaggtggcc tgggagaccg tcgagcgggc 57540cgggatcgac
ccgacctcgt tgcgcggcgc ggacgtcggc gtgttcgccg gggcgggcgc 57600gcagaactac
ggcagcggcc ccggcccggt gcccgagggc ctggagggct acctgggcgt 57660gggcggcgcg
acgagcgtgg tgtccggccg cgtctcctac acgctcggcc tcaccgggcc 57720cgcgctgacg
atcgacaccg cgtgctcctc gtcgctggtg gcgatccacc tggcggtgcg 57780gtcgctgcgc
tcgggcgagt gctcgatggc cctggcgggt ggggtcgcgg tgatgggcga 57840gcccgcggcg
ttcgtggagt tctcccggca gcgcgggctc gccccggacg ggcggtgcaa 57900gtcgttcggc
gcggaggcgg acggcacgac gtgggccgag ggcgcgggac tggtgctgct 57960ggagcggttg
tcggtggcgc gggcgcgcgg gcacgaggtg ctggcggtgc tgcgcgggtc 58020ggcggtcaac
caggacgggg cgtccaacgg cctgaccgcg ccgaacggcc cgtcgcagga 58080gcgggtgatc
cgggcggccc tggccgacgc ggggatcacc ccggacgcgg tggacgcggt 58140ggaggcgcac
ggcaccggca ccaccctcgg tgacccgatc gaggcgcagg ccgtgctggc 58200gacctacggg
caggaccgcg agcagccgct gtggctgggg tcgctgaagt cgaacatcgg 58260gcacgcgcag
gcggcggcgg gcgtcgcgag cgtgatcaag tccgtgctgg cgctgggccg 58320gggcgtgctg
ccccgctccc tgcacgccag caccccgacc ccgcaggtcg actggtcctc 58380gggggcggtg
gagctgctgg cgcgggcgcg ggagtggccg gagaccgggc gtccgcgccg 58440gatcggggtg
tcctcgttcg gggtgagcgg caccaacgcg cacgtggtcc tggagcaggc 58500ccccgagccg
gaacccgcgc gggaggcgga acccgcgcgg gagtccgcgc cagggccgga 58560gtccgttccg
ccgctgaccg gggccacgcc gtggctgctg tccgcccgct cccccgaggc 58620gctggcggac
caggccgccc ggctggtgga cgccgtgccc gccgagtggc gggcctccga 58680cgtgggctgg
tcgctggcca ccacgcgggc cccgctggag cagcgggccg tggtcgtggc 58740gcgggacacc
gcgcgcgggc tcgccgccgc gtccgcgctg gccgccggac gccccgaccc 58800gcacgtggtc
accgggaccg ccgacgtgga cggcaggacc gccttcgtct tccccggcca 58860gggcgcgcag
tgggcgggca tggggcggga actcctggac gcctcgccgg tgttcgccga 58920acgcctgcgc
gagtgcgcgg cggccctgcg cccgtacacc gactgggacc tggtcgaggt 58980gatcacctcg
ggtggcgcgc tggaggacgt ggacgtcgtg cagcccacca gctgggcgat 59040catggtgtcg
ctggccgcgc tgtggcgctc gctcggcgtc cacccggacg cggtgatcgg 59100gcactcgcag
ggcgagatcg ccgccgccac cgtcgcgggc tggctcagcc tccaggacgg 59160cgcgaagatc
gtcgcgctgc gcagccagct gatcgacgag cacctgaccg ggctcggcgg 59220catgatgtcc
gtcgccctgc ccgccgagga catcgacctg accggctacc agggccggtt 59280gtgggtggcc
gcccacaacg gccccaccgc gaccgtggtc gccggggacg ccgacgccct 59340ggcggagctg
cgggacgcgc tggagggcga ggcccgcacc cgcgtgatcc ccgtcgacta 59400cgccagccac
accggccacg tcgacgccat ccgcgaccag ctcgcccgga tgctcgccga 59460cgtcaccccg
cggcccggcg agatcccgtg gctgtccacg gtgaccggcg agtggatcac 59520ccccggcgac
gacgacgccg actactggtt ccacaacctc cgccgcaccg tccacttcgc 59580cgacgggatc
accaccctgc tcgacgccgg gcaccgggcc ttcgtcgagg tctccacgca 59640ccccgtgctc
accccggccg tgcaggaggc cgccgaggcg aacccggcgc tgcgcaccgt 59700cgccgtgggc
accctgcgcc gcgcggacgg cggcgcggag cgggtggtgg cgggcctggc 59760cgagctgctg
gcgcgcgggg tggccgtgga cccggcggcg gtgttccccg gtgcgaggcg 59820ggtcgcgctg
ccgacgttcg cgttccggca cgagacgttc tggctctcgc gggcgctgcc 59880cgacgcgcgg
ccggtgccgc agggcgggca cccgctggcc ccggtggtgg tgagcgatcc 59940gggcacgggc
ggggtgatcc tgtccggccg gatctccgcg gccacccacc cgtggctgct 60000cgaccacgcc
gtcgcgggcg cggtgctgct gcccggcgcg gcgctggccg agctggcggt 60060gcgggccggc
gacgagaccg ggacgcccac cctggaggag ctggtgatcg gcaggccggt 60120ggtgctgccc
gaggacgggg agctgcggct ccaggtggtc gtgggcgccg aggacggggc 60180gcgccgcgag
gtgcgcgcct actcccgcgc cgacgacgcc gcgccgtgga ccgagcacgc 60240gagcggcacg
ctgtcggcga agtcctcgct gcccgccgac gtcccggccg ccccgtggcc 60300gcccgcgggc
gcggagccga tcgcgctgga cgggttctac gaggccatgg caggggccgg 60360ttacgggtac
gggcccgcgt tccgggggct gcgcgcggcc tggcgcgacg gggacgacgt 60420ggtcgccgag
gtggccgtgc cgcgggcgca ggagcaggtg gcgggccggt tcggcatcca 60480cccggcgctg
ctggacgccg ccctgcacgc cgggaacttc tgcttccccg cgcaggacgg 60540cgagcgggcc
acgatgctgc cgttcagctg ggacgacgtg cggttgcacg ccaccggcgc 60600gacgtcggtg
cgggtgcggg cccgcgcggt gggcggccct ggcgcgcccg cgctgaccgt 60660ggcgatcacc
gacccgagcg gggtgccggt ggccggggtg ggcgcgctcg ggatgcgcgc 60720ggtcagcccc
gagcagctgg gcgcgccggg cgtcggcggt gacgcgctgc gggtgctgga 60780gtgggccgag
gtggcggtcg aggcggcgga ccggtgggcc gtgctgggct ccgagcggca 60840cccggacgtg
gacgcctacg cggccgaccc ggaccggccg ggggcgctgc tggtggacgt 60900gggcgcctgg
ctgggcggcg acgacgccgt ggcccgcgcg cacgcgctga ccagcgcggc 60960gctggagctg
gtgcgggact gggcgacccg cggggacctg ggcggtgagc ggctggtgct 61020ggtcacgacc
ggggccgagg acgtgcgcga caccgcgccc cgcgacccgg cgcaggccgc 61080cgtgtggggc
ctggcgcgct cggcccgctc ggagcacccg gaccggttcg cgctggtcga 61140cgcggacgac
cggtccccgg cgacgctcgc cctggcggcc gggtcggcgt tcccggaggt 61200ggtcctgcgc
ggcgagcggg cgcacgcgcc gaggctggcg cgggccgtcc ccggcaggcc 61260ggtggcgctg
gacccggacg gcacggccct gatcaccggc ggcaccggcg ccctgggcgc 61320gctcgccgcc
cggcacctgg tgaccgcgca cggcgtgcgg cgcctgctgc tcaccggccg 61380ccgggggccg
gacgcccccg gcgcggcgga gctggccgag gagctgcgcg ggctgggcgc 61440ggacgtgcgg
gtggaggcgt gcgacgtcgc cgaccgggac gcgctcgccg cgctgctcgc 61500gtcgatcccc
gccgggcgcc cgctcaccgc cgtcgtgcac gcggcgggcg cgctcgacga 61560cgccccggtg
accgacctga ccccggagcg gctgtccgcc gtgctggccc cgaaggtcga 61620cgcgctggcc
aacctggacg agctggtcgg ggacgggccc gcggtgttcg cggtctactc 61680ctcggcgtcc
ggggtgctcg gcacggccgg gcaggcggcg tacgcggcgg ccaacacctt 61740cgcggacgcg
ctggtgcgcc gacgccgggc cgagggccgg gcgggcgtgt cgctggcgtg 61800gggcctgtgg
gcaggcgcca gcgagctgac cggcgacctg gccggtgacc ggctcgcccg 61860cacccgccgg
ggcgggctgg tgccgctgac cgccgccgag ggcatggcgc tgttcgacgc 61920gggcgcggtc
accacgggcg gcccggcgct ggtcgtgccg ctgccgctgg acctggcggc 61980gctgcgcgcc
tccgcgcgcg acgaggcggt gcccgcgctg ctgcgcgcgc tcgtccccgc 62040cgcgcggcgc
tcgctctccc ccgccaccgg gcaggccgcg cccccggccg ggttgcgggc 62100gcgcctggcc
gggctgtcgg gcgacgagca ggaggccgtg ctcaccgagc tggtccgcga 62160cctggccgcc
gccgtgctcg ggcacggcga gaagggcgcg gtgggcccgg acgacgcgtt 62220cttcgagatc
ggcttcgact ccatgacggc cgtgcagctg cggaaccggc tgaacaccgc 62280caccgggctg
cgcctgcccg ccgcgctgct gttcgaccag ccgacgcccg cgatcgccgc 62340cgaggcgctg
cgcgagcgac tggccgccga gcaatcgggc tcagggcaat cgggcgcagg 62400gcagccgggc
gcagggcatt caggcgcagg gcagtcgagc gcagggcgat caggcgcagg 62460gcagtccacc
gacccgaccg acgagaggtg agcaccagca tgatcgacgt ggccgagtac 62520ctgcggcgca
tcggcgtgga gggcggcgtg ccgagcccga cgctggagtc gttgcgggcg 62580ctgcacaagc
ggcacctgat gtccgtgccc tacgacaacg gcggcgcggc cgaccggttg 62640ccgccgaacc
gggggctcgc ggagatcccg ctgccccgtg tgttcgcgca cgtggtgacc 62700ggccgcaacg
gcggggtctg ctacgagctc aaccggctct tccacgccct gctcaccgcg 62760ctgggctacg
aggtgctgat ggtcgcggcg gcgatccggc tggccgacga ccggttcggg 62820ccggacgagg
agcactcgtt caacctggtg cgcctggacg ggcggacctg gctggtggac 62880gtggggttcg
tcggcccgtc ctacctggag ccgctggagc tgtcggcggt cgagcaggag 62940cagtacggct
gcgcctaccg ggtcgtggag cgcggggacg cgcacgtggt ggagcgcagg 63000cccagggacg
gggcgtggca ggcggtgtac cggttccggc cggggcgggc ggaccgggac 63060ggctgggagg
cggtgcggtt ggacgggctg gacgactacg cgcgggactc ggtgctggcg 63120ggcaccacgt
tccggggtcg ggcggcggag aacgggcagc acgtgctgat cggccgccgc 63180tacttcaccg
tgctggacgg ggtggagacg acgcgggtgc tcgtgaagaa ggacgagttc 63240gcccgcgtca
ccgagtcgat catgatcggg gggtgagcgc gtggcgggcg aggtcgagca 63300cgacgtggtg
gtcgtcggct acgggccggt ggggcagctg ctgtcggtgc tgctggcgca 63360gcgcggctgg
cgggtgctgg tgctggagcg ctggccgacg ccgttccggc tgccgcgcgc 63420ggtcgggttc
gacagcgagg cgacccgcgt gctggcctcg gccgggctcg ggcccgcgct 63480ggccgagttc
ggggagcccg cgggcgacta cgagtggcgc accgcgtccg gggagacgct 63540gatcgcgttc
accgtgcggg aggaggggca ctgcggctgg cccgaggcga cctcggccta 63600ccagcccgcg
ctggaggacg cgctgatcgc gcgcggcgag gcgctgccgg gggttcaggt 63660gcggcgcggc
tgggaggtga ccgggctgac cgaccggggc gaccacgtgc gggtggtggc 63720caccgacccc
ggcggggcgc gcgtgaggct gacggcgcgg ttcgcggtcg gctgcgacgg 63780ggcgaacagc
gtggtgcggg cccgcaccgg caccgacgtg accgacctgg acttctcgca 63840cgactggctg
gtgtgcgacg tgcggctgca cgaccggcgc ccggtgacgc cgaacaacct 63900ccaggtgtgc
gacccggcca ggccacgcac cgcggtgtcg gcggggccag ggcaccggcg 63960gtacgagttc
atgcgggtgc ccggcgacga cccggagcgg ttcggcacgc cggagagggc 64020gtgggagctg
ctggcgctgt tcggcgtcgg gcgcggcgac ggggtgctgg accggctggc 64080cgtgtacacg
ttccaggcgc ggtgggcgcg gcggtggcgg gcgggccgga tgctgctggc 64140cggggacgcc
gcgcacctga tgccgccgtt cgccgggcag ggcatgacct ccgggttccg 64200ggacgcggcg
aacctggcgt ggaagctgga cctggtgctg cgcggcgagg ccgggtcggc 64260gctgctggac
agctacacgc tggagcgcgc cgagcacgtg cggcacgccg tgacgatctc 64320ggtgggcctg
gggcgggtgg tgtgcgtggc cgacccggcg gtggctgcgg accgggacgc 64380ggcgatgctg
gcggcgcgcg agcgcgagct gacaccgggc gcgtcggccc ggtcggtgct 64440caagcccctg
gaggacgggg tgctgcaccg ggacggcgac ggcgccctcg cgccgcacgc 64500gggggccgtg
ggcccgcagt ggcgggtggg gcgcggcggg cgggtcgggc tgttcgacga 64560cgtggtgggg
accgggttcg cgctgctcac caggggcggg ctggtggcgg ggccggaggt 64620gcgggcgcgg
ctggacgggc tgggcgcgcg ctacgcgcac ctggtgcccg ccggggcggc 64680ggcggacggg
ccggacgacg tggtcgacgt gagcgggaac tacctgacgt ggctggagga 64740gctggacgcg
gcggcggtgc tgctgcgacc ggacttctac gtgttcggcg cggccgggga 64800cgcggcgggg
ttggccgggc tggtggcgga cctgcgcgcg cggttggggt gacgccccgc 64860aggccccggc
acgtgccgcg ccggggcctg ctcgcgcgtc acgtccggtc gtcggcgagg 64920tgggccaggc
accagtcgag cacctgcgag ggcttgcgga ccaccgcgtc cgggttcgcc 64980gccagcagct
ccgcctcgtc cgtctcgccc cacagcgcgg ccagcgccgg gtagcccgcg 65040gcgcgggcgc
tggccaggtc cgtcagggcg tcgcccacca tcaccacccg gtcggccggg 65100acgtcgagca
ggccggtggc cagcaggagc atgtccggcg cgggcttggg gttcgcgacc 65160tcgtcggagc
cgatgatatg gtcgaacagc cccgccatgc cgagggtggt cagcagcgac 65220cgggcgcgcg
gcccgctctt gccggtgacc acggcggtgc cgaagccgtg ctgccgcagg 65280tccgccagca
gctccggcgc gccctcgaac acctccacct cacccgccag ccggtagctc 65340tcgcggacga
acggaccctc catctccagc ggcaggtcca tgatccgcat gatgtccggg 65400aagtaccgcc
ccaggtgccg gttgtactcc tcgaacggcg cgggcccgtc gccgacgacc 65460tcggcgtagg
cgatctcgaa cgcctgccgc atgacggcga agctgttgac cagcaccccg 65520tcgaggtcga
acagcacggc ccggtcgtag gtcgcgccgg ggacgtgccg gtggggcgcg 65580ggggtcggcg
gggcgagggg gcgcggggcc gcgggcgccg gaaccgcggt cgcggcccgc 65640tcgtccccgg
ctcgggcccg cacgactcgg gggttggtct gtccggtggt catcacgggg 65700ctcccgtcgg
gacgaggtcg accggcgcgt gtcgtcgttc ggcgcaccgc acggtgtcgg 65760cggcgcggta
gacccgttcg atcgcgcccg cgatccagcg cgccccggac gccgcctcgc 65820cgcgcgtggc
ggggtcggcc aggcgggtgg gcaggctcgc gagctgggcg tcgtactcgg 65880cgcccaccgg
ctcggcgggc agctcgaccg gggtggtgcg gccgtcggtg gtgagcagga 65940gccgcgacgg
gccctcgcgg ttcgggctga agccgaaggt gcagcgcagc tccgccgtgc 66000cgccgctgcc
ctccacgcgc acgaccgtgg tgtccagcgc ctggtgcgag gcccaggccg 66060cgcgcacccc
gatcgagatc cccgaccggg tgacgaggaa ccccctggcg gtgtcctcca 66120cgtcgccgac
caccgggtcc accggcgccc cgtcgccgcg ccaggcggcc cggaacgcgt 66180ggtcgttgac
gaagtccgcg gacaccgcgc cggtgacgtg ctccagctcc gcgccctccg 66240ggtcgccggt
ggcgccgcgc agcagcacgc gggcggtgtc gagcaggtgc cagccgaggt 66300cgaccagcgc
gccgccgccg gagcgggtgc ggttggtgaa ccagccgccc cggtccggga 66360tgcccttgga
ccgcacccag gacacgtcga cgtgccgcag cgcgcccagc gacgcggcca 66420cctggcgcag
cgcccgcacg tcggcccggt gccgggcggc gctcccgccc agcagcaccg 66480cgccaccggc
ctgctcggcg gcggtgagcg cggcggcctc ggcggacccc aggcacagcg 66540gcttctccag
gaacaccggg acgccccgcc gcagcagacc ggacgcgacc ggcgcgtgca 66600ggtggttcgg
cacggcgacc acggccaggt cgacctcgtc gcggcgcagg tcctccaccc 66660gctccagcgc
ggtgatcccg cgggagccgg gcacggcggc gcgggcctgc gcggacggct 66720cgaccacggc
gacgacccgg aaggcggggc tgcccagcaa ccggggcagc cacacctccc 66780gcgccaccca
cccgagcccg accaccgcga cccgcaccgg accaccgctc ccggccctcg 66840gcccgtcgct
cacaccacca cccccgctcc ccgcgcccgc caccccgcgc tcacgcgccc 66900gcgaccacgt
cggccacgac ggcggcaagc cggtgcagct gctcctcggt gcccagcagc 66960acccggtggt
gcagccacac gcagtcgcgg gtgatctcct ccgacaccgg gcaccgggcg 67020gccagctcct
cggtggtcag gtcgggcgcg ccggtctccc agaacgcctg ggtgcggtag 67080accgcccgga
acgccatgaa cgccgggatc ccgcgccgca ccagctcgtc caccaccgcg 67140ttgcgccgct
cctcggtgac gccgggcatc cggaacatcg ccatgtagct cgggttgcgg 67200tcgctgcgcg
ggtcgacggt ctgcggcacg acgccgtcga tccccgccag cagcgcggac 67260agcaccggcc
agcgggcctg cctggtcgcg atctgcgagt ccagcctgcc gagctgggcg 67320cgcagcacgg
cggcggagaa ctcgttcatc cggaagttcg agcccgaggt gaggtggaag 67380tagccgcggt
cgcccttggg cctgccgcag ctgtgcagga cgaacgcctt ctcccactgg 67440gcctcgtcct
cgaacagcac ggccccgccc tcgcccgccg tcatcagctt gccgttctgg 67500aagctgaacg
tggcgatcga cccgagctcg ccgacccgct tgccgcgcca gtgggcgccg 67560tgggcgtggg
cggcgtcctg caggaccggc acgccggtgc tcgtggacag cttgtccagc 67620cggtccatgt
cggcgaactg gcccgccatg tgcacgggca tgatcgccga ggtgcgggag 67680gtgacggcgg
cctcggcggc ggcgacgtcc aggcagtagg tgtcggggtc gacgtccacg 67740ggcacggcga
ccgcgccgag gcgctgcacg gcctgcgagg acgagatgaa ggtgaaggcg 67800ggcacgatca
cctcggcgcc ggggcccacg tcgagcacct ggagcgccag ctccagcgcg 67860tgcgtcccgt
tggtgacggc gagcgcgtgg cccgcgccgt ggtactcggc gaactcgcgc 67920tcgaactcgt
cgacctcgct gccgccgacc cgccaccact ggccctggtc cagcgcgcgc 67980agcagggccg
tgcgctcggc gtcgtcgtgc tgcggccagg ccgggaactc gatgcctgcg 68040tccggagaat
tgctcatgag cccctgtccc gtcgttcgcg gaaatggcgc gggggaattc 68100gccgcggcct
gctttcggaa ttcgacgcta ccgattccgc agatcccgac caaccccctt 68160gacctccccc
taatcccccc tgttcccagg ccatcaccgc agcacgcggg cacagcggca 68220cagccgtgcg
cacaatgggg gcgaacggga accggggcgt ccgcgcgccc cggcggcgct 68280ttcggggaaa
ggtgtcaggc gtgggcgagc tgctgctggt gaacgggccg aacctcggca 68340tcctggggcg
ccgcgaggtg tcggtgtacg ggaccgacac gctcgcggac gtcgagaagg 68400cggtcggcga
ggaggtcgcc gggcgcggct ggtcggtccg ctcggtgcag cgcaacggcg 68460agggccagct
cgtggacgag atcgaggcgt cctacgacac ggtgggcgcg atcgtgaacc 68520ccggcgcgct
gatgatggcg ggctggagcc tgcgggacgc gctggcgaac tacccgcgcc 68580cgtggatcga
ggtgcacctg tcgaacgtgt gggcgcgcga gagcttccgg cacgagtcgg 68640tgctggcgcc
gctggcgagc ggtctcatcg cgggcctggg cgcgcgcggc taccggttgg 68700ccgcccgcgc
gctgctggac ctggtggact gaccgccgtc gcgcgcgagc ccggccgcgt 68760gcacggcccc
gcgcagcgag gacaggccgc cgagcagcgc gggccgcacg ggcgcggtcg 68820ggtggccggg
ccgggcgagt gcggcgcagc ggtcggcgac cgcgtcgacg tatccgggca 68880cggcggcggc
gaacccgccc ccgaccacgc acagcgaggg ccgcgccagc tcgccgacgc 68940cgacgagcgc
ggcggccagc gcccgggccc cccgctcgac cgcggcccgc gcccacccgc 69000gcccgtcgcc
gagcgcccgc accaggtcct ccccggtgac cggcgcgccg ccgagcctgg 69060cggcctcggc
gagcacggcc ggtccggacg cgaacgcctg cacgcacccg gcccgcccgc 69120acgggcaggg
cggcccgtcg agcgccacca cgacgtgccc cagctcgcag gacccgcgct 69180cgggtccggg
gaacggcagg ccaccggaca cgacgccccc gccgacgccg gtccccacgc 69240ccgcgtagac
caggtcggcg cacccgtggg cacgggcctc ggcgagcgcg gcgaggtcgc 69300cgtcgtccgc
gacgagcacc ggggcggcca gcccgccgag gaaccccgcg aggtcgacgc 69360cgacccaccc
cggacggctg ggccaggccg tgacgacccc gccgtcgacg gtgccgggga 69420acgcgatccc
gaccccgtcg agcggcgccc cggcccgccc ggcgaggtcg gcgacggcgc 69480ggccgagcag
gtcgaggtcg gcgcgcggat caccgtcccc aggccaccgg aagccctccc 69540gcagcaccag
cggcccgcgt tcgagccgca gcgccacctt ggtcccgccg acgtccaccc 69600cgagcagcgc
cccgctcaca ccccgacctc ccgccgtccg cacccctcgc cgcaccacca 69660cccgcgcggg
ccgccgccca cgacgccgcc cgccacaccg atacccgcgc ggtcctcgct 69720cacgacgccg
ccacaccacc accgcacggg cctgcggtca cgacgccgcc acacctccac 69780ccagcgcacc
accacccgcg cgggccgccg ctcacgaggc caccgctcac gacgccgccg 69840cccgccaggc
cgccaccgcc tcggcccgcc gggcgtcgcc gctctccacc agcgcgaacc 69900ggatcgcccg
cgtggccgcg ttcgccgcct gcagcgcctc gtgcgcgccg ggctcggggt 69960cgctgcgccc
ggtcgccacc tcggtgatgc tgcgcgccgc ctccaggcac gccaggcagg 70020ccgcgacgta
cgcgtccgcc ccgcctcccg cgccgcccag cttctccagc agccgcgtca 70080cgtcggcgtg
cacctcgcgc acggcgtcct ccacccggcc cgcgccgggt ggtgtgccgg 70140ggatgggggt
caccgctgcc tcccggtgcc tgacgcggac ccggtcagcc gagggccggg 70200atgagttcga
cgaaccgacc ctgccacaac cggcgcagct cgcgcgtcaa ctcctcggac 70260ggcgcgccgc
cgaccagctc cgccagggtc gtcacgccgt ccacccggct gagcagcgcg 70320tgcgcctgcg
cggtggtggc ggtgacgggc ccgtggtcgt actccagcga cacctcgtgc 70380accggctcgc
ccgccgccgc gctgcccggc gcgggcgcgg tgcgcacggc caggcgggtc 70440accgggcgca
gcctgggcac cagcgcgccc aggtcctgct cggggcgggc ccgcaccagg 70500aagccctcca
cgaccaggcc gtccaggtcg gtggtcagga agctggtgag cacgtcgtcc 70560aggctctgcg
cgatcggctc ggcgttgttg ttgaacgagg tgttgagcag caccggggtg 70620ccggtcagct
cgccgaaccg cgccaccagc cggtggaagc gctctcccga ctcgggcgtg 70680acgacctgca
cgcgcgcgct gccgtccacg tgcgtgaccg cgcccagctc ggcccgcctg 70740gcgggcagca
ccggcaccac gaacgacatg aactcgtggt gccccagcgc cccggacagg 70800tcgaaccagt
cccgcgcggc ctcggcggtg accacgggcg cgaacggccg gaagctctcc 70860cgcttcttca
ccatggcgtt gatccgcgtc tggttctccg cgggccgggc gtcggcgatg 70920atgctgcggt
gcccgagcgc gcgcggcccg aactcggagc ggccgtgcgc ccagcccagc 70980acctcgccgt
cggcgagcag cttcgccgcg gtctccaccg ggtccaccag cggcgtcacc 71040tccaccaccg
gcgaccagtc cgcgagccgc gcggcgacct gctcgtccgt gccgaggtcc 71100ggcccgagcg
ccgccgacac cagccgcgcc gacggccgct ccagcacgcc cagcgcggcg 71160gctgcggcgt
acgcggcgcc ctcgccggcg cccgcgtcgt gcgaggcggg gtggatgaac 71220acctcgtcga
acagcccggc cttgaggatg cggccgttga gcgtggagtt gtgggcgacg 71280ccgccgccga
acgcgagcgt gcgcagcccg gtgacctcgg cccagtgccc gagcacgtgc 71340agcgcgatct
tctcggtcgc ctcctgcagc gcggcggcga agtcccggtg cgcctggctg 71400aacggctcgt
ccttgcggcg cggccggaac ccggcggcga ccagcgcggg ggtgaccagg 71460ttcggcacct
tggtgttgcc gatcaggtcg tactcgccct tgtcgcgcag cgcgtgcagc 71520ccggagaaga
cctcgcggta ggtcgacggg tcgccggacg gggcgagccc catgaccttg 71580tactcgtcgc
cgaagccgta gccgagcagg aacgtggcgt tcaggtacag cccgccgagg 71640gacttctcca
ccgggtagtc gtgcagcttc tccaggtgcg cgccacgcgc gtggtagacc 71700gtgccggagt
tgtcctcgcc ccggccgtcg aagatcacca ccagcgcctc gtccgcgccc 71760gagtgcaggt
aggacgagta ggcgtgcgcc tcgtggtgcg gaacgtagac gagcttgtcg 71820tcgggcagct
cccagccgag gtcctcgcgc agccgctgct tgatcagctc gcgggagaac 71880cgcagcggca
ccctcgggtg ctcggtgtag acgtggttga gcaccaggtc gaggtggtcc 71940tcggggaagt
agtagccgac cgcgtcgacg tcgtccacgg tcgcgcccgc gagcgccagg 72000cactcgcgga
tggcggtgga cgggaacttg gtcgtcttct tgacgcggtt gagccgttcc 72060tcctccacgg
cggccacgag ctcgccgtcg cgcaccaggg acgctgccgc gtcgtggaag 72120aacagctccg
acatcgacgg gacgaggtcg gtctcggcgg gcgagaagtt cccgttgatc 72180ccgagcacga
gcatgtggca tcaccttgat ccgggaggcg agggctgggg cgcggagggg 72240gtcgccgtca
ggcgcggggc gcgacggcgg cgaggtcggg cgaggtcagc cgcagcgcgg 72300tgggcgcggg
cttcgcggtc ggcacgaggt ggaagcgctc cacgccctcc tcggtgggcg 72360cgggcagctc
ggcggcgcag gcgcagtcgg cgggggcgaa cccggcgaac cggtaggcga 72420tctccatcat
ccggttgcgg tcggtgcgcc ggaagtcggc gaccaggtgc gcgcccgcgc 72480gcgccgcctg
gtcggtgagc caggtcagca gcgtcgaccc ggcgccgagc gagacgacgc 72540ggcacgaggt
ggccagcagc ttgaggtgcc acacccgccg gcgccgctcc agcagcacga 72600tgccgaccgc
gccgtgcggc ccgaaccggt cggacatggc cacgaccagc acctcgtgcg 72660cggggtcggc
gagcaggccg cgcagcgccc ggtcgtcgta atgcacgccg gtggcgttca 72720tctggcttgt
gcgcagggtc agctcctcga cgcgggtcag gtccgcctcg cccgcgcgcg 72780cgacgaccac
ctccaggtcc agggtgcgca ggaactcctc gtcgggcccg gtgaagccct 72840cgcggctggc
gtcgcgctcg aagcctgcgc ggtacatcag ccgccgctgc cgcgagtcgg 72900cggtgaccac
ggcggggctg aactcggggc gctcgggcag ggaggcgacg tcggcctcgg 72960tgtacaggcg
cacctcgggc agggcgcggg ccacctcggc gcgctcgacg gggctgtcgt 73020cgacgaacgc
gatcgtgcgg tgggcgaagc cgagccggtc ggcgatggcg cgcaccgacg 73080ccgacttggc
gccccagccg atctgcggca gcacgaagta gtcggcaagg cccaggcgtt 73140cgagcacggg
ccaggcgtgg tcgtggtcgt tgcggctggc cacggactgc aggacgccgc 73200gcccgtcgag
cgcggtgatc acctcgcgga cccgctcgaa cgggacgacg tcggcgtcct 73260ccaggagggt
gccgcgccac agggtgttgt ccaggtccca gaccaggcac ttgaccgtcg 73320gtgcgggggt
ctcggtcacg gctgctgctc cctgagcgga gttcggctgc gctggtcaag 73380tccgcgcggg
gcccggctgc gcacgtgctg ggcgagcacg agctggcaga tctcggaggt 73440gccctcgatg
atctccatga gcttcgcgtc ccggtgcgcc cgcgccacca cgtgcccgtc 73500gctggcgccc
gcggagccga gcagctgcac cgcgcgcccg gacccggcgg cggcctcgcg 73560cgaggccagg
tacttggcct gcaccgccgc caccgccagg tccggcgagt tcgcgtccca 73620cagcgcgctg
gcgtgctcgc tcgcgcgggc ggcgacctgc tcgccgacgt gcaactcggc 73680caggtgccgg
gcgacgagct ggtggtcggc cagcacgccg ccgccctgct cgcgggtcgt 73740ggtgtgctcg
acggcggcgg ccaggcaggc gcgcaggatg ccgacgcagc cccacgccac 73800cgacacccgc
ccgtaggtca gcgcggcggt gaccaccagc ggcagcggca gcccggtgcc 73860gcccaggacg
tcggcggcgg gcacccgcac cccgtccagg gtgatcccgg agtgcccggc 73920ggcccggcac
ccgctggggt tcggcaccct ctccacccgc acgccgggcg cgtcggcggg 73980cacgacgacc
gcgctcgccc cgccccggta gtgcccgaag accaccagca ggtccgcgta 74040gtgggcggcg
gtgatccacg acttgcggcc ggtcaccacc acctcgccgt cgccggtgtc 74100ggtgatggtc
gtggtcatcg cggacaggtc gctgcccgcg cccggctcgc tgaacccgac 74160cgccgccagc
ccgcccgagg tgagcctgcg caggaaccgc tcgcgctgct cggcggtccc 74220gagcctgcgc
gcggtccacg ccgccatgcc ctgggaggtc atgacgctgc gcagcgagcc 74280gcacagctcc
cccaccgacg cggtcagctc gccgttctcc cggctgccca gcccgaggcc 74340gccgtgcggg
gcgccgacct gggcgcacag cacccccagc ccgcccagct ccaccagcag 74400ctcgcgcggc
agctcgcccg ccaggtccca cccggcggcg cggtcgccga cgcgctcggc 74460gaccagcccg
gccagtgcga cggcgtcgct caccgccccg cctcccgcag ccgcagcacc 74520agcgtggtca
tggtgttgac ggtgcggaag ctgtccaggc ccaggtccgg cccgtcgatc 74580acgacgtcga
aggtcgactc caggtgcacc acgagctcca tcgcgaacat cgaggtgacg 74640gtgccggacg
cgaacaggtc ggtgtccggc tcccaggtct gcttggtgcg ctcggcgagg 74700aacgcctgca
cccgctcggc caccgcgtcg gcggtgagcg cgccgggctg ggaggaggtc 74760gtcacagctg
tgccttcccg tagtcgtaga agccccgccc ggacttgcgc ccgaggtgcc 74820cgtcgcggac
cttgcgcagc agcagctcgc agggcgcgga gcgggggtcg ccggtgcgct 74880cggccagcac
gcgcagcgag tcggccaggt tgtccaggcc gatcaggtcg gccgtgagca 74940gcggtcccgt
gcggtggccg aggcagtcct gcatgagggc gtccacggcc tcgacggagg 75000ccgtgccctc
ctggacgacg cggatcgcgt cgttgatcat cgggtggacg atccggctgg 75060tcacgaagcc
ggggccgtcg ccgacgacga ccggtgtgcg ggccagctcg cccagcacgc 75120ccacgagggt
ctccagcgcg tccgcgccgg tgcgcgcgcc ccggacgacc tcgaccgtgg 75180ggatcaggta
cggcgggttc atgaagtgcg tgccgatcag ccgcgccggg tcggggacgt 75240gcccggccag
ctcgtcgatc gggatcgagg aggtgttgga caccagcggc acgcgcggcc 75300cggtgagcgc
ggcggccccg gccagcacct cggccttgac cggcagctcc tcggtgaccg 75360cctccaccac
cagcgagacg tccgcgacgt cggcgagcga ggtggtggtg agcagctcgc 75420cccgctcgcg
gtcctcgggc agcgcccgca tcagcctggc catgcgcagc tgggcggcca 75480ccgcctcccg
cgcccgcccg accttggccc ggtcggtctc gaccagcacc accggcacgc 75540cgtgcccgac
ggccagggag gtgatcccca ggcccatcgt gcccgcgccg agaacggcga 75600gcaccgtcct
gccgtcctgc tctcccatcg cgctcccccg ccgcggccac cgcggccgcc 75660gtccggtccg
cgcgccgtcc cggcacgcgc attccaccct cgatcgtgtg ccgggaaagg 75720cgcgcccgac
cccctgacct gcccccctga acccccctca acggaaccgg aaatcgaatg 75780tcccgaacgc
gccgtcaaat cgtcgattga cagccgcaga actgttcata gactgtggcg 75840gcagtaccga
tctccgaatt ccacggaaga gtcctccccc atggctcagc agatcagcgc 75900cacctcggaa
atcctcgact acgtccgcgc gacctcgttg cgcgacgacg acgtgctcgc 75960cggtctgcgg
gagcggaccg cggttctccc ggccgcgtcc gcgctgcagg tggccccgga 76020ggaggggcag
ctgctcggcc tgctggtgcg cctggtcggc gcgcgctcgg tgctggaggt 76080cggcacctac
accgggtaca gcacgctgtg catggcccgc gccctcccgc ccggcggacg 76140tgtcgtgacc
tgcgacgtcg tcgcgaagtg gccggacatg ggcaggccgt tctgggagcg 76200ggcgggcgtc
gcggaccgca tcgacgtccg cgtcggcgac gcccgcgcca ccctggccgg 76260cctgcacgcc
gagcacgccg tgttcgacct ggtgttcatc gacgcgaaca agtcggatta 76320cgtccactac
tacgagcgcg cgctgacgct gctgcgcacc ggcggcctgg tcgtcgtgga 76380caacacgctc
tttttcgggc gggtcgccga tccgtccgcg accgatccgg acaccaccgc 76440cgtgcgcgag
ctgaacgcgc tgctgcacgc cgacgagcgg gtcgacatgt gcctgctgcc 76500gatcgcggac
ggaatcacgc tcgccgtgaa gcggtgaacc cgcccgaatc gcgccgaatt 76560cccccggaga
gaaaggccgc cgcagtgttc accgaggacg tggccaccga cctgcccgcc 76620tacccgttcc
tgcgggaccg gggcgactgc ccgttcgcgc cacccccgcg ctacggccaa 76680ttacgggagg
agcagcccgt caccagggtc cgcctgtggg acggcagcac cccgttcctg 76740ctcaccggtc
acgaggtgtg ccgcaccgcc ctgaccgacc cgcgcttcag ctccgacggc 76800gccaaccgcg
cccagccgcg cttcgtgaag ttcgacatcc cggacgacgt gttcaacttc 76860ggcaagatgg
acgacccgga gcacgcgagg ctgcgccgca tggtcgccgg gcacttcgcg 76920agccgccccg
tggaggcgat gcgccccgcg atcaccacga tctgccacgc ccagctgcgc 76980cagctcgtgc
aggcgggctc ccccgccgac ctggtggccc actacgcgtt cccgatcccg 77040tccctggtga
tcggcggcgt gctcggcgtg gcgggccccg gcctggacga gttcgcgcgc 77100gactcgacgc
gcgccctgga cccgtccctg tccgccgagg agatgggcgc cgccatcaac 77160tcgatggtcg
ggttcgtgga cgacctgtgc gcggccaagc gggccgcccc cggcgacgac 77220ctgatcagcc
gcctggtgct ggacttcgag cgcaccggcg agctgacccg gaagcagctc 77280gtcgccaccg
tgatggtcgt gctgctggcg ggctacgaga ccaccgcgaa catgatcgcg 77340ctgggcacga
ccgcgctcct gcgcgacccc gagcagctgg ccttcctgcg cgccgagccc 77400gccggtttcg
ccaacgccgt cgaggagctg ctgcgctggc acaccatcgt ccaggacggc 77460accggccgcg
tggccctgga cgacgtcgag ctggacggcg tgctcgttcc cgcgggctcc 77520ggcgtgatcg
tcaacctgcc cgcggccaac cgcgaccccg acgtcttccc cgatcccgac 77580cgcctcgacg
tgaccaggca caacgcccgg cggcacttcg cgttcggcta cggcgtccac 77640cagtgcgtgg
gcatgacgct ggcgcgcgtc gagctgcaga tcgcgctgga gaccctgctg 77700tgcggcctgc
cgggcctggc gcctgccacg ccgttcgagg acctggactt cgccctggag 77760tccatgaacc
tcggcctgcg ctcgctgccg gtcacgtggt gagcaccgac cgtccaccag 77820gggagagccg
atgacccgca ccacccccac ccccgacctg gccccggagt tcccgatgcc 77880caggtcgccc
gagcacccgt tcgacccgcc ccctcgactc cgcgaggcgc aggaggcggg 77940cggcctgtcg
cgggtgcgcc tgtgggacgg cagcaccccg tggctgatca ccaagcacgc 78000ccaccagcgc
gagctgctgc gcgacccccg cctcagcgcg gacttcctgc gccctggcta 78060ccccagcccg
attcgcatcg aggacaagtc gacgttcatc agcagcttcc cgctcatgga 78120cgaccccgag
cacaaccggc agcgccggat ggtcctgggc ccgttcaccg tccgcaaggt 78180ggaacgcctg
cgcccgttcg tgcagcggat cgtcgacgag aagatcgacg aactcctcgc 78240gggccccaac
ccggtcgacc tggtcaccgc gttcgcgctg cccatcccgt ccctcgcgat 78300cagcgccgtc
ctgggcctgc cctactccga ccacgaggtc ttcgagcgca acagcgccgt 78360gctgatccgc
caggacgtgc ccccgcagga acgggccgag gccagcgagg agctccagca 78420ccacctcgac
cgcgtcctgg gcgacaagat gaccgacccc gccgacgacc tcctctccga 78480cctgggcgca
cgggtgctgg caggcgagat cagcaggccg gaggcggtcg acatgaccgt 78540cctggtgctg
gcgggcgggc acgagaccac cgcgaacatg atcgcgctcg gcaccctcgc 78600gctgctccgg
caccccgacc agctggcgct gctccaggcg ggcgacgacc ccgccctcgc 78660cgagaccgcc
gtcgaggagc tgatgcgcta cctgacgatc tcgcacaccg ggatgcgccg 78720cgtggcgacc
gaggacgtgg agatcgacgg ccaggtgatc cgcgcgggcg agggcgtggt 78780gctggcgacc
tcgatcggca accgcgaccc cgacgtctac gacggcgacc cgcacgtgct 78840ggacctgcgc
aggccggtga agcagcactt cgcgttcagc ttcggcaccc accagtgcct 78900gggccagtcg
ctggcccgca tggagctgca ggtcgtcgtg aacaccctct accgccgcgt 78960cccgaccttg
cgactggcga ccgcgctgga gcgcatcccg ttcaagcacg acgggatcgt 79020ctacggcgtc
tacgagctgc ccgtcacctg gtgaccccgt cccaccagac ctcctgccac 79080gcagacctcc
cgcaagccga ccccgaaagg ccgttcccat gagcgacacc acgctgtccg 79140tgcccgtccc
cgaggaggtc ggcaagctct acgaccagat cctgaaggac gagcacacct 79200acgagcagtt
cgagaagttc aaccaccagc tgcacatcgg ctactgggac gacccgacct 79260cggacgtgcc
catgcgcgag gccgtggtgc gcctgaccga gctgatggtc gagcgcctgc 79320gggtggacgc
cgaggaccgc gtgctggacc tgggctgcgg catcggcggc ccggcgaccc 79380agatcgtgcg
caccaccggc gcacgcgtcg tcggcgtgag catcagcgag gagcaggtca 79440agctcgccac
caggctggcc accgaggcgg gcgtgggcga ccgcgccacc ttccagcgcg 79500ccgacgccat
gcggctgccg ttcgaggacg agtccttcga cgcggtgatg gccctggagt 79560cgatcctgca
catgccgtcc agggagcagg tcctgtccga ggcgcgccgg gtcctgcgcc 79620ccggaggccg
cctggtcctc accgacttct tcgaacgcgc accccgcacg ccggggatgc 79680accccgcgat
cgagggcttc tgccgaaccg cgatgacgac gatggccgac gtggacgact 79740acgtgccgat
gctgcaccgg gtgggcctgc gcgtgcggga gctgctggac atcaccgagc 79800agaccatgga
acgcacttgg cgggagaccc tggagatcgt cagccagaac gaccgcccgg 79860tcgacttcga
cctggcggag ctgttcggcg tggacgagtt cggctgcctg ctggtcgccg 79920cagaccgccc
gtgaggcccg tccccgaggc cgtgggccgc ctgtacgacg acctgctgga 79980ggccgagctg
gaggggggcg cagccgaccc gaacctgcac atcggctact gggacgcgcc 80040ggactcgcca
acgccacgcg cggaggcggt agtgcgcttc accgacgaac acgtccgccg 80100cctgcacgtg
accacgggcg accgagtgct ggacgtgggc tgcggcgtag gcggcccagc 80160cctgcgcgcg
gtggacctga ccggcgccca cgtgaccgga atcagcatca gcgccgccca 80220gatcacccac
gcgacccacc tggccaagtc cgcgggccac gcggacaaca ccaagttcct 80280ccacgcagac
gcgatggccc tcccgttccc ggactcctcg ttcgacgcgg tcatggcgat 80340cgagtccctg
atccacatgc ccgaccgcga gcgggtcctg aacgaggcaa gacgcgtact 80400gcgcccaggc
gggcgactgg tcctcaccga actgttcgaa cgcgccccaa gacccacccg 80460cagacaccca
gcgataaccg agttctgccg agcatcgatg gtgtccctgc ccaacgcaga 80520cgactacccc
gcactactac accgagcagg cctacgccta cgggaactcc tggacatcac 80580cgaccacacc
gtccaacgca acttccgcga actggccgat ctggtaggcg acgcgaaggg 80640cctgctgttc
cacccacgcg acctggtggg cgtcccagaa ttcggctgct tcctagcagt 80700agccgaacac
ccgtaaccac gcggtggcgt cccccacgga cgccaccgcc tcgcgggctg 80760cggggcgagc
gcagcgagcc cgcgcagccc cactcccgcg tccctcttct ccgtgtggcc 80820tggcgcatgt
caaattccca ctgactgcca acagatcatg tgccgtttga gcaggtcagc 80880gacttgtcgc
gcttcggtgc cttaaggccg agctgggatg ggggcactgt ttccggactg 80940agcggggcag
cttggaaggt ggagttcggt gagcagaggc agcacgtccc gtcgcacgta 81000gaggtggttg
tacacgcggt ggcgggacct gcgcagtagg ccgctatccg caagctgctc 81060caagatcagg
agtgcggcgc ggtgcgtata gccgagttcg gcggtcagca tggtgctgtt 81120gagcagtggg
gcgacgagca gcggggcggg aagcgctttg accttcctcc gcccggtgcg 81180catcgcccag
gtgggcgatc gcgcgagcct cacggatcgc ggtcacctca tgcaggctgg 81240cgctcaacct
ggaacgcgcg actgtttcgt ccagacgtgc cagggcggtg taggcgtgca 81300acaaggtctt
gctggtttcg gagcgcagtc tgagccggga ccaggacgac aactccgcga 81360tcctcgcgga
cgggggcggc ctcgtgtctt caccggtggt agttgacctg cgcggggcgg 81420aggtgcccta
ttgctgccgg gacgaggtca tcccccggag cagtttctca gcacgccgtg 81480aatcgagatc
cggggcgctg agcgcggtga acgcctcgtc cagcgagtcg cacgcgcacg 81540tcgtcctgac
atcgggccgc gcatggcccg aggtggtcag cggtgagcgg gaaggcgcgg 81600cagggtgtgt
gcgagacact ccgggactcc gtgcagaagg tcgatcaggc gaaagggttg 81660aactgcgaat
cgcaaagcgg cccggccgca aaggggtcgg gccgcctgcg acgattggtc 81720acgctgctgc
ggcgcggtcc cgccggaact gcttgccgag caggtcgatc cgccccttgt 81780gatcttctgc
cagcgcctcc agaaccgaga gcagtcgtcg ggcgtgcagt gcatggccaa 81840taccatcgtc
gcgtacccca gagggtgtcg ctcccgttca ggggcgacca tttcccacgc 81900ccgcttggcc
tccttggcgg cccggccaag atcgccgagc atcaggtagg tgcccgacaa 81960cccgacaacc
ctgcctgcca acgcggcttc cggcaccccg cgcgcctcgt cggcttccaa 82020cgcccgaaca
ccgtgccaca gcacggcccg cgcgttgccc tcgctcgtct ccagccatcc 82080catgacaccg
tgcgcttcgg ccagtgacca cgatcggctg tcgggatcgg tgttgcacaa 82140cgccagctcc
agcgctcgtt cagcagcgtt accgaccaca gcgcggcgcc gatgtccagc 82200acttcttgcc
ggtacccgcc cacgagtgcg gcggtgcgct gcacggccac gacttcccgc 82260cgatgcacga
tcagccactt gtacgccgcc aaggcgttgt cgaacagcgg cttcgccccg 82320acctcgaagc
cgtcgacgaa cacctcgcgc aaatcaccga gcagtttccc tgcggccaag 82380gtgcgccgat
gcaggtacct gcccaagcgc tccaactctt gcgggaactc ctgctgcaca 82440gcccatcgca
gcagtgcctg ggcttggtct gtctcctgcg cgcgatggcg accggccagc 82500cggtaacgcg
aggaggtgaa ctcggggtgc gtgatgttcg ggtgaagttc agtccacgaa 82560ggctgcgtca
gcaccagcac gccctggttc acccagtccc gcgcgtgttg ggcggtctcc 82620tcgacggtga
ttcccagcat cgcggccaac gacgaggtcg agaccacggg gtccggcagc 82680aggtccagca
atctcagctc ttgcggaatg ggcacaagag tgttgatcat cgatgcccct 82740cccggaggac
ggcgatgatt ggagtggcga acagaagggg aaacgccagt tcgccgggtt 82800ccggcggtcc
acgcgccttc ggccggccac ttggactccg acgggcagaa gttcaccggc 82860aaggactctg
gtgacggtgg agcggtgcac gcccatcgct tcggccaagg cgtagtcgct 82920gtggtaacca
gcgaactgag ctagctttcg catcttgtcg ccgcgcaccc cgacgacctt 82980cttgatcttt
tcggtctcgc tgtcgttgtc gtcgacatgt ccgccgtccg gcgctgacac 83040cgttctcctt
gagatcgccg agctgaatgg gggatgcttc gacgtaaggc gttgcgtatg 83100cgcaacaggt
caggcggcgt cgagtctccc cattaccgag gtttcgcttg atcgccgacg 83160gggcccgcct
cgaagaagtc caatcgagct ggcatcccct tcgattgatc aatagcgcga 83220cgggtgtcgc
tcgacatcgc cccaccgcct gctcctgacg tgccacgagc agggaggagc 83280gacctccctc
gggactgcac cgaccgttcc tccctgtccg ccgattcagt tgcattccgc 83340cacgctaggt
gccggatgcg ggccgaaggg acaacgaagg gacaagtcga acagcccagg 83400tgcgaggtat
cttgaaatag cccgaatcct ccgtcgcgaa gcaggtcgcc atgcccactg 83460acgaacagtc
cgagggtgtc ggagagcgca tagccgtcca acgcaaactg gctggcttga 83520ctcagcaagc
tctggcgaag cgcgcacacg tcagcctcag cctcatcaaa ggggtggaac 83580agggaaggat
tcccgcctct cccgcgctcg tgtcccaggt ctcgcgggcg ctcaaggtcg 83640aggcgacgat
cttgctgggg cagccgtacc gccccgagga tcggagcagt cttcgcgttc 83700actccgtcat
ccccggtctg cgccgagcct tggcggccta ccggttgccc gctgatgagg 83760gcatcagccc
tcgcgggtac gacgagctgg ccgccggtgt agccgccgcg tcgaagatgc 83820gccacgccgc
gacgttggac gtcctggggg ctgaactccc cggcctgctc gacgagatcc 83880gctcggccat
cgacgaggct cggggagttg agcggcagcg cctgttcagc ttgctggcag 83940aggcatacgc
agccgctggt caagtcgcgt ggaagctggg ttacgcggac ctgtcctccc 84000tggcgacgga
gcgcgtggag tgggcggcca aagagtccgg cgatccgctc gcgatgggcg 84060cagcggactt
ctacatcgcc ggtgagctga tcgcagcagc ggagtggcgc ggcgccctct 84120cctacctcga
cggctcccgt cgccgcctgg agcacgtggt gcgcaaggac gacgaggccg 84180ccttgtcgat
ctacggagtc ctgcacctga agtcggggct cgcggcggca cgggccggga 84240aagccgacga
atccgacgcg cacctcgctg aagcccgtgg catcgcggaa agggtgccgc 84300tgggcagtga
ccactaccgg ctcgcgttcg accgggactc ggtcaacatc tggaccgtgg 84360ggctggcagt
ggagcgcatg gacggcacgg aagccgtcaa acgagcccac gggatgcgct 84420tcagcaagac
caccccgcgt gaacgcgtgg gccaccacta catcgatctg gcgcgcggct 84480accagctgca
cggagaccgt gaccgcgccc tgcacaccct tcagatcgcc aggcgaacct 84540caccgcagca
ggtgcgctac cacccgcagg tcagggaaac ccttctcacg ctcgcggaac 84600aggaccgcag
gcgctcggat tccctggcag ggctcgcgcg ctggatcggt atgccggtgt 84660gacaggacgg
cgagctgacg tcgctgttga ggggcagccc cccatcggcc gcccctcaac 84720agcaggtgcc
ggtacgtccc tcacagcgcg acgctgacga tcaggctggc gaacatggcc 84780acggccagcg
cgatcatctg cttgggcgcg ccgccgaaga agagggaccc cacccaccgc 84840agtggtaaac
cggcaccgag caccaacggc caccggccgg cagagcccgt ccccctcttt 84900tttggaggtc
tgccccgccg gcgaggcgtg cccttcagct ctcaagctct ccactctcga 84960tcttgtggtc
cgaacacccc ccgcaccccc actacgatcc ccccatgggg gctctgatca 85020tcgcggtagt
gctgctgctc gtcttcctcg tgcaactcaa gcgggaaccc agacgactgg 85080gcaacggcgt
ctacctgctg atgagcctgg cgttcttcgc cctctggctg ctcaccctcg 85140ccacccccca
gaccaggacg ctggtggtag gcgcggtagt cctgatcgcc ccggtattcg 85200tcaccgtgat
cgccctgttc ctcatcgcca acggcgtcac cctgctgcgc cgcgagggcg 85260tcaaaccagg
caacgccctc tccttcggcg caggcaccgc catcctgtgc gtcgtaggcg 85320gcctgctcct
ggtcctgctc tccgccctgc gcgaaggctc ccccgacccc tgggtgctgg 85380cagcagccgg
ttccctggtc ctcctggccg gctacctggg cttcgccttc accctcttcc 85440tgctctactc
cgtgctctac ggccgagtcc gcaagcgcac cggccacacc gcgatcatcg 85500tcctgggcgc
gggcgtcccc ggcggccgag tgaccccgct cctggcaggc cgcctggacc 85560gcgccctgaa
gctctaccgc cgcgccgcag ccaagggcgc ttcccccgtg gtagtcgcct 85620ctggcggcca
aggcccagac gaaccagcct ccgaagccga ggtcatggcc aactacctcc 85680gcgaacgcgg
catcccggac gaggccctcc tggaagagcg cgagtccacc tcgacctggg 85740agaacctccg
cctctcctcc gccctgctcg ccgaacgcgg cgtgaccggc agactcctgg 85800tcgtcaccag
cagctaccac gtcccccgag ccgcgatcct ctcccgccgc gcaggcctga 85860aggcagacgt
ccgcggcggc cgaaccgcct ggtacttcgt gccgaacgcc ttcctccgcg 85920agttcgccgc
cctcctggtc cagtaccgca ccctcaacgc cctggcagcc tgcaccgcac 85980tctccgtctt
cccgctcctg gcctacggcg tctgaaaagc acgacccggc cgaccggaca 86040ccgcgtcaca
gatccagcgg cgccgacccg aaggccacgt tgaaccggtc gcaccacacc 86100acgacgctgc
gcagccccga caggtccacg tcctccggaa tcaggtagtt ctggttgccg 86160tcggtggcct
tcatgggacc gagcggcagg tagcgcccat cgtcgtactt gccccactcc 86220ccacccgcgg
tcgcgtcgga gagccagatg tgcaggtcgg gcccgtccga ggtggagaac 86280ccatccagcc
gcagcacgcg cgcggccccg ctgcgcagca cggtggcggt gccccgcgtc 86340tcgtgctcct
gggtgacgaa cccacccgtt gccagcaccg tcggctggtc ggcggtcgcc 86400gacgacgtcc
caccgggcgt cgtggcaccg ctgcccgccg ccgccccggt gctcgacgcc 86460ccagccccgg
tgctcacccc cgcaccggtc gagacgctga actcggcggg cagcgcctcg 86520tccgcctcgc
tgcgcgtcca caaccgccac ggctggaaca cccacagccc gacgaccgca 86580gccaccacca
caacccccga caccgcccaa accgctctcc tgcgcaccga accgcgcacc 86640acgtcccctc
ccgttctccg cagacgacct gccaccatgc cacgggtcgc gcccgatgac 86700cacgaccacc
gcgccacacc cgccccacgc agcgactagg ctgcccaccg gggtcgccag 86760ccgatcccga
gcgggttgag caggcagccc accgcagttc gcgctagtgg gatggaggga 86820gcgggccggt
gtccgagctg gatgcagccg cggtggtcac ggtgggttcc gacgtggtgc 86880gcggggtgcc
cgtgctgcgc gtcgccgggg agatcgacac caacgtcgcc gacgaggtcc 86940gccgggcgct
gctgccctgg ctggacgggt tgcgcgggcc aggggtgctc gacctgaccg 87000gggtgaggtt
catggcctcc accgggttgt cgctgctgat cgaggccgcc cggcgcaggc 87060cggcgaagct
ggtgctggcc accgcccagc gcggcgtgct ccggccgctg cagctgaccg 87120ggatgagcgc
gctgctcccg acgcacccca ccgtggacct ggccgtggac gcccagctcg 87180gggccgccct
ggccgggatg cccagcacgg cctgaccacc ctcggtccac gggcggcctg 87240cccgcggacc
acccgcacgg cgccctgggg ggacgagatc acagctggtg gaagacgcga 87300tcctggtccg
cgcgccgcga cgccggtggg cgcgggcgct cccgccacgg cggcggaccc 87360gcccccggtc
cccaccacgg ctccggcacc ggccccgaca ccgacaccga ccccagcccc 87420gcccctgggc
acgaccacac caccaacccc ggtcctgggc gcaggtgtcg ccaccgccac 87480cgccctgacg
ctggcactcg ccggggccgc agccccagcc gacaacagcg cgggaaaggc 87540ggccatcatg
gacgaggtgg acgccccaac caccccaccc accccggccg cgctggacct 87600cacgccccgc
ccacccctgg ccgaggtgcg ccgctggacc ggcgcgctgc tgatcgacgc 87660cgacgaggaa
gcagcggacg acgtgctgct cgtggtcaac gagctggtcg ccaacgccta 87720cgaccacacc
acctccccac tcgccctgcg cctcaccacc acccccgagc acgtgcgcgt 87780ggaggtcgag
gacggctccc ccgacccacc acgcccggac ctcaccgcgg gcctgcgcca 87840gatcggcacg
cgcggacgcg gcctgctgct gatccgccag ctgaccgatc gctggggcag 87900cacgccccac
cccggcggca agaccgtgtg ggcggagctg ccgaacgtcc cggcgacctg 87960agcccgacgc
cccaccaacg aggccacggc ggatctcacg ggaagagcgc ggcggggcac 88020tccgggcgcg
ttggacgccg gcgcactccc cggtgagggg tcgggcggcg gagtggatga 88080gcgtggcggc
gagcagggcc ggtccggcgg acgagacggc catcagcagc ccgccgaccg 88140gggccgcgag
gcgtcgggcg cgggcgccga gcctgcccgg caccgggccg accacggagc 88200tgacgccgag
gacggcggtg caggcgccga gcgcgcgcct gcgggccgcg ccctcgaagc 88260gcacctggac
ggcggtcagc gccccggaga ccatgagcgc cgcgcccgcg ccctggacca 88320cccgcgcggc
caccagcgcc gggccggtgg gggtgaggcc gcaggccggg gaggcggcgg 88380tgaaggtggc
gggcccgacg aggtgggccc agcggcggcc ccggatctcg ccgaggtggg 88440ccccggtgat
cagcaggacg gcgagctgcg gcaggacacc gacacgggca cgaccgcgtc 88500gatgtgcgtc
gcggcggcgc agggcgcgga gacgggcgcg ggcgagcgct gagggcccgc 88560ccggcgccac
tcccccgtca cacctcccgc cgcagcacat cctcctccgt ctcccgccgc 88620accagcaccc
gcgccactcc gtcgcgcacg cccaccacag gcggcctgcc cacggcgttg 88680tagttcgacg
ccagcgcgtg gtggtaggcg cccgtcaccg gcaccgccag caggtccccc 88740gcgcgcacgt
ccgcgggcag cggcacgtcc tcggcgagca cgtcacccgc ctcgcagtgc 88800ctgcccacca
ccgtcaccgg cgcgcgccgc ccgccccggc cgaccaggcg caccgcgtac 88860cggctcccgt
acagcgcggg cctggggttg tcgctcatgc ccccgtccac ggccacgaac 88920acccgcctca
ccccgcgctt gacggcagcc acccggtaca gcgtcacacc agcgcccgcg 88980acgaccgacc
gccccggctc gatcagcagc ctcggcaccg gcacgcgccg cagcgcgcac 89040tcgtggctca
gcgccacccg cacccggtgc gcgaacccgc caaggtcgaa ctccccctcc 89100cccggcaggt
agggcaccgc gaacccgccg ccgaggtcca gctgctcgat ccgcaccccg 89160cacgaggcga
tcagcccgac catccgccgc gccgcctcct cgtacaccgc gacgtgtcgc 89220acctgcgacc
cgacgtggca gtgcagcccc accagcctca gcgacggctg ctcgaccacc 89280cgcagcaccg
cctccagcgc gtccccaccc gccagggaga agccgaactt ctggtcctcc 89340accccggtcg
ccaccgcccg gtgggtgcgc gggtcgacgc cgggggtgac ccggaccagc 89400acgtcctgcg
gccccctggc cagcgcgccc agctgctcga tctcgtcgaa cgagtccacc 89460accacccgcc
cgaccccgta cccgagggcg gccttgaggt cctcgggcgt cttgacgttg 89520ccgtgcagca
gaatccgctc cgccgggaac ccgaccgacc gcgcgatcgc cagctccccc 89580gccgagcaca
cgtccagcga cagcccctcg tccgccaccc accggtacac ctcgcggcac 89640ggcagcgcct
tgcccgcgaa caccacctca gcctccggca gcacctcccg gaacccgcgc 89700gcccgcgccc
ggaccgtgcc ctcgtcgagc acctggcagg gcgtgccgaa ccgggcggcg 89760agctcggtcg
cgggcacccc gccgagcagc agctcccccc gctccagccg ggtccccagg 89820ggccacagcc
ccgcctccag ggccggttcg ccggtcatgc cgacgctggg cagcaactcc 89880gcgagtgtca
tgcccgccag cacacgcccg aaccggccgg ggcgacagcg gcgcgaacgc 89940gtccctgacg
gcgtgccggg cgggattgac gccgccctga cccgaccgcc ccagcccgct 90000ctcgaacccg
gcggaagcac ccccgaaacg cgccggaaac ccgcccgcgc attcccccga 90060acgcctacct
cacggcgatt ttgatgcttt ttttacgccg ggacgccgcg atattcactc 90120ctccgagccg
cgcggggacg ttgacttctc atgcccgacg acgtgatcga ggagagaccc 90180cgaatgtccg
aaacaccggt tttcgccgtt ccacccaggg tggaaagccc ggtacgcccg 90240gccgcgcccg
ccaaccgggt ggggcgctgg ctgctggagc accgggtgca accggcggga 90300cccgcgggca
ccgaccagca cagcacgccc caggcgtggt ggaaggtcat gtgcctgacc 90360ggcgtcgact
acttctcgac cctgtcctac ctgccgggca tcgcggcgct ggcggccggg 90420gcggtctcgc
cgctggcgac gctgctgatc gtcgcgctga ccctgttcgg gatgctgccg 90480atgtaccgcc
gggtggcgca cgagtcgccg cacgggcagg gctcggtggc gatgctggag 90540gacctgctgc
cgttctggcg cggcaagctg ttcgtgctgg tgctgctggg tttcgtggcc 90600acctcgtgga
tcatcacgat caccctgtcg gcggccgacg cgtcggtgca cgcgctggag 90660aacccgcacg
cgcccgcgtt cctgcacggg cacgaggtgc tggtcaccgt ggtgctgctg 90720ctcgtgctgg
gcggggtgtt cctgctgggc ttcaccgagg cggtcagcgt ggccatcccg 90780ctggtcgcgg
tgttcctgct gctcaacgcg gtggtcgtgg tcgccggcgt gctggaggtg 90840atcgcgaacc
cggacgtgct ggacggctgg ttcgcggcgc tgacctccac cggcggcggc 90900ggggtgctgg
gcgtggtcgg cccggccctg ctggcgttcc cgctgctcgt gctcggcctg 90960tccgggttcg
agaccggggt gagcatgatg ccgctggtcg aggcgaaggg cgccgacgac 91020gccgaacgcc
tggcgaaccg cgtccgcaac acccgcaagc tgctcaccac cgccgcgctg 91080atcatgtcgg
tgtacctggt ggccaccagc ttcgtgacca ccctgctcgt gccggtcgag 91140cagttccgcc
ccggcggcga ggccaacggg cgggcgctgg cctacctggc gcacgagctg 91200ctcggcgagt
gggtcggcac ggcctacgac atcagcagcg tgctgatcct gtggttcgcc 91260ggcgcgtccg
cgatggccgg gctgatcaac atcgtgccgc gctacctgcc cgcgtacggc 91320atggccccgg
actggacgcg cgccgtccga ccggtcgtgc tggtctacac ggtgatctgc 91380gtcggcatca
cggtgatctt ccaggccgac gtggacgccc aggccggcgc gtacgcgacc 91440ggcatcctgg
cgatgatggt gtcggcgtcg gtggcggtga ccctgtcggt ggcgcgcgcc 91500gggcggcggg
gcgcggcctc ggcgttcgcg gtgctgaccc tgatcctggt gtacgcgctg 91560gtggagaacg
tgatcgagaa gccggacggc atcacgatct cgttcgtgtt catcgtcggc 91620atcatcgccg
tctcgctggt ctcgcggatc tcgcgcacca ccgagctgcg cgtggagcac 91680atcgagttcg
acgagaccgc gcgcaggctc atcaccgact cgatcgccca cgacggcgcg 91740ctgaccgtga
tcgcgaaccg caggcaggcc ggtgacgtgg ccgagtacgc ggacaaggag 91800gccgagcagc
gcggggtgaa cccggtgccg gggcaggcgg acgtgctgtt cctggagatc 91860gacgtggtgg
acccgtcgga cttcagcgac gtgctggagg tgcgcggcgt ggaggtgggc 91920ggccaccggg
tgctgcgcgc ggacagcccg gcggcgccga acgcgatcgc cgcgatactg 91980ctggcgctgc
gcgactgcac cggggtgcgc ccgcactgcc acttcgcgtg gagcgagggc 92040agcccgctgg
ggcacctgtt ccgctacctg ctggtggggc gcggcgacac ggcgccggtg 92100gtgcgggaga
tcatccgggc gcacgagtcc gacccggagc gcaggccggg catccacgtg 92160ggggcctgag
cgggcacgac ggcggggtgg tccaggcagg cagcgtggtc caggccagtg 92220gggtgctccc
ggccagcaac gtgctcccgg ccggtggggg ctccagggcg ctgcggcggc 92280cgatcgcgcg
ggcgtggtcg gcgaaccgct cgcagtgctc gctgagcagg gccgcgtcga 92340cggcggcgtc
ctcaacgccg cgcagcacgg ccagcacgga ccggggcact caccaaacgc 92400gaagagccac
accaactggg cttcggcgtg ggaggcgcgg tgcagcggtt tgtggtctcg 92460cgctgccgcg
cggcgcgggg gactgggtcg cgagcagcac ctggccgccg tgccgcgcgg 92520cgccccgcgc
caggtcgcac acggcggcca ggtccggcac cggcgcgtcc cgccggtcgt 92580ggaacacgtc
gcgcatcgcg ctctccctcg gaggatcgga tcggaaggcc ctgatcccaa 92640ccgggcgcgc
accccggcga caagccctca cccgccgaac ttgcgctttc cttccgcccc 92700gacccccgcc
cgtcacaaac ccccgtcacc ccgccgtcac tttttgtgat gacgatcagg 92760aaacagtagt
agcccattcg tgacctgcac tgacgcgcag atcaccccac ccgtcaacga 92820aacgtaaaac
cgcctggtca ccccgtcaaa gacccgtcag caccccgctc acggcgtttt 92880ccccgttgca
cccttttggc gtcgcggtcc ccacgaacgg gggccgctcg gagtcgggaa 92940gggagcacgc
tcatggccga cctggcctac gcgtcgctgc tcatcgctgt gttcggactg 93000ctcgtcctcg
gcattcgcgg actggggcgg ctctgatggg cggcacggga gtcgtggcca 93060acgccgtcgg
tggcgtgctg gccctgctgc tcatcgggta cctgttcgtc gcgctgatca 93120ggccggagaa
gttctgatgt cctcgaccac ggcgggcctg ctccaggtcg ccctgctcat 93180cgccgcgctg
gccgccgcct accggccgtt cggcgactac atggcccgcg tctacaccga 93240cgccaagcac
accaaggtcg agcgcctgct ctaccgcgca gcccgcgtcg accccgactc 93300gcagcagcgc
tggggcacct acgcgcaggg cgtgctcggc ttctccctcg tcggcgtggc 93360cctgctgtac
ctgatgcagc gagtgcagcc ctggctgccg ttcgaccacg accggggcgc 93420ggtctcgccc
ggcatggcgt tcaacaccgc cgcctcgttc gtggccaaca cgaactggca 93480gtcctacgtc
ccggagaccg tcctcggcca caccgtgcag atggccgggc tgaccgtgca 93540gaacttcgtc
tccggcgcgg tcggcatggc cgtcgccgtg gcgctggtgc gcggcttcac 93600ccgcgagggc
tccgaccggc tcggcaactt ctgggtcgac ctcaccaggg gcaccctgcg 93660cgtcctgctg
cccgtgtcgt tcgtgttcgc catcgtgctg gtcgcgaccg gcgtcgtgat 93720gagtctgaag
gcgggcgtgg acgtggacgg ccagcaggtc gccatcgccc cggccgcctc 93780gcaggaggcc
atcaaggagc tcggcaccaa cggcggcggc atcttcaacg ccaactccgc 93840ccacccgttc
gagaacccca acggctggtc gaacctggtc gagatcttcc tgatcctgct 93900gatcccggtc
tcgctcaccc gcaccttcgg caccctggtc ggcaaccgca agcagggcta 93960cgtgctgctc
agcgtcatgg gcgtgctgtg gaccgcgatg ctcgcggtca tctgggcggc 94020cgaggcgcac
ggcctgcgcc ccctggaggg caaggagctg cggttcggcg tccccggcag 94080cgccctgttc
gccaacacca ccaccgccac ctccaccggc gcggtcaacg ccatgcacga 94140cagcctcacc
ggcctgggcg gcggcgcgac gctgctgaac atgctgttcg gcgagatgac 94200gccgggcggc
gtcggcaccg gcctgtacag catcctggtg atggcgatca tcgcgatgtt 94260cctggccggt
ctgatggtcg ggcgcacccc ggagtacctg ggcaagaagc tgggccgccg 94320cgaggtgacc
tgcgccgcgc tgtccatcct ggcgatgccc gcgctggtgc tggtcggcgc 94380cgggatctcg
gcggtgctgc cgtcgacggc cgggtacctg aacaaccccg gcgagcacgg 94440cctgtccgag
atcctctacg cctacgcgtc ggcctcgaac aacaacggca gcgcgttcgc 94500gggcatcacc
gtgaccagcg actggttcca gtcctcgctc ggcgtctgca tgttgctcgg 94560ccggttcgtc
ccgatcatcg cggtgctgtg cctggccggt tcgctcgccc ggcagaagcg 94620cgccccgcgg
accgcgggca cgctgcccac ggacagcccg ctgttcgcct cgctgctggt 94680cggcgcgatc
gtgctcgtcg ccgccctcac cttcgtcccc gccctcgccc tcggccccat 94740cgcggaggca
ctgctgtgac caccaccgac acccgccagc ccgcccccga ggacacgggc 94800gcgcggcccc
cggccaagcc cgtcccgtcg ggcgtgttcg ccccgcgcca gctgctcacg 94860tccctgccgg
acgcgctgcg caagctccac ccccgccacc agctgcgcaa ccccgtgatg 94920ttcgtggtgt
gggcgggctc ggtcctggtc acggtcttcg ccgtcaccga cccgaacccg 94980ttcacgatcg
cggtcgcgct gtggctgtgg ttcaccgccc tgttcgccaa cctcgccgag 95040gccgtcgccg
aggggcgcgg caaggcgcag gccgagtcgc tgcgcaggac taagaccgac 95100gcgctggccc
gcctgaccga cggccgcacc gtgcccggca ccgagctgaa ggtcggcgac 95160ctggtcgtgg
tcgaggccgg tgaggtgatc cccggcgacg gcgacgtggt cgagggcatc 95220gccaccgtcg
acgagtcggc gatcaccggc gagtccgcgc ccgtggtgcg cgagtccggc 95280ggcgaccggt
gcgcggtcac cggcggcacc accgtgctgt cggaccggat cgtcgtgcgc 95340gtcaccagca
agccgggcga gacgttcgtg gaccggatga tcgcgctggt cgagggcgcg 95400cagcggcaga
agacgccgaa cgagatcgcg ctgacgatcc tgctgtccac gctcacgatc 95460atcttcctgc
tcgcggtgct cgcgctccag ccgttcgcgg tgtactccgg cggcgagcag 95520tcggtgatcg
tgctgaccgc gctgctggtg tgcctgatcc ccaccacgat cggcgcgctg 95580ctgtccgcga
tcggcatcgc gggcatggac cgcctggtgc agcgcaacgt gctggccacc 95640tcgggccgcg
ccgtcgaggc ggccggtgac gtggacacgc tgctgctgga caagaccggc 95700accatcacct
ggggcaaccg ccgcgccacc gagctgatcc ccgcgcccgg cgtcacgctg 95760gacgagctgg
tggacgccgc ccggttgtcg tcgctggccg acggcacccc cgagggccgc 95820agcgtggtcg
agctgtgcgc gaccgggcac ggccgctccc ccgagcccac cgacgcggag 95880aagaccggcg
agttcgtgcc gttcaccgcc cagacccgga tgagcggcat cgacctggac 95940ggccgcagcg
tccgcaaggg cgccgcgacc gcgttcaccc tcaccgactc ggtcaagtcc 96000acggtggacg
agatcagcgg cgacggcggc accccgctgg tggtcgccga cggcgagcgg 96060gtgctcggcg
tgatccggct gtccgacgtg gtcaagcccg gcatgaagga gcggttcgcc 96120gagctgcgcg
ccatgggcat ccgcacggtc atggtcaccg gcgacaaccc gctgaccgcc 96180agggcgatcg
cggccgaggc gggggtcgac gactacctcg ccgaggccaa gcccgaggac 96240aagatggccc
tgatccgcaa ggagcaggag ggcggcaagc tggtcgcgat gaccggcgac 96300ggcaccaacg
acgcgccggc gctggcccag tccgacgtgg gcgtggccat gaacaccggc 96360acctcggccg
ccaaggaggc cgggaacatg gtggacctgg actccgaccc caccaagctc 96420atcgagatcg
tggagatcgg caagcagctg ctgatcacgc ggggcgcgct gacgacgttc 96480tcggtcgcca
acgacctggc gaagtacttc gcgatcctgc ccgccatgtt cgccgcgatc 96540cacccgcagc
tggacaagct caacgtcatg ggcctggcca cgccgcagtc ggcgatcctg 96600tcggcggtca
tcttcaacgc gctgatcatc gtggtgctga tcccgctggc gctgcgcggc 96660gtgcgctaca
agccctccag cgcgagctcg ctgctgcggc gcaacctgct ggtgtacggc 96720gtcggcggca
tcatcacgcc gttcgtcggc atctggctca tcgacctgct cgtccgcctc 96780atccccggaa
tcgggtgaac tccgtgaacg cgttcgtgaa gcaggccctg gccggtctgc 96840gcgtcctgct
ggtgctgacc gtcatcaccg gcgtgctcta ccccgccgcc gtctggctcg 96900tctcgcgggt
gcccggcctg cacgccaacg ccgaggccac cggcaccgag ctggtcgtgg 96960cgccgcgcga
gggcgacggc tggttccagc cgcgcccgtc gatggcgacg ctgcccgcgt 97020cgggcgggtc
caacaagggc gagcgcaacg ccgactacga cgcggtgatc gccgagcgcc 97080gcaccgagat
cgcccggcgc gagggcgttg cggaggacgc cgtgccgcag gacgcggtga 97140ccgcctcggc
ctccgggctg gacccgctga tcagcgccga gtacgcggcg atccaggtgc 97200cgcgcgtggc
gcgggagcgc ggggtgtcgg aggacgccgt gcgggcgctg gtcgccgagg 97260cgtcggtggg
ccgctcgctc gggttcgtgg gcgagccggg cgtcaacgtc accgccctca 97320accgggccgt
cgacgcggcg gagtgagacc gaccgggggc cgtcctcgcg gcggcccccg 97380gtcttcccca
tttctctgat ctcgggagcg ggcgggaccg tggacaagcg caagcgcggc 97440gaactgcgca
tctacctggg cgcggcgccg ggcgtcggca agaccttcgc gatgctcggc 97500gaggcgcacc
gccgccgggg gcgcggcgcg gacgtcgtcg tcgccctggt cgagacgcac 97560ggccgcgagc
gcaccgccac catggtcgac ggcctggagg tgctgccccg caaggaggtc 97620cagcaccggg
ggaccacgat caccgagatg gacgtggacg cggtgctggc ccgcgcgccc 97680gagatcgccg
tggtggacga gctggcgcac accaacgccc ccggctcccg caacgccaag 97740cgctggcagg
acgtcgagga gctgctggac gccggcatcg acgtgctgtc cacgctcaac 97800atccagcacc
tggagtcgct caacgacgtg gtgcgccgca tcacccgcgt cgagcagcgc 97860gagaccatcc
ccgacgaggt ggtgcgccgc gccgagcagg tggagctggt cgacctgacc 97920ccggaggcgc
tgcgccgccg cctggcgcac ggcaacgtct acgccgcgca caagatcgac 97980gccgcgctgg
gcaactactt ccgggtcggg aacctgaccg cgctgcgcga gctggcgctg 98040ctgtgggtgg
ccgaccaggt ggacgtggcg ctccagcggt accgcaccga gcagcgcatc 98100accgacacct
gggaggcccg cgagcgggtc gtggtcgcgg tgaccggcgg cgcggagagc 98160gagaccctga
tccgcagggc ccgccgcatc gccgcgcgcg ccggggcgga gctgctggtg 98220gtgcacacca
tgcgcggcga cggcctcgcg ggttccgcgc cggagtcgat ccggacccgc 98280gtcgggctca
ggtgctcgac ggtgctcttc aacgtggtct cctcgtaacg ggacgtgcgg 98340aacaccccgc
agcgcccagg gtcgggcggc tgacgggatt cgcctgagtc taggcgaggc 98400cgcccccggc
cggggtggca ccccgcgacc gggtggttca cgtgcgggtg cgcgcgcccg 98460gcgcggcgcg
cgcggtgcga gaggtgggcc gtccggcggc gcgcggtttt ccgacatggc 98520gcgcgcacga
aatagttttc ggcgggtcgg gcgccgtcga atcgactcgg ggtcgggttt 98580tccgcgccac
cccggaagcg gacgaaccgg gcgggcgaac cgggcgggcg gtgcgcggac 98640aacgggcgcg
accgccgcgg tgcgccggtt tgggcagcct ttaccgccct ccaggtcacc 98700cattccgccg
ttgcggggaa catccgcgta ccagtggccc ccggcggaca cgcggcccag 98760cacccgctag
gccgttcgca ggacgtcgtg gtgcaccggg agcgtgaaac cgaacgtaac 98820cggacagcgg
cgggctcaag tggggtaaca ctggcgccgc agcgcactct tacccacagc 98880gacgaacgcg
gcggaacgct accctttaca ggtgaagtga ggccattcgg agcaccggtg 98940cgcagaaaac
tttcacgccc ggagatgact ccactcgccg tagtccatta gtgtgggatt 99000ccggtaccgt
tgcgccgcag gccgcaagaa ggcggccagg aaagacgatt aactcatccg 99060ggcgccccgc
cgtcgtgcac gtgaacgcga cgggcgaccg ggaacggaac gagcgagaca 99120tgtcatcgcg
ctctttacca cctaccagaa aaggtgccga tgaccccgat gaagaccatt 99180ccgccgattc
ccccgaacac gcgggcgtcc gcccgtccgc cgctcgggca accgcacgac 99240gggcttgcgc
gccacccgga accccagggc ccggccgcga ggcgatgttg acccgacccc 99300cggccaccag
ccgggacagg ccgaccaccg ccacgcgcgg aacccacggc gaaccgcctc 99360tcgccgtgat
ccaccacgga ccgttgggga gttccatgga gacccgtcaa cttctggcgt 99420tcaccacagt
ggtgcagacc ggcagcttca cgaaggccgc cgccacgctg aactgctctc 99480agcccacgat
caccaccagg atcaaggcgc tggaggagac cctcggcgtc gccctgttcc 99540gcaggttgcc
gcgcggcatc cagatgacct ccgccggggt cgagctgctg ccgttcgcgc 99600gcaacatcat
cacgctcacc gacaaggccc gcaaggcgat caccatgaac ggggagccgc 99660acgggcacct
cgtgataggc agcgcccaga gcctcaccga ctaccggctc ttacccctga 99720tcgagtacat
gtgctggcgc tacccgagcg tccagatctc gctgcactcg cgaacaaccc 99780ggtcgaacct
ggccgccgtg cgcgagggca ggttggactg cgcgttcttc atcggcccgg 99840tcgagcagcg
ggacggtctg gagacgacgg tgctgtgccc cgaaccgctg gtgatggtcg 99900cgggccccgg
ccacgcgctg gcgcggtcgg gcgcggtcac cgaggcggac ctgcggggca 99960gcacgctggt
cagggccgag aacggggcga gctaccacga gcagttcgag cgggcgctcg 100020ggctgcacga
ggccgagtcg cgatcgccgg tgctggccct ggactcggtc gacgcggcca 100080agcgggcggt
cgcctcgggg ctgggcatct cgctggtgcc ggaggtcacg gtcgccgcgg 100140agctggcgga
cggcaggctc agccgcatcg gctggacccc gccgttccgg gtgttcaccc 100200agttcgcgtg
gcgccaggac aactcggcga acccgtcggt gaccgcgctg gtctcggcgg 100260cggcgcaggt
ggtgagcgag caggtggccg cgacacccgc gtagggcgtc gacgtgcagg 100320gtcgtggatg
cggagcggcc ccctcgtgct gcgcagaggg ggccgagacc gtcggggcga 100380caggatttga
acctgcgacc ccccgctccc aaagcgggtg cgctaccaaa ctgcgccacg 100440ccccggtcac
caggagctta gcgcgacgcg ctaagctgtt ttcagcaccc acccggtggg 100500cgctgcgcgg
gtgtagctca atggtagagc cccagccttc caagctggtc atgcgggttc 100560gattcccgtc
acccgctcca ccagatcc
1005881827DNAArtificialPrimer 18ccscgggcgn ycngsttcga cngygag
271928DNAArtificialPrimer 19cgtcncggan
nccggagcac atgccctg
28201421DNAActinosynnema pretiosum 20atatactagt cacgtcaccg gcgcggtgtc
cgcggacttc gtcaacgacc acgcgttccg 60ggccgcctgg cgcggcgacg gggcgccggt
ggacccggtg gtcggcgacg tggaggacac 120cgccaggggg ttcctcgtca cccggtcggg
gatctcgatc ggggtgcgcg cggcctgggc 180ctcgcaccag gcgctggaca ccacggtcgt
gcgcgtggag ggcagcggcg gcacggcgga 240gctgcgctgc accttcggct tcagcccgaa
ccgcgagggc ccgtcgcggc tcctgctcac 300caccgacggc cgcaccaccc cggtcgagct
gcccgccgag ccggtgggcg ccgagtacga 360cgcccagctc gcgagcctgc ccacccgcct
ggccgacccc gccacgcgcg gcgaggcggc 420gtccggggcg cgctggatcg cgggcgcgat
cgaacgggtc taccgcgccg ccgacaccgt 480gcggtgcgcc gaacgacgac acgcgccggt
cgacctcgtc ccgacgggag ccccgtgatg 540accaccggac agaccaaccc ccgagtcgtg
cgggcccgag ccggggacga gcgggccgcg 600accgcggttc cggcgcccgc ggccccgcgc
cccctcgccc cgccgacccc cgcgccccac 660cggcacgtcc ccggcgcgac ctacgaccgg
gccgtgctgt tcgacctcga cggggtgctg 720gtcaacagct tcgccgtcat gcggcaggcg
ttcgagatcg cctacgccga ggtcgtcggc 780gacgggcccg cgccgttcga ggagtacaac
cggcacctgg ggcggtactt cccggacatc 840atgcggatca tggacctgcc gctggagatg
gagggtccgt tcgtccgcga gagctaccgg 900ctggcgggtg aggtggaggt gttcgagggc
gcgccggagc tgctggcgga cctgcggcag 960cacggcttcg gcaccgccgt ggtcaccggc
aagagcgggc cgcgcgcccg gtcgctgctg 1020accaccctcg gcatggcggg gctgttcgac
catatcatcg gctccgacga ggtcgcgaac 1080cccaagcccg cgccggacat gctcctgctg
gccaccggcc tgctcgacgt cccggccgac 1140cgggtggtga tggtgggcga cgccctgacg
gacctggcca gcgcccgcgc cgcgggctac 1200ccggcgctgg ccgcgctgtg gggcgagacg
gacgaggcgg agctgctggc ggcgaacccg 1260gacgcggtgg tccgcaagcc ctcgcaggtg
ctcgactggt gcctggccca cctcgccgac 1320gaccggacgt gacgcgcgag caggccccgg
cgcggcacgt gccggggcct gcggggcgtc 1380accccaaccg cgcgcgcagg tccgccacca
gcctaggata t 1421211423DNAActinosynnema pretiosum
21atatcctagg caccacgtcg tgctcgacct cgcccgccac gcgctcaccc cccgatcatg
60atcgactcgg tgacgcgggc gaactcgtcc ttcttcacga gcacccgcgt cgtctccacc
120ccgtccagca cggtgaagta gcggcggccg atcagcacgt gctgcccgtt ctccgccgcc
180cgaccccgga acgtggtgcc cgccagcacc gagtcccgcg cgtagtcgtc cagcccgtcc
240aaccgcaccg cctcccagcc gtcccggtcc gcccgccccg gccggaaccg gtacaccgcc
300tgccacgccc cgtccctggg cctgcgctcc accacgtgcg cgtccccgcg ctccacgacc
360cggtaggcgc agccgtactg ctcctgctcg accgccgaca gctccagcgg ctccaggtag
420gacgggccga cgaaccccac gtccaccagc caggtccgcc cgtccaggcg caccaggttg
480aacgagtgct cctcgtccgg cccgaaccgg tcgtcggcca gccggatcgc cgccgcgacc
540atcagcacct cgtagcccag cgcggtgagc agggcgtgga agagccggtt gagctcgtag
600cagaccccgc cgttgcggcc ggtcaccacg tgcgcgaaca cacggggcag cgggatctcc
660gcgagccccc ggttcggcgg caaccggtcg gccgcgccgc cgttgtcgta gggcacggac
720atcaggtgcc gcttgtgcag cgcccgcaac gactccagcg tcgggctcgg cacgccgccc
780tccacgccga tgcgccgcag gtactcggcc acgtcgatca tgctggtgct cacctctcgt
840cggtcgggtc ggtggactgc cctgcgcctg atcgccctgc gctcgactgc cctgcgcctg
900aatgccctgc gcccggctgc cctgcgcccg attgccctga gcccgattgc tcggcggcca
960gtcgctcgcg cagcgcctcg gcggcgatcg cgggcgtcgg ctggtcgaac agcagcgcgg
1020cgggcaggcg cagcccggtg gcggtgttca gccggttccg cagctgcacg gccgtcatgg
1080agtcgaagcc gatctcgaag aacgcgtcgt ccgggcccac cgcgcccttc tcgccgtgcc
1140cgagcacggc ggcggccagg tcgcggacca gctcggtgag cacggcctcc tgctcgtcgc
1200ccgacagccc ggccaggcgc gcccgcaacc cggccggggg cgcggcctgc ccggtggcgg
1260gggagagcga gcgccgcgcg gcggggacga gcgcgcgcag cagcgcgggc accgcctcgt
1320cgcgcgcgga ggcgcgcagc gccgccaggt ccagcggcag cggcacgacc agcgccgggc
1380cgcccgtggt gaccgcgccc gcgtcgaaca gcgtctagaa tat
14232247DNAArtificialPrimer 22atatactagt cacgtcaccg gcgcggtgtc cgcggacttc
gtcaacg 472342DNAArtificialPrimer 23atatcctagg
ctggtggcgg acctgcgcgc gcggttgggg tg
422442DNAArtificialPrimer 24atatcctagg caccacgtcg tgctcgacct cgcccgccac
gc 422542DNAArtificialPrimer 25atattctaga
cgctgttcga cgcgggcgcg gtcaccacgg gc
4226161DNAActinosynnema pretiosum 26ctcgccgacg accggacgtg acgcgcgagc
aggccccggc gcggcacgtg ccggggcctg 60cggggcgtca ccccaaccgc gcgcgcaggt
ccgccaccag cctaggcacc acgtcgtgct 120cgacctcgcc cgccacgcgc tcaccccccg
atcatgatcg a 16127161DNAActinosynnema pretiosum
27tcgatcatga tcggggggtg agcgcgtggc gggcgaggtc gagcacgacg tggtgcctag
60gctggtggcg gacctgcgcg cgcggttggg gtgacgcccc gcaggccccg gcacgtgccg
120cgccggggcc tgctcgcgcg tcacgtccgg tcgtcggcga g
1612822PRTActinosynnema pretiosum 28Val Ala Gly Glu Val Glu His Asp Val
Val Pro Arg Leu Val Ala Asp1 5 10
15Leu Arg Ala Arg Leu Gly202930DNAArtificialprimer 29ggtcactggc
cgaagcgcac ggtgtcatgg
303036DNAArtificialprimer 30cctaggcgac taccccgcac tactacaccg agcagg
36311595DNAActinosynnema pretiosum 31cctaggcgac
taccccgcac tactacaccg agcaggccta cgcctacggg aactcctgga 60catcaccgac
cacaccgtcc aacgcaactt ccgcgaactg gccgatctgg taggcgacgc 120gaagggcctg
ctgttccacc cacgcgacct ggtgggcgtc ccagaattcg gctgcttcct 180agcagtagcc
gaacacccgt aaccacgcgg tggcgtcccc cacggacgcc accgcctcgc 240gggctgcggg
gcgagcgcag cgagcccgcg cagccccact cccgcgtccc tcttctccgt 300gtggcctggc
gcatgtcaaa ttcccactga ctgccaacag atcatgtgcc gtttgagcag 360gtcagcgact
tgtcgcgctt cggtgcctta aggccgagct gggatggggg cactgtttcc 420ggactgagcg
gggcagcttg gaaggtggag ttcggtgagc agaggcagca cgtcccgtcg 480cacgtagagg
tggttgtaca cgcggtggcg ggacctgcgc agtaggccgc tatccgcaag 540ctgctccaag
atcaggagtg cggcgcggtg cgtatagccg agttcggcgg tcagcatggt 600gctgttgagc
agtggggcga cgagcagcgg ggcgggaagc gctttgacct tcctccgccc 660ggtgcgcatc
gcccaggtgg gcgatcgcgc gagcctcacg gatcgcggtc acctcatgca 720ggctggcgct
caacctggaa cgcgcgactg tttcgtccag acgtgccagg gcggtgtagg 780cgtgcaacaa
ggtcttgctg gtttcggagc gcagtctgag ccgggaccag gacgacaact 840ccgcgatcct
cgcggacggg ggcggcctcg tgtcttcacc ggtggtagtt gacctgcgcg 900gggcggaggt
gccctattgc tgccgggacg aggtcatccc ccggagcagt ttctcagcac 960gccgtgaatc
gagatccggg gcgctgagcg cggtgaacgc ctcgtccagc gagtcgcacg 1020cgcacgtcgt
cctgacatcg ggccgcgcat ggcccgaggt ggtcagcggt gagcgggaag 1080gcgcggcagg
gtgtgtgcga gacactccgg gactccgtgc agaaggtcga tcaggcgaaa 1140gggttgaact
gcgaatcgca aagcggcccg gccgcaaagg ggtcgggccg cctgcgacga 1200ttggtcacgc
tgctgcggcg cggtcccgcc ggaactgctt gccgagcagg tcgatccgcc 1260ccttgtgatc
ttctgccagc gcctccagaa ccgagagcag tcgtcgggcg tgcagtgcat 1320ggccaatacc
atcgtcgcgt accccagagg gtgtcgctcc cgttcagggg cgaccatttc 1380ccacgcccgc
ttggcctcct tggcggcccg gccaagatcg ccgagcatca ggtaggtgcc 1440cgacaacccg
acaaccctgc ctgccaacgc ggcttccggc accccgcgcg cctcgtcggc 1500ttccaacgcc
cgaacaccgt gccacagcac ggcccgcgcg ttgccctcgc tcgtctccag 1560ccatcccatg
acaccgtgcg cttcggccag tgacc
15953230DNAArtificialprimer 32cctaggaacg ggtaggcggg caggtcggtg
303331DNAArtificialprimer 33gtgtgcgggc
cagctcgccc agcacgccca c
31341541DNAActinosynnema pretiosum 34gtgtgcgggc cagctcgccc agcacgccca
cgagggtctc cagcgcgtcc gcgccggtgc 60gcgcgccccg gacgacctcg accgtgggga
tcaggtacgg cgggttcatg aagtgcgtgc 120cgatcagccg cgccgggtcg gggacgtgcc
cggccagctc gtcgatcggg atcgaggagg 180tgttggacac cagcggcacg cgcggcccgg
tgagcgcggc ggccccggcc agcacctcgg 240ccttgaccgg cagctcctcg gtgaccgcct
ccaccaccag cgagacgtcc gcgacgtcgg 300cgagcgaggt ggtggtgagc agctcgcccc
gctcgcggtc ctcgggcagc gcccgcatca 360gcctggccat gcgcagctgg gcggccaccg
cctcccgcgc ccgcccgacc ttggcccggt 420cggtctcgac cagcaccacc ggcacgccgt
gcccgacggc cagggaggtg atccccaggc 480ccatcgtgcc cgcgccgaga acggcgagca
ccgtcctgcc gtcctgctct cccatcgcgc 540tcccccgccg cggccaccgc ggccgccgtc
cggtccgcgc gccgtcccgg cacgcgcatt 600ccaccctcga tcgtgtgccg ggaaaggcgc
gcccgacccc ctgacctgcc cccctgaacc 660cccctcaacg gaaccggaaa tcgaatgtcc
cgaacgcgcc gtcaaatcgt cgattgacag 720ccgcagaact gttcatagac tgtggcggca
gtaccgatct ccgaattcca cggaagagtc 780ctcccccatg gctcagcaga tcagcgccac
ctcggaaatc ctcgactacg tccgcgcgac 840ctcgttgcgc gacgacgacg tgctcgccgg
tctgcgggag cggaccgcgg ttctcccggc 900cgcgtccgcg ctgcaggtgg ccccggagga
ggggcagctg ctcggcctgc tggtgcgcct 960ggtcggcgcg cgctcggtgc tggaggtcgg
cacctacacc gggtacagca cgctgtgcat 1020ggcccgcgcc ctcccgcccg gcggacgtgt
cgtgacctgc gacgtcgtcg cgaagtggcc 1080ggacatgggc aggccgttct gggagcgggc
gggcgtcgcg gaccgcatcg acgtccgcgt 1140cggcgacgcc cgcgccaccc tggccggcct
gcacgccgag cacgccgtgt tcgacctggt 1200gttcatcgac gcgaacaagt cggattacgt
ccactactac gagcgcgcgc tgacgctgct 1260gcgcaccggc ggcctggtcg tcgtggacaa
cacgctcttt ttcgggcggg tcgccgatcc 1320gtccgcgacc gatccggaca ccaccgccgt
gcgcgagctg aacgcgctgc tgcacgccga 1380cgagcgggtc gacatgtgcc tgctgccgat
cgcggacgga atcacgctcg ccgtgaagcg 1440gtgaacccgc ccgaatcgcg ccgaattccc
ccggagagaa aggccgccgc agtgttcacc 1500gaggacgtgg ccaccgacct gcccgcctac
ccgttcctag g 1541
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