FIELD OF THE INVENTION 10002] The invention relates to materials and methods for biosynthesis of fostriecin, fostriecin derivatives and analogs, and other useful polyketides. The invention finds application in the fields of molecular biology, chemistry, recombinant DNA technology, human and veterinary medicine, and agriculture.

BACKGROUND OF THE INVENTION [0003] Polyketides are complex natural products that are produced by microorganisms such as fungi and mycelial bacteria. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including: adriamycin, epothilone, erythromycin, mevacor, rapamycin, tacrolimus, tetracycline, rapamycin, and many others. However, polyketides are made in very small amounts in microorganisms and are difficult to make or modify chemically. For this and other reasons, biosynthetic methods are preferred for production of therapeutically active polyketides. See PCT publication Nos. WO 93/13663; WO 95/08548 ; WO 96/40968 ; WO 97/02358; and WO 98/27203 ; U. S. Pat. Nos.

4,874, 748; 5,063, 155; 5,098, 837; 5,149, 639; 5,672, 491; 5,712, 146 and 6,410, 301 ; Fu et al., 1994, Bioclienzistry 33: 9321-26; McDaniel et al. , 1993, Science 262: 1546-1550; Kao et al. , 1994, Science, 265: 509-12, and Rohr, 1995, Angew. Chem. Int. Ed. Engt. 34: 881-88, each of which is incorporated herein by reference.

[0004] Biosynthesis of polyketides may be accomplished by heterologous expression of Type I or modular polyketide synthase enzymes (PKSs). Type I PKSs are large multifunctional protein complexes, the protein components of which are encoded by multiple open reading frames (ORF) of PKS gene clusters. Each ORF of a Type I PKS gene cluster can encode one, two, or more modules of ketosynthase activity. Each module activates and incorporates a two- carbon (ketide) unit into the polyketide backbone. Each module also contains multiple ketide- modifying enzymatic activities, or domains. The number and order of modules, and the types of ketide-modifying domains within each module, determine the structure of the resulting product.

Polyketide synthesis may also involve the activity of nonribosomal peptide synthetases (NRPSs) to catalyze incorporation of an amino acid-derived building block into the polyketide, as well as post-synthesis modification, or tailoring enzymes. The modification enzymes modify the polyketide by oxidation or reduction, addition of carbohydrate groups or methyl groups, or other modifications.

[0005] In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker regions. These regions collectively can be considered to define boundries of the various domains. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKSs by various available methodologies. Using this method, new polyketide synthases (which produce novel polyketides) can be produced.

[0006] It will be recognized from the foregoing that genetic manipulation of PKS genes and heterologous expression of PKSs can be used for the efficient production of known polyketides, and for production of novel polyketides structurally related to, but distinct from, known polyketides (see references above, and Hutchinson, 1998, Curr. Spitz. Microbiol. 1: 319-29; Carreras and Santi, 1998, Curr. Opirz. Biotech. 9: 403-11; and U. S. Pat. Nos. 5,712, 146 and 5,672, 491, each of which is incorporated herein by reference).

[0007] One valuable class of polyketides includes fosteriecin and its analogs. Fostriecin (CI- 920) is a structurally novel phosphate ester produced by Streptomyces pulveraceus having potent antitumor activity. Fostriecin's antitumor activity is believed to result from selective inhibition of protein phosphatase 2A (PP2A) and protein phosphatase 4 (PP4). Both synthetic and naturally produced analogs of fostriecin with similar activities have been described. See, e. g., Lewy et al. , 2002, "Fostriecin : Chemistry and Biology"Current Medicinal Chemistry 9: 2005- 2032, and references cited therein, for additional information regarding fostriecin and its analogs.

The chemical structure of fostriecin and congeners PD 113,270 and PD 113, 271, as reported by Lewy et al., 2002, is shown below: lid x. g ! « S i4 y J J a : d,.. . i : v ; : 1Q yI I. 2n m *a, Pa ot =OR l. t : i ! n "T''% . s. '...-8' m 1. Fostriecin 2. PD 113, 270 3. PD 113, 271 [0008] Phase I clinical trials of fostriecin were halted due to the unpredictable chemical purity and storage instability of the compound. Accordingly, there is a need for methods for producing fostriecin and both known and novel analogs with sufficient purity and, preferably, with superior storage stability. Fostriecin is synthesized by a modular PKS and modification enzymes.

[0009] There is a need for recombinant nucleic acids, host cells, and methods of using those host cells to produce polyketides including but not limited to fostriecin and fostriecin analogs.

These and other needs are met by the materials and methods provided by the present invention.

SUMMARY OF THE INVENTION [0010] The present invention provides recombinant nucleic acids encoding polyketide synthases and polyketide modification enzymes. The recombinant nucleic acids of the invention are useful in the production of polyketides, including but not limited to fostriecin and fostriecin analogs and derivatives, in recombinant host cells.

[0011] In nature, the biosynthesis of fostriecin is performed by a modular PKS, the fostriecin polyketide synthase, and polyketide modification enzymes. Nucleic acids encoding the PKS, modification enzymes, and other polypeptides, have been cloned and characterized. The present invention provides polypeptide, modules, and domains of the fostriecin polyketide synthase, and corresponding nucleic acid sequences encoding them and/or parts thereof. Such compounds are useful, for example, in the production of hybrid PKS enzymes and the recombinant genes that encode them. The present invention also provides post-synthesis modification enzymes, and other proteins involved in fostriecin biosynthesis, and corresponding nucleic acid sequences encoding them and/or parts thereof.

100121 The present invention provides these nucleic acid sequences in isolated, synthetic or recombinant form, including but not limited to isolated form sequences incorporated into a vector or the chromosomal DNA of a host cell.

[0013] The present invention also provides recombinant host cells that contain the nucleic acids of the invention. In one embodiment, the host cell provided by the invention is a Streptomyces host cell that produces a fostriecin modification enzyme and/or a domain, module, or protein ofthe fostriecin PKS. Methods for the genetic manipulation of Strepto7nyces are described in Kieser et al,"Practical Streptomyces Genetics, "The John Innes Foundation, Norwich (2000), which is incorporated herein by reference in its entirety.

[0014] Accordingly, there is provided a recombinant PKS wherein at least 10,15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of fostriecin polyketide synthase. In an embodiment at least an entire domain of a module of fostriecin polyketide synthase is included. Representative fostriecin PKS domains useful in this aspect of the invention include, for example, KR, DH, ER, AT, ACP and KS domains. In one embodiment of the invention, the PKS is assembled from polypeptides encoded by DNA molecules that comprise coding sequences for PKS domains, wherein at least one encoded domain corresponds to a domain of fostriecin PKS. In such DNA molecules, the coding sequences are operably linked to control sequences so that expression therefrom in host cells is effective. In this manner, fostriecin PKS coding sequences or modules and/or domains can be made to encode PKS to biosynthesize compounds having antibiotic or other useful bioactivity other than fostriecin.

[00151 In one aspect, the invention provides a recombinant DNA molecule comprising a sequence encoding at least one domain, and optionally one or more modules, of fostriecin polyketide synthase polypeptide. In an embodiment, the recombinant DNA molecule includes a sequence encoding an open reading frame encoding a polypeptide encoded by fosA, fosB, fosC, fosD, fosE or fosF or encoding a conservative variant of such a polypeptide. In an embodiment, the recombinant DNA molecule encodes a modified fostriecin polyketide synthase polypeptide in which at least one fostriecin PKS domain is inactivated.

[0016} In one aspect, the invention provides a recombinant DNA molecule that encodes a chimeric polyketide synthase (PKS) module composed of at least a portion of fostriecin PKS and at least a portion of a second PKS for a polyketide other than fostriecin.

[0017] DNA molecules of the invention may be integrated into a host cell chromosome, or into a recombinant vector such as an expression vector in which the DNA molecule is operably linked to a promoter.

[0018) In one aspect, the invention provides a host cell comprising a recombinant DNA molecule as described above.

[0019] In one aspect, the invention provides a recombinant Streptomyces pulveraceus cell in which at least one domain-encoding region of an endogenous fostriecin polyketide synthase gene is deleted or otherwise inactivated. In an embodiment, the domain has been replaced by a different PKS domain. Also provided is a recombinant Streptomyces pulveraceus cell in which at least polypeptide-encoding ORF of the fostriecin polyketide synthase gene cluster is deleted or otherwise inactivated.

[0020] In one aspect, the invention provides an isolated, synthetic or recombinant DNA molecule having a sequence encoded by the insert of pKOS279-117. 1F70 ; pKOS279-117. 3F45; pKOS279-117. 2F15, or pKos279-117. 5F58. The DNA molecule may contain sequence encoding a complete fostriecin PKS module or domain.

[0021] The invention provides a method of producing a polyketide by culturing a cell under conditions under which the cell produces the polyketide, where the cell contains a recombinant polynucleotide synthase that contains at least one domain from the Streptomyces pulveraceus fostriecin polyketide synthase, and where the cell does not make the polyketide in the absence of the recombinant polynucleotide. In one embodiment, the domain is encoded by a subsequence of SEQ ID NO : 1 or a sequence that hybridizes under stringent conditions to a subsequence of SEQ ID NO : 1. In one embodiment the cell is not Streptomyces pulveraceus. In an embodiment, the polyketide is fostriecin, PD 113,270 or PD 113, 271.

[00221 The invention provides a method of producing a polyketide by recombinantly modifying a gene in the fostriecin PKS gene cluster of a cell comprising the gene cluster to produce a recombinant cell, or obtaining a progeny of the recombinant cell and growing the cell, or progeny, under conditions whereby a polyketide other than fostriecin is synthesized by the cell. Non-limiting examples of such modifications include (a) substitution of a fostriecin AT domain with an AT domain having a different specificity ; (b) inactivation of a domain of a fostriecin polyketide synthase module, where the domain is selected from the group consisting of a KS domain, an AT domain, an ACP domain, a KR domain, a DH domain, and an ER domain ; or, (c) substitution of KS domain, an ACP domain, a KR domain, a DH domain, or an ER domain with a domain having a different specificity.

[0023] The aforementioned methods can also include the step of recovering the synthesized polyketide. The recovered polyketide may be chemically modified and/or formulated for administration to a mammal.

[0024] These and other aspects of the present invention are described in more detail in the Detailed Description of the Invention, below.

BRIEF DESCRIPTION OF THE DRAWINGS [0025] Figure 1A, IB and 1C show the organization of the fostriecin PKS biosynthetic gene cluster.

[0026] Figure 2 shows hypothetical roles for the nine modules of the fostriecin polyketide synthase complex (modules 0-8) ) by showing hypothetical PKS-bound intermediates, the product released from the PKS (in brackets) and the result of post-PKS modification enzymes (symbolized by three arrows).

[0027] Figure 3 shows the approximate relationship of cosmids from"overlap family I," encoding the fostriecin PKS gene cluster as estimated during cloning.

DETAILED DESCRIPTION OF THE INVENTION [0028] The present invention provides recombinant materials for the production of polyketides including, but not limited to, fostriecin and its derivatives and analogs. In an aspect, the invention provides recombinant nucleic acids encoding at least one domain of a fostriecin polyketide synthase. In another aspect, the present invention provides recombinant nucleic acids encoding an enzyme involved in fostriecin biosynthesis or post synthesis modification. Methods and host cells for using these nucleic acid sequences to produce or modify a polyketide in recombinant host cells are also provided. Given the valuable properties of fostriecin and its derivatives and analogs, means to produce useful quantities of these molecules in a highly pure form is of great value. The nucleotide sequences of the fostriecin biosynthetic gene cluster encoding domains, modules and polypeptides of fostriecin polyketide synthase, and modifying enzymes, and other polypeptides can be used, for example, to make both known and novel polyketides. Further, the fostriecin modifying enzymes can be used to modify other polyketides and produce derivatives with enhanced solubility and/or bioactivity. The compounds produced using methods of the invention may be used, without limitation, as antitumor agents or for other therapeutic or research uses, as intermediates for further enzymatic or chemical modification, as agents for in vitro inhibition of protein phosphatase and/or for other therapeutic, industrial and agricultural purposes.

[0029] The polynucleotides encoding fostriecin PKS domains, modules and polypeptides, and encoding fostriecin modifying proteins of the present invention were isolated from Streptomycespulveraceus as described in Example 1. Tables 1-4, and Figure 1 describe the genes or open reading frames of the fostriecin polyketide synthase gene cluster and the encoded polypeptides, modules and domains. These tables and figure also describe the characteristics of non-coding sequences and sequences encoding other genes of the fostriecin gene cluster, including genes encoding regulatory proteins, transport proteins, and others.

[0030] It will be understood that each reference herein to a nucleic acid sequence is also intended to refer to and include the complementary sequence, unless otherwise stated or apparent from context. Provided with the nucleic acid sequences disclosed herein, it will be trivial for the reader to immediately determine the sequence of a complementary stand based on base-pairing rules (e. g. , A: T, A: U, C: G). Similarly, provided with the nucleic acid sequences disclosed herein one of skill can easily, by reference to the genetic code, identify open reading frames and the amino acid sequences of encoded polypeptides. l0031] Table 1, below, describes the positions of fostriecin polyketide synthase polypeptides, modules and domains with reference to the DNA sequence set forth in Table 3 (SEQ ID NO : I)..

"Complement"indicates that the polypeptide sequence is encoded by the complement of SEQ ID NO: 1. Abbreviations used in the table, and elsewhere in the specification, include: ketosynthase ("KS") domain or activity; acyltransferase ("AT") domain or activity; acyl carrier protein ("ACP") domain or activity ; ketoreductase ("KR") domain or activity, a dehydratase ("DH") domain or activity; enoylreductase ("ER") domain or activity; thioesterase ("TE").

TABLE 1<BR> Fostriecin polyketide synthase ORFs, Modules and Domains Position in SEQ ID NO:1 ORF # aa coding strand(nucleotide pair) fosC 3542 Modules 3-4 complement(56750..67378) KS3 complement(65783..67063) AT3 complement(64382..65431) DH3 complement(63770..64357) KR3 complement(62039..62839) ACP3 complement(61757..62014) KS4 complement(60401..61678) AT4 complement(59054..60067) KR4 complement(57368..58105) ACP4 complement(57014..57271) fosD 1738 Module 5 complement(51497..56713) KS5 complement(55328..56605) AT5 complement(53942..54994) KR5 complement(52241..52918) ACP5 complement(51809..52066) fosE 3537 Modules 6-7; complement(40820..51433) KD6 complement(50024..51334) AT6 complement(48608..49648) DH6 complement(47993..48574) KR6 complement(46151..47017) ACP6 complement(45854..46114) KS7 complement(44474..45754) AT7 complement(43058..44098) KR7 complement(41429..42229) ACP7 complement(41093..41350) fosF 1932 Module 8 and TE; complement(34979..40774) KS8 complement(39428..40672) AT8 complement(38003..39079) KR8 complement(36212..37009) ACP8 complement(35912..36169) TE complement(34979..35911) fosA 3414 Modules 0-1 complement(17358..27602) KS0q complement(26019..27278) AT0 complement(24722..25640) ACP0a complement(24414..24671) ACP0b complement(24039..24296) KS1 complement(22701..23990) AT1 complement(21336..22394) DH1 complement(20715..21302) ER1 complement(18813..19685) KR1 complement(17952..18797) ACP1 complement(17631..17891) fosB 1880 Module 2 complement(11623..17265) KS2 complement(15883..17163) AT2 complement(14455..15576) DH2 complement(13855..14424) KR2 complement(12247..13026) ACP2 complement(11920..12177) [0032] In one aspect of the invention, purified and isolated DNA molecules are provided that comprise coding sequences for one or more domains or modules of a Streptomyces pulveraceus fostriecin polyketide synthase. Examples of such encoded domains include fostriecin polyketide synthase KR, DH, ER, AT, ACP, and KS domains. In one aspect, the invention provides DNA molecules which sequences encoding one or more polypeptides of fostriecin polyketide synthase are operably linked to expression control sequences that are effective in suitable host cells to produce fostriecin, its analogs or derivatives, or novel polyketides. In one aspect, the complete set of synthase-encoding genes is provided.

100331 In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one domain, alternatively at least one module, alternatively at least one polypeptide, involved in the biosynthesis of a fostriecin.

[0034] In one aspect, the invention provides an isolated or recombinant DNA molecule encoding a polypeptide or portion thereof, including a PKS module or domain, encoded in the Streptomycespulveraceus fostriecin polyketide synthase gene cluster sequence.

[0035] In one aspect, the invention provides an isolated or recombinant DNA molecule encoding a complete polypeptide, module or domain comprising an amino acid sequence encoded in SEQ ID NOS: 1,23, 27 or 33, or a conservatively modified variant thereof. In one aspect, the invention provides an isolated or recombinant DNA molecule encoding a subsequence from a polypeptide, module or domain comprising an amino acid sequence encoded in SEQ ID NOS: 1, 23,27 or 33, or a conservatively modified variant thereof. The subsequence may comprise a sequence encoding a catalytically active fragment (having an activity characteristic of the domain, e. g. , AT, KR, KS, DH, ER, ACP, TE activity) of a PKS module or domain. The DNA molecule may comprise a sequence encoding a polypeptide involved in post- synthesis modification of the fostriecin precursor or encoding another polypeptide of the fostriecin gene cluster. lQ036] In one aspect, the invention provides the present invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes an open reading frame, module or domain having an amino acid sequence identical or substantially similar to an ORF, module or domain encoded by an ORF of the fostriecin polyketide synthase cluster sequence. A polypeptide, module or domain having a sequence substantially similar to a reference sequence may have substantially the same activity as the reference protein, module or domain (e. g. , when integrated into an appropriate PKS framework using methods known in the art).

[0037] In an embodiment, the invention provides a nucleotide sequence that encodes a polypeptide, such as a conservatively modified variant of a polypeptide, module or domain involved in the biosynthesis of a fostriecin, and comprises at least 10,20, 25,30, 35,40, 45, or 50 contiguous base pairs identical to a sequence of SEQ ID NOS: 1,23, 27 or 33. In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain that comprises at least 10,15, 20,30, or 40 contiguous residues of a corresponding polypeptide, module or domain comprising a sequence of SEQ ID NOS: 1, 23,27 or 33.

[0038] It will be understood that, due to the degeneracy of the genetic code, a large number of DNA sequences encode the amino acid sequences of the domains, modules, and proteins of the fostriecin PKS, the enzymes involved in fostriecin modification and other polypeptides encoded by the genes of the fostriecin biosynthetic gene cluster. The present invention contemplates all such DNAs. For example, it may be advantageous to optimize sequence to account for the codon preference of a host organism. The invention also contemplates naturally occurring genes encoding the fostriecin PKS and modifying (or"tailoring") enzymes that are polymorphic or other variants.

[0039] As used herein, a conservatively modified variant of a protein or fragment (e. g., domain) has substantial sequence identity to a reference amino acid sequence or is encoded by a DNA substantial sequence identity to a reference nucleic acid sequence.

[0040] The terms"substantial identity,""substantial sequence identity,"or"substantial similarity"in the context of nucleic acids, refers to a measure of sequence similarity between two polynucleotides. Substantial sequence identity can be determined by hybridization under stringent conditions, by direct comparison, or other means. For example, two polynucleotides can be identified as having substantial sequence identity if they are capable of specifically hybridizing to each other under stringent hybridization conditions. Other degrees of sequence identity (e. g. , less than"substantial") can be characterized by hybridization under different conditions of stringency."Stringent hybridization conditions"refers to conditions in a range from about 5°C to about 20°C or 25°C below the melting temperature (Tm) of the target sequence and a probe with exact or nearly exact complementarity to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e. g. , Berger and Kimmel, 1987, Methods In Enzymology, Vol. 152: Guide To Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed. , Vols. 1-3, Cold Spring Harbor Laboratory). Typically, stringent hybridization conditions for probes greater than 50 nucleotides are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8. 3, and temperatures at least about 50°C, preferably at least about 60°C. As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.

Exemplary conditions include hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04 pH 7.0, 1 mM EDTA at 50° C. ; wash with 2xSSC, 1% SDS, at 50° C.

[0041] Alternatively, substantial sequence identity can be described as a percentage identity between two nucleotide or amino acid sequences. Two nucleic acid sequences are considered substantially identical when they are at least about 70% identical, or at least about 80% identical, or at least about 90% identical, or at least about 95% or 98% identical. Two amino acid sequences are considered substantially identical when they are at least about 60%, sequence identical, more often at least about 70%, at least about 80%, or have at least about 90% sequence identity. Percentage sequence (nucleotide or amino acid) identity is typically calculated using art known means to determine the optimal alignment between two sequences and comparing the two sequences. Optimal alignment of sequences may be conducted using the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2 : 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U. S. A. 85 : 2444, by the BLAST algorithm of Altschul (1990), I. lllol. Biol. 215: 403-410 ; and Shpaer (1996) Genetics 38: 179-191, orby theNeedleham et al. (1970) J. Mol bol 48: 443-453; and Sankoff et al. , 1983, Time Warps, St) ingEdits, and Macromolecules, The Sheory and Practice of Sequence Comparison, Chapter One, Addison-Wesley, Reading, MA; generally by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI; BLAST from the National Center for Biotechnology Information (http://www. ncbi. nlm. nih. gov/). In each case default parameters are used (for example the BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad.

Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands).

[0042] As discussed in Example 1, the gene cluster sequences disclosed herein were determined from the inserts of cosmids pKOS279-117. 1F70, pKOS279-117. 3F45, pKOS279- 117. 2FI5, and pKos279-117. 5F58. Accordingly, the invention provides an isolated or recombinant DNA molecule comprising a sequence from the insert of one or more of these cosmids. In an embodiment, the isolated or recombinant DNA molecule encodes a polypeptide or portion thereof, such as a module or domain. In an embodiment, the isolated or recombinant DNA molecule comprises at least 10,20, 30,40, 50 or 100 basepairs having a sequence of the cosmid insert.

[0043] The invention methods may be directed to the preparation of an individual polyketide.

The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. The resulting polyketides may be further modified to convert them to other useful compounds. Examples of chemical structures of that can be made using the materials and methods of the present invention include PD 113,270 and PD 113, 271 b other known analogs, such as those described in Lewy et al. , 2002,, "Fostriecin : Chemistry and Biology"Current Medicinal Chemistry 9: 2005-2032 and the references cited therein, and novel molecules produced by modified or chimeric PKSs comprising a portion of the fosteriecin PKS sequence, molecules produced by the action of polyketide modifying enzymes from the fosteriecin PKS cluster on products of other PKSs, molecules produced by the action on products of the fosteriecin PKS of polyketide modifying enzymes from other PKSs, and the like.

[0044] As noted, in one aspect the invention provides recombinant PKS wherein at least 10, 15,20, 30, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of fostriecin polyketide synthase.

[0045] In one aspect, the invention provides a recombinant polyketide synthase derived from a naturally occurring PKS. A PKS"derived from"a naturally occurring PKS contains the scaffolding encoded by all the portion employed of the naturally occurring synthase gene, contains at least two modules that are functional, and contains mutations, deletions, or replacements of one or more of the activities of these functional modules so that the nature of the resulting polyketide is altered. This definition applies both at the protein and genetic levels.

Particular embodiments include those wherein a KS, AT, KR, DH, or ER has been inactivated (e. g. , by deletion or other mutation), mutated to change its activity, and/or replaced by a version of the activity from a different PKS or from another location within the same PKS.

Embodiments include derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been inactivated (e. g. , by deletion or other mutation) wherein any of these activities has been added or mutated so as to change the ultimate polyketide synthesized. There are at least five degrees of freedom for constructing a polyketide synthase in terms of the polyketide that will be produced. See, U. S. Pat. No. 6,509, 455 for a discussion.

[00461 As can be appreciated by those skilled in the art, polyketide biosynthesis can be manipulated to make a product other than the product of a naturally occurring PKS biosynthetic cluster. For example, AT domains can be altered or replaced to change specificity. The variable domains within a module can be deleted and or inactivated or replaced with other variable domains found in other modules of the same PKS or from another PKS. See e. g. , Katz & McDaniel, Med Res Rev 19 : 543-558 (1999) and WO 98/49315. Similarly, entire modules can be deleted and/or replaced with other modules from the same PKS or another PKS. See e. g., Gokhale et al. , Science 284 : 482 (1999) and WO 00/47724 each of which are incorporated herein by reference. Protein subunits of different PKSs also can be mixed and matched to make compounds having the desired backbone and modifications. For example, subunits of 1 and 2 (encoding modules 1-4) of the pikromycin PKS were combined with the DEBS3 subunit to make a hybrid PKS product (see Tang et al., Science, 287: 640 (2001), WO 00/26349 and WO 99/6159).

[0047] It will be appreciated that an amino acid sequence of a protein or domain can be changed without eliminating or substantially changing the function or activity of the wild-type protein or domain, for example, by making conservative substitutions of amino acids. The present invention encompasses polypeptides that are conservatively modified variants of a polypeptide encoded in SEQ ID NO : 1 and retain the activity of the wild-type polypeptide. Such polypeptides can be identified by routine screening methods. For example, a polypeptide having a substitution or combination of substitutions relative to wild-type can be prepared by mutation of DNA encoding the fostriecin cluster polypeptide or domain, and the effect (if any) of the sequence modification can be assessed by expressing the protein in a suitable host cell under conditions in which fostriecin is produced in the cell when the unmodified protein is expressed.

This assay can be carried out in Streptomyces pulveraceus by modification of endogenous genes or, alternatively, polynucleotides modified in vitro can be expressed in heterologous hosts as described elsewhere herein. Production of fostriecin at a level not less that 60% of the level produced by the wild-type sequence, preferably at least 80%, and most preferably not less than 95% of the level produced by the wild-type sequence is indicative that the modified polypeptide or domain has the same activity as the unmodified parent. The invention includes such modified polypeptides and the nucleic acid sequences encoding them.

[00481 In other embodiments, a domain or other region of a fostriecin polyketide synthase polypeptide can be removed or otherwise inactivated or replaced with a different PKS domain.

[0049] Mutations can be introduced into PKS genes such that polypeptides with altered activity are encoded. Polypeptides with"altered activity"include those in which one or more domains are inactivated or deleted, or in which a mutation changes the substrate specificity of a domain, as well as other alterations in activity. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. (See, e. g., Kuiel, T. A. ProcNatl Acad Scì USA (1985) 82: 448 ; Geisselsoder et al. BioTecA2niques (1987) 5: 786. ) Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. (See Zoller and Smith, Methods in Ehzymology (1983) 100: 468). Primer extension is effected using DNA polymerase. The product of the extension reaction is cloned, and those clones containing the mutated DNA are selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. (See, e. g., Dalbie-McFarland et al. Proc Natl Acad Sci MM (1982) 79: 6409). PCR mutagenesis can also be used for effecting the desired mutations.

Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can be accomplished by several different techniques known in the art, e. g., by inserting an oligonucleotide linker randomly into a plasmid, [0050] In addition to providing mutated forms of regions encoding enzymatic activity, regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS synthase can be recovered, for example, using PCR techniques with appropriate primers. By"corresponding"activity encoding regions is meant those regions encoding the same general type of activity--e. g., a ketoreductase activity in one location of a gene cluster would"correspond"to a ketoreductase-encoding activity in another location in the gene cluster or in a different gene cluster; similarly, a complete reductase cycle could be considered corresponding--e. g. , KR/DH/ER could correspond to KR alone.

[0051) If replacement of a particular target region in a host polyketide synthase is to be made, this replacement can be conducted iii vitro using suitable restriction enzymes or can be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene. One such system involving plasmids of differing temperature sensitivities is described in PCT application WO 96/40968. Another useful method for modifying a PKS gene (e. g. , making domain substitutions or"swaps") is a RED/ET cloning procedure developed for constructing domain swaps or modifications in an expression plasmid without first introducing restriction sites. The method is related to ET cloning methods (see, Datansko & Wanner, 2000, Proc. Natl. Acad. Sci. U. S. A. 97, 664045 ; Muyrers et al, 2000, Genetic Engineering 22 : 77-98).

The RED/ET cloning procedure is used to introduce a unique restriction site in the recipient plasmid at the location of the targeted domain. This restriction site is used to subsequently linearize the recipient plasmid in a subsequent ET cloning step to introduce the modification.

This linearization step is necessary in the absence of a selectable marker, which cannot be used for domain substitutions. An advantage of using this method for PKS engineering is that restriction sites do not have to be introduced in the recipient plasmid in order to construct the swap, which makes it faster and more powerful because boundary junctions can be altered more easily.

[0052] In a further aspect, the invention provides methods for expressing chimeric or hybrid PKSs and products of such PKSs. For example, the invention provides (1) encoding DNA for a chimeric PKS that is substantially patterned on a non-fostriecin producing enzyme, but which includes one or more functional domains, modules or polypeptides of fostrìecin PKS ; and (2) encoding DNA for a chimeric PKS that is substantially patterned on the fostriecin PKS, but which includes one or more functional domains, modules, or polypeptides of another PKS or NRPS.

[0053] With respect to item (1) above, in one embodiment, the invention provides chimeric PKS enzymes in which the genes for a non-fostriecin PKS function as accepting genes, and one or more of the above-identified coding sequences for fostriecin domains or modules are inserted as replacements for one or more domains or modules of comparable function. Construction of chimeric molecules is most effectively achieved by construction of appropriate encoding polynucleotides. In making a chimeric molecule, it is not necessary to replace an entire domain or module accepting of the PKS with an entire domain or module of fostriecin PKS: subsequences of a PKS domain or module that correspond to a peptide subsequence in an accepting domain or module, or which otherwise provide useful function, may be used as replacements. Accordingly, appropriate encoding DNAs for construction of such chimeric PKS include those that encode at least 10+ 15,20, 40 or more amino acids of a selected fostriecin domain or module.

[0054] Recombinant methods for manipulating modular PKS genes to make chimeric PKS enzymes are described in U. S. Patent Nos. 5,672, 491 ; 5,843, 718; 5, 830, 750; and 5, 712, 146; and in PCT publication Nos. 98/49315 and 97/02358. A number of genetic engineering strategies have been used with DEBS to demonstrate that the structures of polyketides can be manipulated to produce novel natural products, primarily analogs of the erythromycins (see the patent publications referenced supra and Hutchinson, 1998, Curr Opin Microbiol. 1 : 319-329, and Baltz, 1998, Trends Microbial 6: 76-83). In one embodiment, the components of the chimeric PKS are arranged onto polypeptides having interpolypeptide linkers that direct the assembly of the polypeptides into the functional PKS protein, such that it is not required that the PKS have the same arrangement of modules in the polypeptides as observed in natural PKSs. Suitable interpolypeptide linkers to join polypeptides and intrapolypeptide linkers to join modules within a polypeptide are described in PCT publication WO 00/47724.

[0055] A partial list of sources of PKS sequences for use in making chimeric molecules, for illustration and not limitation, includes Avermectin (U. S. Pat : No. 5,252, 474; MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256; MacNeil et al. , 1992, Gene 115: 119-25) ; Candicidin (FRO008) (Hu et al., 1994, Mol. Microbiol. 14: 163-72); Epothilone (U. S. Pat. No. 6,303, 342) ; Erythromycin (WO 93/13663; U. S. Pat. No. 5, 824, 513; Donadio et al. , 1991, Science 252: 675- 79; Cortes et al. , 1990, Nature 348: 176-8) ; FK-506 (Motamedi et al. , 1998, Eur. J. Biochem.

256: 528-34 ; Motamedi et al. , 1997, Eur. J. Biochem. 244: 74-80) ; FK-520 (U. S. Pat. No.

6,503, 737; see also Nielsen et al., 1991, Biochem. 30: 5789-96) ; Lovastatin (U. S. Pat. No.

5,744, 350); Nemadectin (MacNeil et al., 1993, supra) ; Niddamycin (Kakavas et al. , 1997, J.

Bacteriol. 179: 7515-22); Oleandomycin (Swan et al. , 1994, Mol. Gen. Genet. 242: 358-62; U. S.

Pat. No. 6, 388, 099; Olano et al., 1998, Mol. Gen. Genet. 259: 299-308); Platenolide (EP Pat.

App. 791,656) ; Rapamycin (Schwecke et al., 1995, Proc. Natl. Acad. Sci. USA 92: 7839-43); Aparicio et al. , 1996, Gene 169 : 9-16); Rifamycin (August et al., 1998, Chemistry & Biology, 5 : 69-79); Soraphen (U. S. Pat. No. 5,716, 849 ; Schupp et al. , 1995, J. Bacteriology 177: 3673-79); Spiramycin (U. S. Pat. No. 5,098, 837); Tylosin (EP 0 791,655 ; Kuhstoss et al. , 1996, Gene 183: 231-36 ; U. S. Pat. No. 5,876, 991). Additional suitable PKS coding sequences remain to be discovered and characterized, but will be available to those of skill (e. g. , by reference to GenBank).

100561 The fostriecin PKS-encoding polynucleotides of the invention may also be used in the production of libraries of PKSs (i. e. , modified and chimeric PKSs comprising at least a portion of the fostriecin PKS sequence. The invention provides libraries of polyketides by generating modifications in, or using a portion of, the fostriecin PKS so that the protein complexes produced by the cluster have altered activities in one or more respects, and thus produce polyketides other than the natural fostriecin product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method. By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native PKS cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities. Expression vectors containing nucleotide sequences encoding a variety of PKS systems for the production of different polyketides can be transformed into the appropriate host cells to construct a polyketide library. In one approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected for successful transformants. Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide. A variety of strategies can be devised to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide. The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity. See, for example, [0057J Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of candidate polyketides. The polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like. The supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand.

Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can be included.

10058] As noted above, the DNA compounds of the invention can be expressed in host cells for production of proteins and of known and novel compounds. Preferred hosts include fungal systems such as yeast and prokaryotic hosts, but single cell cultures of, for example, mammalian cells could also be used. A variety of methods for heterologous expression of PKS genes and host cells suitable for expression of these genes and production of polyketides are described, for example, in U. S. Patent Nos. 5,843, 718 and 5,830, 750; WO 01/31035, WO 01/27306, and WO 02/068613 ; and U. S. patent application nos. 10/087,451 (published as US2002000087451) ; 60/355,211 ; and 60/396,513 (corresponding to published application 20020045220).

[0059] Appropriate host cells for the expression of the hybrid PKS genes include those organisms capable of producing the needed precursors, such as malonyl-CoA, methylmalonyl- CoA, ethylmalonyl-CoA, and methoxymalonyl-ACP, and having phosphopantotheinylation systems capable of activating the ACP domains of modular PKSs. See, for example, US Patent 6, 579, 695. However, as disclosed in U. S. Patent No. 6,033, 883, a wide variety of hosts can be used, even though some hosts natively do not contain the appropriate post-translational mechanisms to activate the acyl carrier proteins of the synthases. Also see WO 97/13845 and WO 98/27203. The host cell may natively produce none, some, or all of the required polyketide precursors, and may be genetically engineered so as to produce the required polyketide precursors. Such hosts can be modified with the appropriate recombinant enzymes to effect these modifications. In one embodiment the host cell is a bacterium. In another embodiment the host cell is a fungus, such as a yeast cell. Suitable host cells include Streptomyces, E. coli, yeast, and other prokaryotic hosts which use control sequences compatible with Streptomyces spp.

Examples of suitable hosts that either natively produce modular polyketides or have been engineered so as to produce modular polyketides include but are not limited to actinomyetes such as Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces fradiae, Streptomyces ambofaciens, and Saccharopolyspora erythraea, eubacteria such as Escherichia coli, myxobacteria such as Myxococcus xanthus, and yeasts such as Saccharomyces cerevisiae.

[0060] In sone embodiments, any native modular PKS genes in the host cell have been deleted to produce a"clean host, "as described in US Patent 5,672, 491.

[00611 Host cells can be selected, or engineered, for expression of a glycosylatation apparatus (discussed below), amide synthases, (see, for example, U. S. patent publication 20020045220"Biosynthesis of Polyketide Synthase Substrates"). For example and not limitation, the host cell can contain the desosamine, megosamine, and/or mycarose biosynthetic genes, corresponding glycosyl transferase genes, and hydroxylase genes (e. g., pi, cKj megK, eryK, megF, and/or eryF). Methods for glycosylating polyketides are generally known in the art and can be applied in accordance with the methods of the present invention; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in WO 98/49315, incorporated herein by reference. Glycosylation with desosamine, mycarose, and/or megosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention. Alternatively and as noted, glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases. In addition, synthetic chemical methods may be employed.

[0062] Alternatively, the aglycone compounds can be produced in the recombinant host cell, and the desired modification (e. g., glycosylation and hydroxylation) steps carried out in vitro (e. g. , using purified enzymes, isolated from native sources or recombinantly produced) or in vivo in a converting cell different from the host cell (e. g. , by supplying the converting cell with the aglycone).

[0063] Modification or tailoring enzymes for modification of a product of the fostriecin PKS, a non-fostriecin PKS, or a chimeric PKS, can be those normally associated with fostriecin biosynthesis or"heterologous"tailoring enzymes. Tailoring enzymes can be expressed in the organism in which they are naturally produced, or as recombinant proteins in heterologous hosts.

In some cases, the structure produced by the heterologous or hybrid PKS may be modified with different efficiencies by post-PKS tailoring enzymes from different sources. In such cases, post- PKS tailoring enzymes can be recruited from other pathways to obtain the desired compound.

[0064] In some embodiments, the host cell expresses, or is engineered to express, a polyketide"tailoring"or"modifying"enzyme. Once a PKS product is released, it is subject to post-PKS tailoring reactions. These reactions are important for biological activity and for the diversity seen among polyketides. Tailoring enzymes normally associated with polyketide biosynthesis include oxygenases, glycosyl-and methyltransferases, acyltransferases, halogenases, cyclases, aminotransferases, and hydroxylases.

[0065] In the case of fostriecin biosynthesis, tailoring enzymes include P450 hydroxylases for addition of hydroxyl groups. The PKS is expected to initially produce hydroxyls at C3, C5, C9 and Cl 1, with the C9 hydroxyl further modified by phosphorylation, the C5 hydroxyl further reacting to help create the 6-membered lactone ring, and the C3 hydroxyl being removed by dehydration in the creation of a double bond between C2 and C3. In addition hydroxyls at C8 and CIS (and C4 in PD 113,271) are expected to be introduced by post-PKS-acting accessory proteins. The fostriecin gene cluster encodes three cytochrome-P450-hydroxylase homologs (FosG, FosJ and FosK). Based on apparent homology between FosJ and the PlmT4 P450 hydrolase encoded in the Streptor, lyces phoslactomycin synthase gene cluster, apparent homology between FosK and the PlmS2 P450 hydrolase encoded in the Streptomyces phoslactomycin synthase gene cluster, evidence that PlmS2 is responsible for cyclohexyl modification at C18 but not C8 of the polyketide phoslactomycin, and the presence of hydroxyls at the tertiary C8 of fostriecin and the tertiary C8 of phoslactomycin, FosJ may produce the C8 hydroxyl of fostriecin. FosG and/or FosK are expected to modify the C4 and C8 positions, with perhaps a specific P450 for each site. The phosphorylation of the hydroxyl group at C9 is predicted to be accomplished by FosH, a distant homolog of homoserine kinases. ORF7 encodes a type II thioesterase.

[0066] The P450 hydroxylases and kinase of the fostriecin PKS gene cluster can be expressed heterologously to modify polyketides produced by non-fostriecin polyketide synthases or can be inactivated in the Fostriecin producer.

[0067] In addition to biosynthetic accessory activities, secondary metabolite clusters often code for activities such as transport and regulation. FosI appears to be a permease having a transport function. ORF1 and ORF3 are putative transcriptional regulators. ORF1 is a homolog of MarR-family transcriptional regulators, including SC07709, SC07639 and SC00447 from Streptomyces coelicolor. ORF3 is a homolog of LuxR family transcriptional regulators.

[00681 ORF2 is a homolog of a conserved family, including SC7708, SC6340 and SC5938 from Streptomyces coelicolor, and SAV1967 and SAV0886 from S. avermitilis. ORF4 is a homolog of a conserved family, including PlmT2 from the phoslactomycin biosynthetic cluster, SAV4898 from Streptomyces avernzitilis and SC04633 from S. coelicolor. ORF5 is a homolog of BorL from the borrelidin biosynthetic cluster. ORF6 encodes a homolog of the product of plu4507 from Photorhabdus luminescens subsp. laumodii TTO1, and has some similarity to 3- hydroxy-3-methylglutaryl coenzyme A reductases. ORF8 encodes a homolog of chaperone protein HtpG (heat shock protein HtpG) from Streptomyces coelicolor.

[0069] Tables 2 and 4 describe the characteristics of open reading frames of the fostriecin polyketide synthase gene cluster. Table 2 shows the position of each ORF relative to SEQ ID NO : 1, as well as identifying certain homologous proteins.

TABLE 2<BR> ORFs Encoding Additional Polypeptides Encoded in the Fostriecin polyketide synthase Cluster amino Position in SEQ ID NO:1 % ORF acids putative function @oding strand(nucleotide pair) homology identity SC0770.9 (142 aa; 43% MarR-family identity/137 aa), orf1 165 transcriptional SEQ ID NO 1(72775..73272) SC07639 and SC00447 regulator from Streptomyces coelicolor 43%/137aa Sc7708 (216 aa; 48% identity/192 aa), orf2 213 complement(72055..72696) SC6340 and Sc5938 from Streptomyces coelicolor 48%/216aa LuxR-family orf3 967 transcriptional complement(68498..71401) PikD (Streptomyces regulator venezuelae) 29%/977aa PlmT2 from the orf4 295 unknown complement(67600..68487) phoslactomycin biosynthetic cluster 41%/212aa ORF4 from the mitomycin P450; possible C8 or C biosynthetic cluster fosG 409 complement(33643..34872) C4-hydroxylase in Streptomyces lavendulae 48%/395aa PlmT5 from the fosH 316 polyketide kinase complement(32552..33502) phoslactomycin biosynthetic cluster 43%/259aa PlmS4 from the phoslactomycin biosynthetic cluster fosI 444 polyketide export complement(31111..32445) [COG0477: Permeases of the major facilitator superfamily] 50%/431aa PlmT4 from the P450; possible C4- or fosJ 420 complement(29742..31004) phoslactomycin C8-hydroxylase biosynthetic cluster 54%/397aa PlmS2 from the P450; possible C18- fosK 398 SEQ ID NO 1 (28443..29639) phoslactomycin hydroxylase biosynthetic cluster 57%/404aa BorL from the orf5 538 complement(7892..9508) borrelidin biosynthetic cluster 30%/536aa plu4507 from homology to 3-hydroxy- photorhabdus orf6 781 3-methylglutaryl complement(5550..7895) luminescens subsp. coenzyme A reductases laumondii TTO1 39%/774aa thioesterase (TEII orf7 258 complement(3840..4616) AveG (Streptomyces family) avermitilis) 52%/238aa chaperone protein htpG orf8 633 (heat shock protein complement(1424..3325) HtpG (Streptomyces htpG) coelicolor) 79%/638aa *fosG, H, @, J and K were previously called ORFs 1, 2, 3, 4 and 5 [0070J It will be apparent to the reader that a variety of recombinant vectors can be utilized in the practice of aspects of the invention. As used herein, "vector"refers to polynucleotide elements that are used to introduce recombinant nucleic acid into cells for either expression or replication. Selection and use of such vehicles is routine in the art. An"expression vector" includes vectors capable of expressing DNAs that are operatively linked with regulatory sequences, such as promoter regions. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[00711 The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes may be chosen to contain control sequences operably linked to the resulting coding sequences in a manner that expression of the coding sequences may be effected in an appropriate host. Suitable control sequences include those which function in eukaryotic and prokaryotic host cells. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This can be done individually, or using a pool of isolated encoding nucleotide sequences, which can be inserted into host vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies.

[00721 Suitable control sequences for single cell cultures of various types of organisms are well known in the art. Control systems for expression in yeast are widely available and are routinely used. Control elements include promoters, optionally containing operator sequences, and other elements depending on the nature of the host, such as ribosome binding sites.

Particularly useful promoters for prokaryotic hosts include those from PKS gene clusters which result in the production of polyketides as secondary metabolites, including those from Type I or aromatic (Type II) PKS gene clusters. Examples are act promoters, tcln promoters, spiramycin promoters, and the like. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, are also useful. Additional examples include promoters derived from biosynthetic enzymes such as for tryptophan (trp), the , 8-lactamase (bla), bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U. S. Patent No. 4,551, 433), can be used.

[0073] As noted, particularly useful control sequences are those which themselves, or with suitable regulatory systems, activate expression during transition from growth to stationary phase in the vegetative mycelium. The system contained in the plasmid identified as pCK7, i. e. , the actI/actIII promoter pair and the actII-ORF4 (an activator gene), is particularly preferred.

Particularly preferred hosts are those which lack their own means for producing polyketides so that a cleaner result is obtained. Illustrative control sequences, vectors, and host cells of these types include the modified S. coelicolor CH999 and vectors described in PCT publication WO 96/40968 and similar strains of S. lividans. See U. S. Patent Nos. 5,672, 491; 5,830, 750, 5,843, 718 ; and 6,177, 262, each of which is incorporated herein by reference.

10074l Other regulatory sequences may also be desirable which allow for regulation of expression of the PKS sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0075] Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid. Alternatively, several polyketides are naturally colored, and this characteristic provides a built-in marker for screening cells successfully transformed by the present constructs.

[0076] The various PKS nucleotide sequences, or a mixture of such sequences, can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements or under the control of a single promoter. The PKS subunits or components can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunits so that hybrid or chimeric PKSs can be generated. The design of such restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site- directed mutagenesis and PCR. Methods for introducing the recombinant vectors of the present invention into suitable hosts are known to those of skill in the art and typically include the use of CaCk or other agents, such as divalent cations, lipofection, DMSO, protoplast transformation, conjugation, and electroporation.

[0077] Thus, the present invention provides recombinant DNA molecules and vectors comprising those recombinant DNA molecules that encode all or a portion of the fostriecin PKS and/or fostriecin modification enzymes and that, when transformed into a host cell and the host cell is cultured under conditions that lead to the expression of said fostriecin PKS and/or modification enzymes, results in the production of polyketides including but not limited to fostriecin and/or analogs or derivatives thereof in useful quantities. The present invention also provides recombinant host cells comprising those recombinant vectors.

[0078] Suitable culture conditions for production of polyketides using the cells of the invention will vary according to the host cell and the nature of the polyketide being produced, but will be know to those of skill in the art. See, for example, the examples below and WO 98/27203"Production of Polyketides in Bacteria and Yeast"and WO 01/83803"Overproduction Hosts For Biosynthesis of Polyketides." [0079l The polyketide product produced by host cells of the invention can be recovered (i. e., separated from the producing cells and at least partially purified) using routine techniques (e. g., extraction from broth followed by chromatography).

100801 The compositions, cells and methods of the invention may be directed to the preparation of an individual polyketide or a number of polyketides. The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. It will be understood that the resulting polyketides may be further modified to convert them to other useful compounds. For example, an ester linkage may be added to produce, a"pharmaceutically acceptable ester" (i. e. , an ester that hydrolyzes under physiologically relevant conditions to produce a compound or a salt thereof). Illustrative examples of suitable ester groups include but are not limited to formates, acetates, propionates, butyrates, succinates, and ethylsuccinates.

[00811 The polyketide product can be modified by addition of a protecting group, for example to produce prodrug forms. A variety of protecting groups are disclosed, for example, in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York (1999). Prodrugs are in general functional derivatives of the compounds that are readily convertible in vivo into the required compound. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in"Design of Prodrugs, "H. Bundgaard ed. , Elsevier, 1985.

[0082] Similarly, improvements in water solubility of a polyketide compound can be achieved by addition of groups containing solubilizing functionalities to the compound or by removal of hydrophobic groups from the compound, so as to decrease the lipophilicity of the compound. Typical groups containing solubilizing functionalities include, but are not limited to: 2- (dimethylaminoethyl) amino, piperidinyl, N-alkylpiperidinyl, hexahydropyranyl, furfuryl, tetrahydrofurfuryl, pyrrolidinyl, N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl, morpholinyl, N-alkylaziridinylmethyl, (1-azabicyclo [1. 3.0] hex-1-yl) ethyl, 2- (N- methylpyrrolidin-2-yl) ethyl, 2- (4-imidazolyl) ethyl, 2- (l-methyl-4-imidazolyl) ethyl, 2- (l-methyl- 5-imidazolyl) ethyl, 2- (4-pyridyl) ethyl, and 3- (4-morpholino)-l-propyl. Solubilizing groups can be added by reaction with amines. Typical amines containing solubilizing functionalities include 2- (dimethylamino)-ethylamine, 4-aminopiperidine, 4-amino-1-methylpiperidine, 4- aminohexahydropyran, furfurylamine, tetrahydrofurfurylamine, 3- (aminomethyl)- tetrahydrofuran, 2- (amino-methyl) pyrrolidine, 2-(aminomethyl)-1-methylpyrrolidine, 1- methylpiperazine, morpholine, 1-methyl-2 (aminomethyl) aziridine, 1-(2-aminoethyl)-1- azabicyclo- [1. 3. 0] hexane, 1-(2-aminoethyl) piperazine, 4-(2-aminoethyl) morpholine, 1-(2-amino- ethyl) pyrrolidine, 2-(2-aminoethyl) pyridine, 2-fluoroethylamine, 2,2-difluoroethylamine, and the like.

[0083] In addition to post synthesis chemical or biosynthetic modifications, various polyketide forms or compositions can be produced, including but not limited to mixtures of polyketides, enantiomers, diastereomers, geometrical isomers, polymorphic crystalline forms and solvates, and combinations and mixtures thereof can be produced.

[0084] Many other modifications of polyketides produced according to the invention will be apparent to those of skill, and can be accomplished using techniques of pharmaceutical chemistry.

[0085] Prior to use the PKS product (whether modified or not) can be formulated for storage, stability or administration. For example, the polyketide products can be formulated as a "pharmaceutically acceptable salt. "Suitable pharmaceutically acceptable salts of compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonic acid, or the like. Where the compounds carry one or more acidic moieties, pharmaceutically acceptable salts may be formed by treatment of a solution of the compound with a solution of a pharmaceutically acceptable base, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, ammonia, alkylamines, or the like.

[0086] Prior to administration to a mammal the PKS product will be formulated as a pharmaceutical composition according to methods well known in the art, e. g. , combination with a pharmaceutically acceptable carrier. The term"pharmaceutically acceptable carrier"refers to a medium that is used to prepare a desired dosage form of a compound. A pharmaceutically acceptable carrier can include one or more solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co. , Easton, PA, 1975) and Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe ed. (American Pharmaceutical Assoc.

2000), disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[00871 The composition may be administered in any suitable form such as solid, semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 edition, Lippicott Williams & Wilkins (1991). In an embodiment, for illustration and not limitation, the polyketide is combined in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use. The carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi- solid, or liquified form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.

[00881 It will be appreciated by those of skill that recombinant polynucleotides and polypeptides of the invention have a variety of uses, including, but not limited to, those described above and including use as probes and primers (e. g. , for gene amplification or targeting) or as enzymes, or components of enzymes, useful for the synthesis or modification of polyketides.

Recombinant polypeptides encoded by the fostriecin PKS gene cluster are also useful as antigens for production of antibodies. Such antibodies find use for purification of bacterial (e. g., Streptomyces pulveraceus) proteins, detection and typing of bacteria, and particularly, as tools for strain improvement (e. g. , to assay PKS protein levels to identify"up-regulated"strains in which levels of polyketide producing or modifying proteins are elevated) or assessment of efficiency of expression of recombinant proteins. Polyclonal and monoclonal antibodies can be made by well known and routine methods (see, e. g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York; Koehler and Milstein 1075, Nature 256: 495). In selecting polypeptide sequences for antibody induction, it is not to retain biological activity; however, the protein fragment must be immunogenic, and preferably antigenic (as can be determined by routine methods). Generally the protein fragment is produced by recombinant expression of a DNA comprising at least about 60, more often at least about 200, or even at least about 500 or more base pairs of protein coding sequence, such as a polypeptide, module or domain derived from a fostriecin polyketide synthase (PKS) gene cluster. Methods for expression of recombinant proteins are well known. (See, e. g. , Ausubel et al. , 2002, Current Protocols In Molecular Biology, Greene Publishing and Wiley-Interscience, New York.) EXAMPLES [0089] The following examples are provided to illustrate, but are not intended to limit, the present invention.

Example 1 Cloning and Sequencing of Gene Cluster for Fostriecin Biosynthesis Growth of organism and extraction of genomic DNA.

[0090] For genomic DNA extraction, a spore stock of Strepto7nyces pulveraceus subsp. fostreus ATCCC 31906 was used to inoculate 35 ml of Tryptitone Soy Broth (TSB) liquid media. After two days growth in 30°C, a 10 ml portion of the cell suspension was centrifuged (10,000 x g). The pellet was suspended into 3.5 ml of buffer 1 (Tris, 50 mM, pH7. 5 ; 20 mM EDTA, 150 llg/ml RNase (Sigma-Aldrich) and lmg/ml of lysozyme (Sigma) ). After incubation of the mixture at 37°C for 30 min, the salt concentration was adjusted by adding 850 PI 5 M NaCl solution, then the mixture was extracted two times with phenol: chlorofOrm : isoamylaclohol (25: 24: 1, vol/vol) with gentle agitation followed by centrifugation for 10 min at 3500 x g. After precipitation with 1 vol of isopropanol, the genomic DNA knot was spooled on a glass rod and redissolved in 500 ul of water Genomic library preparation [0091] Approximately 10 pg of genomic DNA was partially digested with Sau3A1 (1 hr incubation using dilutions of the enzyme) and the digested DNA was run on an agarose gel with DNA standards. One of the conditions used was found to have generated fragments of size 30- 45 kb. The DNA from this digestion was ligated with pSuperCos-1 (Stratagene), pre-linearized with BamHI and XbaI and the ligation mixture was packaged using a Gigapack XIII (Stragene) in vitro packaging Kit and the mixture was subsequently used for infection of Escherichia coli DH5a employing protocols supplied by the manufacturer.

Identification of fostriecin biosynthetic gene cluster [00921 To find the gene cluster for fostriecin biosynthesis, cosmids from 11X95 E.coli transductants resulted from the above ligation mixture were sequenced with using convergent primersT7cos (5'-CATAATACGACTCACTATAGGG) [SEQ ID NO: 21] and T3cos-1 (5'- TTCCCCGAAAAGTGCCAC) [SEQ ID NO: 22]. After BLAST analysis, the sequences revealed 28 cosmids carrying DNA fragment encoded type I or type II PKS (polyketide synthase) genes at either of ends or both ends. Based on sequence and restriction enzyme maps of the 21 cosmids most likely related to modular PKS, most could be assigned to two major groups ("overlap family 1"and"overlap family 3"). Since overlap family 3 carries genes (homologous to gdmI and K) for methoxymalonyl-ACP, which is not needed for the biosynthesis of fostriecin, we focused on overlap family 1 (See Figure 2). Based on the relation of among these cosmids, we chose to sequence pKOS279-117.1F70, pKOS279-117.3F45, pKOS279-117.2F15, and pKos279-117.5F58 from the overlap family 1. Other cosmids in this family were pKOS279- 127. 11F54 ; pKOS279-127. 10F6 ; pKOS279-127. 1OF75 ; pKOS279-127. 3F46 ; pKOS279- 127. 5F58.

DNA Sequencing [0093] In initial sequencing efforts the sequence of inserts of three cosmids (pKOS279- 117. 1F70, pKOS279-117. 3F45 and pKOS279-117. 2F15) was determined. The results of this sequencing effort are provided in the appended sequence listings (which are part of and incorporated into this specification) as SEQ ID NOs: 23,27 and 33. Small gaps in the sequence indicated as"x"or"n. "Complete or partial open reading frames (ORFs) encoded by these sequences can be determined by reference to the genetic code and are also provided in SEQ ID NOs: 24-26,28-32 and 34-36. Complete sequencing was carried out using (pKOS279- 117. 1F70, pKOS279-117. 3F45, pKOS279-117. 2F15, and pKos279-117. 5F58).

TABLE3 FOSTRIECIN SYNTHASE GENE CLUSTER from Streptomyces pulveraceus subsp. fostreus AT31906 source 1.. 18774 from pKos279-117.1F70 source 18651.. 29679 from pKos279-117. 3F45 source 28694.. 29679 from pKos279-117. 2F15 source 29683.. 53913 from pKos279-117. 3F45 source 29683.. 66484 from pKos279-117. 2F15 source 58636.. >73984 from pKos279-117. 5F58 2968. 0.. 29682 is an unsequenced fragment of putative hairpin terminator" 2521 GGCGGATCGT CTCCAGGGGG TCGCGCCAGT CGTGGCTGAC GTGCTTGTAC AGCTCGTGGT 2581 ACTCGTCGTC GGAGACCTCG TCGCGCGAGC GTGCCCACAG CGCCTTCATC GAGTTCAGCG 2641 TCTCGGGTTC GGGCGTTTCC TCGCCGTCGG TCGCCTGCGG GAGGAGCCGG ACCGGCCAGG 2701 TGATGAAGTC GGAGTACCGC TTGACGATCT CCTTGATCTT CCAGGCGGAG GTGTAGTCGT 2761 GCAGTTGGTC GTCGGCGTCG GCCGGCTTGA GGTGGAGCGT GACGGCACTG CCCTGCGGCA 2821 CGTCGTCGAC CGTCTCCAGG GTGTACGTGC CCTCACCGCG CGACGACCAC CGCGTGCCGC 2881 TGCGCTCCCC GGCACGCCGG GTCACCAGGG TCATCTCGTC GGCCACCATG AAGCCGGAGT 2941 AGAAACCGAC GCCGAACTGT CCGATGAGCC CCTCGGCCCC GGCCGCGTCC TGCGCCTCCT 3'001 TCAGCTCCTG GAGGAAGGCG GCCGTGCCCG AATTGGCGAT GGTGCCGATG AGCTTGCCGA 3061 CCTCGTCGTA CGACATCCCG ATGCCGTTGT CCCGCACGGT GAGCGTACGG GCCTTCTGGT 3121 CGAGCTCGAT CTCGATGTGC GGGTCGGACG TGTCGGCGTC GAGCCCGTCG TCCCGCAACG 3181 CGGCGAGACG CAGCTTGTCG AGCGCGTCGG AGGCGTTGGA GACGAGCTCG CGCAGGAAGA 3241 CGTCCTTGTT CGAGTAGACC GAGTGGATCA TCAGCTGGAG CAGCTGGCGT GCTTCTACCT 3301 GGAACTCGAA CGTTTCGGTC GCCATGCTTC GTATTCCTCA CAGGTTCCTG GGTGGCCGAA 3361 TCGGGCGACA GCCACTGTAA GACACCAAGT CGGCGCATTG TCACCGCCGT TCGCCGCGCG 3421 GCGTCCGCAT CTGCGTCTGC GTCTGCGTCA GACCTCGCCG TGGGCGCGCC TGCCCCGGCC 3481 GTCCCGCCAG GACGTGGGGC AGGTGCCCCG CGCGGTCGCG GCCCCGGCGT CGGCGAACAC 3541 GGGCGCTCCA GCCGCCTTCG GAAGGCATCC CGGATGCGGA GCGGAGACCT TCGAACACGC 3601 CGGTCCTGAC CCGGTCGCAC GCCCCTGCTC CGGCTCGCTC CCGGGGATCC GGCACAATCG 3661 CACCGCGGAC CGACGGCCGC ATCCGCCTTC CTGCTGTGGT GCCGGACGGA GCCGTGGGAT 3721 CTGCGCTCCT ACGGCCACGT CGTCAAGCTG GAGCAGGAAC GTCTCGCCTA CCGGGCCCGC 3781 CGCACTCCCG CTTCGGCCGC TCCTGTCGCC GCAAGGCCCC GCCGACGCCC ACCGCCCCAT 3841 CACACGTACG GAGGGGCGAC CAGCAAGGTG TTGCGGATCG CCTCGACGAT CTCAGGCTGG 3901 TGCTGCACCA GGTAGAAGTG GCCGCCCGGC AGCAGTGTCA GGTCGAACGA ACCGGCGGTG 3961 TGGTCGGCCC ACAGCTTCAT CTCGCCCTCG TCGACCTTCG GGTCCTGCGC GCCGAGGAAC 4021 CCGCGGATCG GACAGGCCAC AGGCGGGCCG GGCACGTAGC GGTAGGTCTC GATGAGGCGG 4081 TAGTCGTTGC GCAGCGGGGG CATGATCATC TCGATGATCT CGGGATCGTC GAACATCCGG 4141 CTGTCCGTGC CCTCCAGGCC GCGCAGTTCG GCCAGCACGT CTTCCTGCGA CATGGCGTGC 4201 ACCCGCTCGC CGCGGTTGAC CGACGGGGCG CCGCGGCCAG ACGCGAAGAG CGCCGTCACC 4261 GGCGTCGTGG AGTCCTCGAG GAGGCGGATG ACCTCGAAGG CCACCGCCGC CCCCATGCTG 4321 TGCCCGAAGA ACGCCGTCGG TACGGCCGGT TCTCCGCACA GCGCCTCGGC GACGTGGGCG 4381 GCGAGGTCTT GCAGCGTGGC GGGGAACGGG TCGGCCCTGC GGTCCTGCCG CCCCGGGTAC 4441 TGGACGGCGA CGATGTCGAT CTCCGGGGCC AGGGCACGCG CGAGCGGCAT GTAGAAGCTG 4501 GCGGAACCGC CCGCGTGCGG CAGGCAGACG AGCCGGTGGC GGGCGTCCGG GGCGTTCGTG 4561 TAACGGCGCA GCCACGCCTG CCGGTCCGTG GACGGTTTCG GGGTCGGGGC GTACATCAAG 4621 GTTTCTCCAG AGTGCGGAAG GCGAAGCGCC GAGGCGGAGG TCGCCCGCGG CGGTGGGTGC 4681 GTCAGTGGGC GACCGCGGCC CCCGGTTCGG TGACCGCGGT GTGCTGCTCC ATGACCCGGC 4741 TCGGACGCGC CGGCCCTGTC ACCGGCGCCC GAGCCTGTGT GTCCACTCCC AGCAGGCCGG 4801 CCACCTCGTC GGCGACGGAC GTCGCGTCGC GGCCGACGCA GTCGACCCGG TGCACCGTGA 4861 CGCGGCCGGA GAGCCCGTCG AGCACGTCGT CGTAGGCGTG GGCGAGCCCG GTGAGCAGCC 4921 GGGCGTCCTC GTGCAGTTCG GTGCCGTTCC GGCGGCTGCG GGTGCGCCGC AGTGCCTCGT 4981 CGACCGGCAG TCGGAGCCGT ACCACGGCGT CCGGCGGGGC GGTCGAGAAC ATGTCGGCCA 5041 GGCGCTGCGC CAGTTCCTCG CGGGGCGCGG CGGCGAGCCG CAGCAGGTCC AGGCCGAGCG 5101 CCCACGGATC CGTACCGCCC GAGCAGGCCT CCAGCCAATC CCGCACCTCG CGCGCGGTCT 5161 CCGGACCCTG CTCCGCCCAC CAGCGCCGGA CGGCGTCGCC GGGATCGGGA TCGCCCGCGA 5221 TCCGCGCGAA CATCGGCAGA TAGACGAGGG GGTCGACCAG CGGGTGCCTG TCCGCCAGGA 5281 CGATCGCGCC ACGCTGCGTG GCGCGGCGCT CGGCCGGGGC GTACTGGCAC AGTTGCAGAT 5341 AGAGCACCGC GACCTTCAGC GGCGAACTGC CGATCAGGTC CGCGGCGGCG GACGCCCGCG 5401 CCAGGTGCAG GGAGCGCGTC GCCTCGGGGC TGTCCGGGTC GTCGTGCGCG CGGATCGCGT 5461 GGACGACGGA AACGCCGGGC ACGGTGCTCA GCAGCCGCGC CACCGTCGTC TTGCCGGTGC 5521 CGTCGATGCC CACCAGGGCG GCCCGCATCT CACTCACCGT CCCGGGCGGC TTCCAGGTTC 5581 CACAGGTGGA ACGTGGTGTC CAGCACGGCC GGGAGGAACG GCAGTTCCCG GTGGCTGTGC 5641 GCGGCGGTGT ACAGCTGGAG GCGGCTCAGG AGCATCTCAC GCAGCGCGAG CCCGTAGCCG 5701 CGCCGCCACT GTTCCGGCTG CGGCACGGTC GCGTCCGGTC CGGCCGCGGC GGCGACCGCG 5761 GCGCGGTGCA CCTCCAGATG GTGGTCGACC TCGTCGGTGG TGCTGTGCGG GGTGAGGGTG 5821 AAGGCCAGCA GTTCGGCCAG GTCGCGCTGG GGCACTGCGA CGGTGGCCAG CTCCCAGTCG 9301 AGGGACCGAA CGCGTTCTGC GTGCGGATCG GCAGGGCCTT GCGGAACTCC TCGGCGCCGC 9361 TTCGCTCGTT CAGGCCGTGC TCGCGCAGGT AGCTCGTCGC ACCGTTCGCC GCGAGGAGTT 9421 CCGCGAGGAC CGTCTGCTGG GTCTGCTCGG GGTGGTCGAG CGTGGCGAGG AACCTGTGGT 9481 GTTCGGTCAG AATGCGTTCG GTGTGCAAGC TTTCCCTCCA GCGCGGTCGG AGAAGAGGGC 9541 TCCGTACAGG CGGAGCCGCT CGGGGTCGGC GGTGTCGTGC GTGATCCGGC TCTCGACGCG 9601 GTGGGCACGG GCCGGGTCGA GCGCGGCGCG CAGCCGCGGT GCGTCGAGCG CCGGGCGGTG 9661 CGTCACGTGC ACGGTGTCGC CGGGACGGAC CAGACCGCGC AGCGCGGCCT CGGCGAAGAG 9721 GTGATGGGTG TGGACGAGGT AGTCCACGAC GGTGACGTCC GCGGTCGCCA TCACCGCCTC 9781 GGCCCGGAAT CCGGTGTCGG GGCCGGGCAG CCGCACGGTG AACGGCTCCG GCGCGGGGGC 9841 CGGTGCCGGA GCCGTGAAGG CGCGCAACAG CCGTGCCAGG TTCTTCAGTT GCGCCGTCGG 9901 CACCGGGCCG TACCAGCTCA GCGCGTTGTC GGGGGTCGCG TACCCGTCCG GGAACCGGGC 9961 CGCCACCGTG GCGGGCCGGC CCGCCAGGAA GATCGTGCGG GTGAACGTCC GCAGCCATGC 10021 GTCGGCCGGC TGCTCGGGGA GCGAGAGCGC GAAGTCGAGG ACGCCGCGGA CGAACGACTG 10081 CGGGGTCAGG GTGTCCACCA CGACCGCCAC GGTGTGGAAG TGGTGTCCCG GTTGGTCGTC 10141 GAGGCGGGCC AGGCGGGCCG CCGCGGAGGC TTGCAGCAAC GTCTGGAGCG AACCATCCGG 10201 GTCTTCGACG TCCGGGACGT CGATGTCCGG GAGCGCGGGG TCGCTCACGC GGGGTGCGGT 10261 CTGTCCGGTC ATACGAGCAC CTCGCGCAAC AGGGCGGGCA GCGGCGCCGG GCGCGGTCCG 10321 AGCAGCGAGC AGGCCAGGCG GTGCGGGTCG ACATAGCCGT GCGGGCTGAG CGGCAGTACC 10381 GGGTCCTCCC GTTCGTCCGA CGGCCCCAGG GCCAGGATCC GGGCCGCGCG CCCGACGCAG 10441 CGGCGCAGCA GGTCGATCCG GAAGGACTGG CTGCACTGCC GGTCGAGCAG ACCGACGAAG 10501 TAGAGGCGCA TGCCGAGCCG GTAGGCGTCC GCGTCCGCCG ACCCGGAGAT GGCCGACAGG 10561 TCCGAGAGCG TCGACCCGGT CCACCCGCCG TACTTGCTGG AGACGACCTT GCCGCCGATC 10621 GGCACCCGGG TGAGCGGGAG CCGGGTGGTG TCGGCGCCGA ACTCGGCCAG GATCCGGTCA 10681 AGGAGCAGGT AGTCGACGGC GAGCCCTTCG TCGAAGAGCA GGAGGAAGAG CCTGCCGGGC 10741 GCGACGGCGG GCAGCAGCGA GCGCAGGATC GGCAGCAGGT AGTTCGCGTG GCCGTCCGTC 10801 GAGACGATCT GCCGGATCGG CACGCCCCAG CGGGCGCCGT CCAGGTAGAC GGGCCCGCCG 10861 TGCGGCCTGT GGTCGATGAG CAGCCCGCGG TCCGCGAGGG CCGCCAGGGC GCGCTGCTCC 10921 GACCAGGTCA GCGGGCGGGT GTCGGTCAGG CCGGGGTCGC GCACGTGCAG CAGGTCGAGT 10981 TCGGTGCGCC ACAGCTCCAG CAGGCGGTGG GAGGCCGGAT GGATCCAGCC GTCCCGCTCG 11041 ATCCGGGCGA AGTAGGGGTC GAGTGCGCGC GCGTCGGTCG TCGGACGGCC GCGGTGGAAC 11101 TCGACGTAGC GCCGGCCGAT CGCGGTCTCG TCCTCGCCGG ACCAGTCGGT GTCCGGTTCC 11161 GTGCGGTCCA GGTGGTTCCA GAACGCCGTG GTCTGGGTCG TCAGCGTGGA CATCCGCGGA 11221 TTCCAGACCA GGGTGGTGGG GCCGAGGGTG GCCGTCGCCT TGAACAGGGC GTCGGCCCAC 11281 AGCAGGCCTT TGACATGGGT GGGGGTCAGC GGCTTGGTGG GGGTGATCGT CACCGGGGCG 11341 ATCACGAACT CCCTCGCGGC GCGGCGGGCG CCGGGATGCG CGCGGTGTGC GCGGCTCGCG 11401 GGTGTGGGCG GGCCGGTCGT CGCGGTTCGT ACGGTGCTCG GGGTGCGCAC GGTGCTCGAG 11461 GTGCGCAGGG TGGTGCTCAT GGATGGCTCC TGTCGATGTC TGCCGCGACC GGGCGGACGA 11521 GGTCGTGCGC GGCGCGCGAC ATGCCGGTCG CGGCGCAGGA CGTGCCGGTT GCGGCGCGCG 11581 CCGCGTGGGC CGTGACGCGC GCCGCGTTCG AATCGACAGC GGTCACGACG TGGTCGGCCG 11641 GGACGGGTCG GACGCCGTCC CGTCGGAGGC CCCGTATTCG GCGTCGAGGA ACTGCATGAG 11701 GTCGCTCGCG CTCGCCGTCT CCAGGTCATC GGTGCCGGAG TTCCTGCCGG CTCCCCCGGC 11761 TCCGGTGCCC GTGCCGGAGA GCCGGTCCAG GAGCGCGGAG AGACGGGTGG CGAGCGCGGC 11821 CCGGGTGTCT GCCGGTGTGA ACGGCGAGAG CACCTTGGCC TCAAGGCGGT CGAAATCCTC 11881 GAGGACCGGA CCGTCGTCGG CGGGGGCTTC GTCGAGGTCC AGTTCCGTGA GCAGCCGCTC 11941 GGCGAGCGAC TCGGGCGTGG GGTGGTCGAA CGCCAGCGTG GCCGGCAGCC GGGCGCCGAC 12001 GAGTTCGCCG AGCCTGTTCC GCAGCTCCAC GGACATCATC GAGTCGAAGC CGAGTTCCCG 12061 CAGCGCCCGG TCCGCCTGCA CCTGTTGCGG GTCGCCCCGC CCGATGAGCT GCGCCACGTG 12121 GGTGCGCACG ACGTCGAGCA GTACGGGCAG CCGCTCGTCC TCGGGCAGCG CGCCCACCCG 12181 GTCCTTCAGC GCGCCCGCGG TCGTGCCCGC GCCGGGGAGG GCGGCGGACG CTGCGTCCCG 12241 GCCGCCGCCC GCCGTGTCCG CGGGCGCGAA TCCGCTGAGC AGCGGGCTGG GGCGAAGGAC 12301 GGTGAACGCG GGCAGGAAGC GCGACCAGGC CACGTCCACC ACGACCGGGT CGGTGTCGCC 12. 361 CGGCACGGCG CGCTCGAGCG CTTCGACGGC GCTGGGCACG GCGAGCGGGA GCAGCCCGCG 12421 CTTGCGCAGT TCCCGGTCCT CGTACTCGGT GACCATGCCG CCGCCCGACC AGGGCCCCCA 12481 GGCGATGGAG ACGGCGGACG CGCCCCGCTC GCGGCGTCGG CGGGCGAGCG CGTCGAGGCA 12541 GGCGTTGCCC GCGGCGTAGG CGGCCTGGTC CGCGGCGCCC CAGATCCCGG CGATGGAGGA 12601 GAACATGACG AACTCGTCGG CGTCGGGCAG CAGTTCGTCG AGCAACAGCG CGCCGTTCAC 12661 CTTCGCCGCC ATGTCGGTCT CGAAAGCCTC GGGGTCGACG TCCGCGATGC GCGCGTACCG 12721 GATCACGCCG GCGGCGTGGA CGACCGTACG CACCGGGTCG TCCTCGGCAT CAAGACGGGA 12781 GAGCAGTTGC GTGACGGCGG TGCGGTCGGT GATGTCGATC GCCGCGGCTT CGGCCCGGAC 12841 GCCCGATTCG CCGAGTTCTG CCAGGAGTTC GGCGGCACCG GGTGACTCGG CGCCGCGGCG 12901 GGAGAGCAGC ACCAGGCGGT CGGCCGTGCC GAGAGCGGCC AGGCGGCGGG CCACGTGCGC 12961 GCCCAGGCCG CCGGTTCCGC CGGTGATCAG GACGGTGCCC TTGGGCTGCC AGGTTCCCGC 13021 GTCGGCCGCG GGTGCGGTGC CGAGCCGGCG GGCCCACAGG CGCCCGTCGC GCACCGCGAG 13081 CTGGTCCTCG GCGCCATCGC CGGTGAGCAC GCCGGCGAGC AGACGTGCGG TGGTCCGCCC 13141 GGCGTTGTCG TCAGCCGTCC CGAGGTCGGC CGTCTCCGGA ACGTCGACGA GTCCGGCCCA 13201 GCGCGAAGGG AGTTCGAGGG CGGCGACCCG GCCGAGCCCC CAGGCGCCCG CCGCGGCCAC 13261 GTCCGTCGCC GGGTCGTCGA CACCGACGCC GACCGCGCCG CTGGTCACGC ACCAGACCGG 13321 CGCGTCGAGC CGCTCGGTCT CCCGCAGCGC GGCGAGCAGT TCGGAGGCGT CGGCGGGGCA 13381 GACGAGCACG CCTTCGACCG GTGTCGAGGC CGATGCATCC CATTCGGTGG ACGTGAGGAC 13441 GTGCTCGAAC AGCTCGGCGA GTCCGGGGCG TGCGGTGCCG AAGAGCAGCC AGGTGCCGGG 13501 CACGCGCTCG GCGATCCGCG CCTCGCTCAG CGGCTCCCAC CGCACGGCGA AGGTCGCGGC 13561 CTGTCCGACG GCCCCTTCGG CCGCCTGGAG CTGTGTGCGC TCCACGGCGC GCAGGATCAG 13621 CGATTCCATG GCGAGGACCG GGGTTCCGGC GGCGTCCGTG ATCCACACCG AGGTCGCCTC 13681 CGGCCGCGGG CGCAGCCGTA CGCGGACGCG CCGCACGTCC GTGGCGAAGA GGCTGACGCC 13741 GCCGAAGGAG AAGGGCAGGC GCACCTCGTC GTCGGTCTCG TAGAAGCTGC GGGTGATGGG 13801 CAGGGCGTGC AACGAGGCGT CCAGCAGAGC CGGGTGGGCG CCGAAGCCGT AGGGCTGGTC 13861 CTCGGGCAGC ACGACCTCCG CGAACAGATC GTCGCCGCGC CGCCACAGCG CCTTGACCGA 13921 CCGGAAGGCG GGGCCGTACT CGTAGCCGCG CTCGGCGAGG TCCGGGTAGA ACGTGTCACC 13981 GGGGATCTGT TCCGCGCCCG CGGGCGGCCA CACCGCGCCG GTCCAGTCGG GCGTGAAGCC 14041 GTCGGTGTCC ACGCGGGAGG CGGTCACGAC GCCGGTGGCG TGCAGCGTCC AGTCCTCGCC 14101 CGGCGTACGC GTGCGGATCA GCAGTTCGCG CTCGCCGCCC TGGTCGGGTG CGACCCACAC 14161 CTGGAGGTCG CGGGCCCGGC CGCCCGGGAA CACCATCGGG GCGCGCAGGA CGAGTTCTTC 14221 GACGCGGCCC GCGCCGACCG CGCGCGCGGC CTCCAGCGCC AGTTCCACGA ATCCGGTGCC 14281 GGGCAGCAGC AGGGTGCCCA TGACGGCGTG GTCGGGCAGC CACGGGTCCG TGCCGGGCGC 14341 GAGGCGGCCG GAGAACAGGA CGCCGCCCCC GCCGGGGAGG TCCGTGCGCT GCGACAGCAT 14401'CGGGTGCGGC AGCGCGTCGG CCCCGGCGCC GGGGCCGGTA CCCGAGGACG GGGCCGTCAG 14461 CCAGTAGTTC TCGTGCTGGA AGGGGTAGGT GGGCAGGTCG AGGTGCGGGA CCGTGGCCCT 14521 GCCGCGCTCG TGTCCGCGGC CGTCGGCGGT ATCGGCGTTG TCGGCGTCCG CCTCGGCATG 14581 GTCGGCGAAC CAGGTGACCG GCGTCCCGGT CACGTGCAGG GTCGCAAGGG CCCTGAGGAA 14641 CGTGTCGGGC TCGGGCTGCC CGGCACGCAG CGTCGGCACG AGCGCCGCGG GAGCGGCGGA 14701 CGCGTCCTCC AGTGTCTCGG CGGCGAGCGG CGCGAGTGTG GGGTGCGCGG TGAGTTCGAG 14761 GTAGCGGGTG GTGCCGAGTC CGTGCAGGGT GGTGACGGCG TCGGCGTGGC GGACGGTGTG 14821 GCGCAGCTGT TCGGTCCAGT GGTCGGCGGT CGTGATCCGG TCCTGCTCGG CGAGCAGTCC 14881 GGTGAGCGTC GAGACGATGG GAATGCGCGG CGCCCGGTAC GTGAGCCCCG CCGCGATCCG 14941 GCGGAACTCG TCCAGGATCT GGTCCTGGTG CGGACTGTGG AAGGCGTGGC TGACGGTCAG 15001 CCGCCTGCTC CTGATCCCCC GCTCCGCCAG CTGTGCGGCG ATGTCCGCGA GGACCTCGGG 15061 ATCACCGGAC AGGACGGTCG ACTCCGGCGC GTTGACCGCC GCGAGCGAGA CCACGTCCTC 15121 ACGCCCCGCC ACGAGACCCC GCGCGGTGGC CTCGCCGGCC TGGAGGGCGA GCATGGTGCC 15181 GGGCGTGGTG ATCTGCTGCA TCAGACGGGC CCGGTGGAAG ACCAGCGTGG CCGCATCAGC 15241 CAAAGAGAGC ATCCCGGCGG CATGCGCCGC GGACAGCTCA CCGATCGAAT GCCCCACCAG 15301 ATGGTCCGGC CGCACACCGA ACGACTCCAG CAGCCGGAAC AACGCGGTGT GCAGAACGAA 15361 CAACGCCGGC TGCGTGAACG CGGTCCGGTT CAGCAGTTCC GCCCCCTCCG ACCCCGGCCC 15421 CGCGAACATG ACCTCACGCA GCGAGCGGCC CAGCAGCGGA TCGAAGACCG CACACGCCTC 15481 ATCGACGGCA GCGGCGAACA CGGGATACGA CGCATACAAC TCCCGCCCCG CACCCGGGCG 15541 TTGACTGCCC TGACCGGAGA ACAGGAACGC AGTCCTGCCC GTGGTGACCT GCCCGCGGAC 15601 GAGGCTCGGG TGCCCGGAGC CCGACGCGAG CGCCGACAAC GCCTCGGTCA GTCCGGCACG 15661 GTCGGCGCCG ATGAGGGCGG CGCGCTGCTC GAACTGCGAC CGGGTCGTCG CGAGCGCGCG 15721 GGCGAGGTGA CCGGTGCCGG TCTGCGGGCG GGCGGCGAGG AACTCCGTCA GGCGGTCGGC 15781 CTGCGCGCGC AGCGCGTCGG GGCTTTTCGC GGAGACCAGC CATACGGCGG GGTCTGCGGA 15841 GTCGGCTTCG GCCTCGTACG CGGTCCGCTC CTTGACCGGA GGCTCTTCGA GGATCAGGTG 15901 CGCGTTGGTA CCACTGATCC CGAACGACGA CACGGCGGCA CGACGCGGCC GCTCCCCCGC 15961 CTCCCAGACG ACCGGCCCGG TCAACAACCT GACCTCACCC GCCTCCCAGT CCACATGCGG 16021 CGAAGGCTCG TCCACATGCA AGGTCCTGGG AAGCACCCCG CCCCGCATCG CCATGACCAT 19441 GGGATACGAG CGAGGGTGTC CCGCTCCGCC ACGGCCTGGG GGCCGAAGTT GCCGGTGAGC 19501 AGGCCGAACA CCCGGTCGCC GACGGCCAGA TCGGACACGC CGGGGCCGGT CTCGGTCACC 19561 ACACCCGCGC CTTCGAGCCC GAGGATGTCG TGGTCCGGCG GTGTGTCGCG GGCGAGGACC 19621 GGGGCGCGGA GATCGACTCC GGCCGCGCGC ACCGCGATCC TCACCTGCCC GGTGCCGAGC 19681 GGGGCGCGCT GGGCCGCCCC GACCTGCTCG GCCACGTCGG CCGGTGCGGG CCACGGGACG 19741 TCGGCGTCCT GCGCGGCGCG AGCGAGTCGC GGTACGAGCA CCTCGCCGTC GCGCACCGCG 19801 AGTTGCGGTT CGCCGGTGCG CAGGGCGGCG GGCAGGGCGG CGTATCCGTC GGACGGGTCG 19861 CCGTCGATGT CGACGAGGGT GAAGCAGCCC GGGTTCTCGG TCTGGGCGCT GCGCACCAGG 19921 CCCCACGCGG TCGCCTGCGG CAGGCCGAGT GCGTCCGGCG CCGTCGCCGC GGCCGTCGCA 19981 TCGTGCGTGA CGAGGACCAG GCGGCCGACC GTGAGGGCGG GCGCGTCGAG CCAGGACGTG 20041 AGGAGGGCGA GCGTGCGGCG GGCGACGGCG TGCGCGCCGG CCGCACCTCC TTCGTGCTCT 20101 CCCGTCCGCT CTCCTTCGTG GGCCGGAAGT TCGGCTCCGT GCGCAGCGAG ATCGGCGAGC 20161 ACGACGGACG GGTGCGGGTC ACCGTTCTCG ATGCCGTGGA CCAGGGCGTC GAGGTTCGGG 20221 TACGTGCGGA TGTCGACCGC CTCGGCCGCC AGCGTGGAGA CCGGATCGGG CACGGGGCTC 20281 CGACCGATGA GCGCCCACTG CTCCGGCGCG TCCGGAGCCG CGGCGCCGAC CGGTGCCGCT 20341 GCCGGTGTGG CGGACCACTC GACGTGGTAC AGCGACCGGG GTCCGCCCGC CTCCGTGCCG 20401 GAGCCGTCGC TCAACCGGTC CAACACCACG GGACGCAGGG ACAGTTCCCC TGCGGTCAGT 20461 ATGAGGGCGC CGGACGGATC GCTCGCGACG ACGCGGACGG TGGTCGGCCC GGTCCGGGTG 20521 AACCGGACCC GCAGCGCGGT GGCGCCCAGC GCGTGCAGGG CGACATCGCC CCAGGAGAAG 20581 GGCAGCAGCG TGCCCGAGGC ATCGGTTCCG GACACCCCGT CCGCGAGCAG TCCGCTGCGC 20641 AGCAGGGCGT GCAGCGAGGC GTCGAGCAGC GCCGGGTGCA CCGAGAACCG GTCCACGTCG 20701 TCCGAGGCGG CGGCATCGTC ACCCAGCTCG ACCTGTGCGA ACACGTCATC GTCCGTGCGC 20761 CACGCGGCGG TCAGCAACCG GAAGTCGGGG CCGTACTCGT AGCCGGTCAG GGCCAGAGCG 20821 GGGTAGAGGT CCGTCAGGTC GACCGGCGCC GCGTCGGCGG GCGGCCACTG CGGTGCACGG 20881 TCCGCGGCGT CGGGCGCCTC GGTGGGCCCC AGGGCTCCGC TCGCGTGCCG GGTCCAGGAG 20941 GACGAGCCGG AGGCGTCGTC GCCGGCGGGC GCCGGGCGCG AATGGACGGC GAAGGCGCGC 21001 AGGCCGGACT CGTCCGCCTC CTGGACGGTC ACCCGGATGT CGACGCCGCG CTCGCCGGGC 21061 AGCACCAGCG GTGCCTGAAG CGCGAGTTCC GCGACAGCCG GGTGCTCCCC CGCGCCGTCC 21121 GACGCGGCAT GCAGCACCAG ATCGAGCAGG GCGGTGCCGG GCAGCAGCGT CGTGCCGTGG 21181 ATCGCATGAT CGGCGAGCCA GGGGTGCGTG AGCGTGCCGA TACGCCCGGT GTGGACGAAG 21241 CCGCCGCCCT CGGGCAGTTC GACGGCCGCC GCGAGCAGCG GATGCGGCGT GCCCGTCAGC 21301 CCGGCCTGCG TGACATCGGC GCGCGGCGCG GGCGGCGTGA GCCAGTAACG CTCGCGCTGG 21361 AAGGCGTACG TGGGCAGTTC GGGCAGAGCA GCGGAGCGAG GGCCGGGGAG TGCCGGCCAG 21421 GCGACGTCGG CACCGCTCGT GTGCAGGGTC GCGAGGGCGC GCAGCAGGGC GTCGTGCTCC 21481 GGCCGTCCGT GGCGCAGCAC CGGGACCAGA GCCGCGGGGC TCTCCTCCAG GGTCTCGGCG 21541 ACCAGCGTGG CCAGCGTCGG AGTGGGAGTG AGTTCGAGGT AGCGGGTGGT GCCGAGCCCG 21601 TGCAGGGTGG TGACGGCATC GGCGTGGCGG ACGGTGCGGC GCAGCTGTTC GGTCCAGTAG 21661 TCGGCGGTCG TGATCCGGTC CTGCTCGGCG AGCAGTCCGG TGAGCGTCGA GACGATGGGA 21721 ATGCGCGGCG CCCGGTACGT GAGCCCCGCC GCGATCCGGC GGAACTCGTC CAGGATCTGG 21781 TCCTGGTGCG GACTGTGGAA GGCGTGACTG ACGGTCAGCC GCCTGCTCCT GATCCCCCGC 21841 TCCGCCAGCT GTGCGGCGAT GTCCGCGAGG ACCTCGGGAT CACCGGACAG GACGGTCGAC 21901 TCCGGCGCGT TGACCGCCGC GAGCGAGACC ACGTCCTCAC GCCCCGCCAC GAGACCCCGC 21961 GCGGTGGCCT CGCCGGCCTG GAGGGCGAGC ATGGTGCCGG GCGTGGTGAT CTGCTGCATC 22021 AGACGGGCCC GGTGGAAGAC CAGCGTGGCC GCATCCGCCA AAGAGAGCAT CCCGGCGGCA 22081 TGCGCCGCGG ACAGCTCACC GATCGAATGC CCCACCAGAT GGTCCGGCCG CACACCGAAC 22141 GACTCCAGCA GCCGGAACAA CGCGGTGTGC AGAACGAACA ACACCGGCTG CGTGAACGCG 22201 GTCCGGTTCA GCAGTTCCGC CCCCTCCGAC CCCGGCCCCG CGAACATGAC CTCACGCAGC 22261 GAGCGGCCCA GCAGCGGATC GAAGACCGCA CACGCCTCAT CGACGGCAGC GGCGAACACG 22321 GGATACGACG CATACAACTC CCGCCCCGCA CCCGGGCGCT GACTGCCCTG ACCGGAGAAC 22381 AGGAACGCAG TCCTGCCCAC CGTGGCCCGA CCACGTACCA CCATGGGATG CCCGGCACCC 22441 GAGGCAAGCG CGGACAGCGC CTCGGCGAGT GCGTCCCGGT CCTGGGCGAC GACCGCCGCC 22501 CGGTGGTCGA AGTGCGTACG GCCGGTGGCC AGGGCCCGAG CGGCCCGGCG GATGCCGACC 22561 TCCGTCCGGG TCCTGGCGAA CTCGGCCAGC CGGCCGGCCT GTTCCCCGAG CGCGTCGGCT 22621 TTCTTCGCGG AGACCAGCCA TACGGCGGGG TCTGCGGAGT CGGCTTCGGC CTCGTACGCG 22681 GTCCGCTCCT TGACCGGAGG CTCTTCGAGG ATCAGGTGCG CGTTGGTACC ACTGATCCCG 22741 AACGACGACA CGGCGGCACG ACGCGGCCGC TCCCCCGCCT CCCAGACGAC CGGCCCGGTC 22801 AACAGCCTGA CCTCACCCGC CTCCCAGTCC ACATGCGGCG AAGGCTCGTC CACATGCAAG 22861 GTCCTGGGAA GCACCCCGCC CCGCATCGCC ATGACCATCT TGATCACACC ACCCACACCC 22921 GCCGCGGCCT GCGTGTGCCC GATGTTCGAC TTCAGCGAAC CGAGCCACAA CGGACGACCC 22981 TCCGACCGAC CCTGTCCATA GGTGGCCAGC AACGCCCGTG CCTCGATCGG GTCACCGAGC 23041 GCCGTTCCCG TCCCATGAGC CTCGACGGCG TCGACATCGG CGGCCTCCAG CCCCGCGTCG 23101 GCCAGGGCCT GACGGATCAC CCGCTGCTGC GACGGGCCGT TCGGCGCGGT CAGACCATTG 23161 CTCGCACCGT CCTGATTGAC CGCCGAGCCG CGAATGACCG CGAGCACACG GTGCCCGTTG 23221 CGCCGCGCGT CACCGAGACG TTCGAGCACC AGCATGCCCA CACCCTCGGC CCATGAGGTG 23281 CCGTCGGCGG CGGCGGAGAA CGACTTGCAC CGTCCGTCGC CCGCGAGGCC GCGCTGCCTC 23341 GCGAATTCGA CGAACATGCC GGGGCTCGCC ATGACGGCGG CGCCGCCTGC GAGCGCCAGT 23401 TCGCATTCGC CGTTACGCAG CGACCGGGCC GCGAGGTGCG CGGCGACGAG CGACGACGAG 23461 CAGGCGGTGT CCACCGTCAT CGCGGGGCCC TCGAACCCGA AGGTGTAGGC GATGCGTCCG 23521 GAGGCCACGC TGACGGTGCT GCCGGTCAGC AGATAGCCGC CGACGCTTCC CGCCGTCTCG 23581 GGGACCGTCT CGTGCAGCCG GGGGCCGTAT TCCATGGCCG TCGCGCCGAC GAACACGCCG 23641 GTGCGGCTTC CGGCCAGGCC GGTCGGGTCG ATGCCGGCCC GCTCCACCGC CTCCCAGGAG 23701 GTCTCCAGCA GGAGACGCTG CTGCGGGTCG ACGGCCAGGG CCTCGCGCGG CGAGATGCCG 23761 AAGAACTGCG CGTCGAACCG GTCGGCCTCG TAGAGGAAAC CGCCCTCGCG CGCGTAGGTC 23821 CGGCCCGGTG CGTCGGGGTC CGGGTCGTAG AGCCCCTCCA GGTCCCAGCC ACGGTTCTCC 23881 GGGAACACGT CGATCGCGTC GGCGCCCTCG GCGACAAGCT GCCACAGCGC TTCGGGGGAA 23941 TCGGCGGCGC CGGGGTAACG GCAGGCCATG CCGACGATCG CGATCGGCTC GTCGGAGACC 24001 GAGGCCGCCG ACGTGGCGTC GCTCTGCGTG GTACCGGCCA GCTCCGCTCC CAGCACTCGT 24061 GCCAGGGCTC GTGGCGTCGG ACTGTCGTAC AACAGGGTTG CCGGCACGCT CAGTTGGAGC 24121 ACAGCGGCCA GCCGCTCGCA CAGGTCCTCC GCGGACTGCG ACTCCAGGCC GAGATCGTTG 24181 AAGGAGCGGG CCAGGTCCAC TTCGCGCGGA TCGGAGTGAC CGAGCACCGC CGCCGCCTCG 24241 TCACGGATGA GGTCCAGCAA CTGCTCGTCA CGCCCGGCAG GAGCGGCCTC GACCAGCCCT 24301 CGCAGCCAGT CGGAGTCCCG CACGGACGCG GGTGCGGAGA CAGCGGACGT CTTCTCCGGG 24361 CGGGCCGTCA CGGCCGATGC GGCCGACGCG GTGGCACGCC GCGCGGCACC CTTCAACTCC 24421 AGACCCAACA CCTGCGCGAG GACCTTCGGC GTCGGGCTCT CGTACAACAG CGTTGCGGGC 24481 AGACGCAGTT GCAGCACGGA ACCCAACCGC TCGACCAACT CCACACCCGA CGCCGACTCC 24541 AGGCCGAGGT CCTTGAACGA GCGGGCGAGA TCCACTTCGC GCGGATCGGA GTGACCGAGC 24601 ACCGCCGCCG CCTCGTCACG GATGAGGTCC AGCAACTGCT CGTCACGCCC GGCAGGAGCG 24661 GCCTCGACCA GCCCTCGCAG CCAGTCGGAG TCCCGCACGG ACGCAGACGC GGAGACAGGG 24721 GGCCCGGAGG CGGGCACAGC GGCGCCAGCG GGAGCAGCAG GGTTCGGCGT CGGAACGGCG 24781 GCAGCGCCCT GGCGTGCCAC GGGCGCGGAC GTCGGCGTGG GCTCGGGCCA ATACCGGCGC 24841 CGGTCGAAGG CGTAGCCGGG CAGTTCGACG CGGCGCGCCG CCGGAAGGCC GTAGAGCGCC 24901 GGCCAGTCGA CGGCAGCGCC CCGCACGTGC GCGGCGGCGA GCGAGGACAG CAGCCGCGGC 24961 CGGCCGCCGT CGCCGCGGCC CAGGGCGGGA ATGCCGACCG CGCCCGCGGC GTCGAGGAGT 25021 TCCAGGATCT CGGGCGGCAG CACGGCGTGC GGGCCGACCT CGATGAAGAC GGTGTGCCCG 25081 TCGTCCATCA GTTCCTCGAC GGCCGGATGG AAGGGCGCCG GCTGCCGGAA GTTGCGGTAC 25141 CAGTGGTCCG CGTCCAGGGC GGCGGTGTCC ACCGGACCGC CGAGGGTCGT CGACTGGAAG 25201 CGCGTCCCGC TCGGCCTGGG CTCGATGCCG CTCAGCTCGT CGAGGAGCGC GTCGCGCACC 25261 GCCTCGGCCT GCTCGCCGTG CGCACGGCCG CGTGCGACGA CGAGAGCCGC GGCGTCCTGG 25321 AGGGTGAGCG CGCCGATGCT GTACGCGGCG GCGATTTCGC CCGCGGCGTG GCCGAGCACG 25381 GCATGGGGCT GGACGCCGAG CGTGCGCCAG GTGTGCGCGA GGGCGGTCGT GACGGCGAAC 25441 AGCACGGGCT GGACGTGGTC GGGGGTGTCC GGCAGGGTCT CCGGACCGGT GAGGTGGTCG 25501 ACGAGGGACC AGCCGGTGAG CGGGTCGAGG GCCGCGGCGG CGGCCTCCAC GTGCTCGCGG 25561 AAGACGGGCA GGGTCGCCAT CAGGTCGCGG ACCGTGCCGG CCCACTGCAC GCCCTGGCCG 25621 GGAAACACGA ACACGGTCTT CGGCCCGGTG CCGGGCGTGG TGCCGGTCCC GGGGTCCGGC 25681 GCGGTGCCGC GCAGCAGGCC GTCCGAGGGG CGGCCCTGCG CGAGGGTGCG CAGCTCGGAG 25741 AGCAGGGCGG ACCGGTCGCC GCCGAACGCG GCGGCGCGGT GCTCGTGATG CGTGCGAATG 25801 GTGGCCAGGC CGCGCGCCAC GGTGGCGGCG TCCAGCTCGG GGTGCTGCTC CAGGTGCGCG 25861 GCGAGGGCCG CGGCCTGACC GCGCAGCGCC GCCTCGCTGC GCGCCGAGAT CAGCCACGGC 25921 GACGCGACGT CCTGCGGGAC GGCAACGGGA ACAGGAACGG TCGCGGGCGG GGCCGCCGAT 25981 GACGTCGCGG GTGACGGCGC CGACGACTCG GCCGGGGCGT CGCAGAGCAC GACGTGGCAG 26041 TTGGTGCCGC CCATGCCGAA CGAGCTGACC CCTGCGACGA TCGGACCGTC CGGGCGCGGC 26101 CACGGTGTCA GCCCGACCTG GACGCGCAGG CTGAGCTCGT CGAAGGCGAT CTTCGGGTTC 26161 GGCGTCACGA AGTTCAGGCT CGGCGGCAGC TTCCGGTGCC GGATCGCCAG AGCCGTCTTG 26221 ACCAGGCCCA CCACGCCCGA AGCACCCTCG AGATGCCCGA TGTTGGTCTT CGCGGAGCCC 26281 ACCAGCAGCG CGTTGTCGGC CACCCGGCCC ACGCCCGCGC CGAAGGCGGT GCCGAGCGCC 26341 GCCGCCTCTA TCGGGTCGCC CACGGCGGTG CCCGTGCCGT GCAGTTCCAC GTACTGCACG 26401 TCGCCGGGGG CCACGTCCGC CTGCCGGCAG GCGGCGCGCA GCAGCTCCGT CTGCGCGGGC 26461 GCGCTGGGCA CCGTCAGTCC GTCGGTGGCG CCGTCGTTGT TGACCGCGCT GCCCCGGATG 26521 ACGCAGTACA CGAAGTCGCC GTCGGCACGG GCCTGTTCCA GCGGCTTGAG TACGACCAGT 26581 GCGCCGCCCT CGCCGCGAAC GTACCCGTTG GCCCGCGCGT CGAAGGTGTG GCAGCGGCCG 26641 TCGGGCGAGA GGG. CGCCGAA GCTCATCGAC GCGGCCATGC CCTCCGGCGC GGCGATCAGG 26701 TTCACGCCGC CCGCCAGCGC CACCCGCGAC TCGCCGCGGC GCAGGCTCTC GCAGGCGAGG 26761 TGCACGGCGA CCAGCGACGA GGCCTGCGCG GCGTCCACGG TCATGCTGGG GCCGCGCAGG 26821 CCCAGCGTGT ACGACACACG GTTCGCGATG AGCCCACGGC CCATGCCGGT CATCGTGTGC 26881 TGGTTGAAGG AGGCGGTTCC GGCCCGGGCG ACGACGCTGC GGTAGTCGTC CCAGATCGCT 26941 CCGACGAACA CTCCGGTGCC GCTGCCGCCG AGCGAGGCGG GGACGATCGC GGCGTCCTCC 27001 AGCGCCTCCC AGCTCAGTTC CAGCATCAGC CGCTGCTGCG GGTCCATCGC CCGTGCCTCG 27061 TGCGGCGAGA TACCGAAGAA CCCGGGGTCG AAGGTGTCGA TCCGGTCCAG GTAGGCGCCG 27121 TACCGGGCCG CACCGGTCGG CGTGCCGGCC GCGTCGGGCC AGCGGTCGGC GGGCGTCTCG 27181 CCCACCGCGT CCACGCCCTC GCTCAGCAGC CGCCAGAAAG TCGCAGGGTC CGGAGCCGCC 27241 GGCAGCCGAC AGGCCATACC GACGACGGCG ATGGGCATGA ACCCTTCAGA CATGAAGCGC 27301 ACCCTCGACG GATATGGAGG AGTCGCCGCG GTCCGCGGCG GGCCGGCCGA GCGTCTCCAA 27361 CGACGCAAGG AAGTCGAGCT CTTCCAGCGG ACGATCGGTG AAGAAGCGCG CGATCGTCTC 27421 GGCCCACTCG GCGGCACGCC CGAGGAACAC GAGATGGTCG GCCTCCGGGA TGACGGCGAA 27481 CAGCGCACCC TCGATCTCGG CGGCCAGGGC GCGCGCGCCC TCCATGGTGG CGAAGGTGTC 27541 GTGCTCCCCG ACCAGGCACA GGCTGGGTAC GCCGCTGATG CCGCCGGGCA GGACGGCATC 27601 ATCCTGGAGG AGGAGGTCCG AGACGTTGAG GTAGCCGGGA AGATCGGCCT CCTCGATCGT 27661 GCTGAAGCGA CCGTTGAGGG CCCTGCGGAC GATTTCACGG TTGCGCACCG TGACCGCGGG 27721 GTCCAGGCAC ATGAGCAGGT CCAGCAGACC TTCGGCGAAC TCGGCGAAGC GGCCCGCCGT 27781 GAGGATCGGA TACATCTGCG TCATCCGCTC CCGGTTGCGC GGCGGCCAGT CGGCGGCCCC 27841 GGCCAGGACG AGACGGGAGA CCCGCGACGG GCTCTGCTGG GCGTAGCGGT AGGCCGCCGG 27901 GAATCCGTTG CTGATCCCCA GCAGGTTCAC ACGCGGCAGC CCCAGCTCGT CGATGAGGTG 27961 GGCGAGCGCT TCGGTCTGGA CGTCATAGCC GCCTTCGGCG GGCACGGGGT CCGCCGTGCG 28021 CGAGCCCGGC AGATCCACAC AGACGATGGT GGCCGTGTCC TGCCAGTACT TGTCGAACCG 28081 CCGGTAGCTG AACTTGTCCT GGTACGCACC GGACAGCACA ACAAAAGGCT CGGTGACCGG 28141 TGCTTCGCAT TCGACCATCC GGTAGCTGAA CGCAAGACCT TTGTAATCAA GCTCTTCGAC 28201 CTGCTCGCGC GGATTTCCGG CGCCCACGGA TCAGCTCCTC GAATTTCGGG CGGATGTGCA 28261 CGGACGGACA ACGGATACAC GTCCGTGCAT GAGCCCGATC TTTGTCGCCG GCCAGGGCAC 28321 CGACAACCCC TATTTCCCCC CTTAGCCGAA CCGGCTTGCC GGATCGGAGC TGGTCGGAGC 28381 TGCGAGATGA GTCCCGATAC GAATCCTCTC CAGATTCACC CCCTGGCACA CGACCCATCG 28441 ACATGTATTC TCGGCGTATG CCCATCATCG AACTTGCCGA ATACGGGCCA GACTTTCTCG 28501 CAGATCCTTA CCCGTATTAC GCGAAACTCC GCGAGGAGGG ACCCGTGCAC GAGGTACGGG 28561 CCCCGGACGG CTATCGATTC TGGCTGATCG TCGGATATGC CGAGGGGCGC GCCGCCCTGA 28621 CCGATTCGCG GCTGGTCAAG GCACGCGACA CGATGGCGAC GTCCGAGGCG TCGCCACTGG 28681 GCAAGCATGT GCTGATCGCC GACCCGCCGG ACCACACCCG GCTGCGCAAG CTGATCTCCC ; 28741 GGGAGTTCAC CGTGCGGCGG GTGGACAACC TGCGCCCGCG CATCCAGGAA CTCACCGACG 28801 ACTTGCTGGA CGTCATGCTG CCCGCGGGGC GGGCCGACCT GGTGGAGGCG CTGGCCCGGC 28861 CGCTGCCGAT CGCCGTGCTG TGCGAACTGC TCGGAGTGCC GAACGCCGAC CGGGACGAGT 28921 TCCACTCCTG GGCCAAGGGC ATCCTCGCGC CGCAGAACCC GACCGAGACG CACACGGCCG 28981 TCAAGGCCTT GATGAGTTAT CTCGACGACC TGATCGAGGA CAAGCGGCAC GGAGAGCCCA 29041 CCGGTGACCT GCTGTCGGGT CTCATACGCA CCAGCATCGA GAACGGCGAC CGCCTCTCCT 29101 CGGAGGAAGT GCGCTCCACG GCCTTCCTCC TGATGATCGC CGGACACGAG ACGACGGCGA 29161 ACCTCATCTC CAACGGAACG CGGGCGCTGC TCACGCACCG GGACCAACTG GACCTGCTGC 29221 GCTCCGACAT GGACCTCCTC GACGGCGCCG TCGAGGAGAT GCTCCGCTAC GACGGCTCGC 29281 TGGAGAGCAC GACCAAGCGG TTCACCGGTG TGCCGGTCCA GATCGGCGAC ACGGTCATCC 29341 CGCCGGGCGA GACGGTGCTG GTCAGCCTCG CGTCGGCGGA CCGCGACCCG GCGAACTTCG 29401 ACGACCCCGA CCGCTTCGAC ATCCGTCGCG GCACCCCGGC CGGCGTCGGC CACCTCGCGT 29461 TCGGGCACGG GATCCACTAC TGCCTGGGAG CCTCACTCGC CCGCGCAGAG GGCCGGATCG 29521 CGTTCCGCGC GCTGCTGGAG CGCTGCCCCG ACCTCGAACT CGACCCCGAG GCACCGCCGT 29581 TCGAGTGGAT GCCGGGCGTT CTCGTCCGCG GCGTGCAGCG GTTGTCGCTG CGCTGGTAGG 36421 ACTCTTCTCC GGCCATGCCG CCGCCGCTCC AGGGCCCCCA GGCCAGCGTG GTGGCGGCGG 36481 CGCGGCGGGC GCGGCGGCGC TCGACCAGCG CGTCCAGGAA GGCGTTCCCG GCCGCGTACG 36541 CACCGCCGCG CGTGCTGCCC CAGGTCCCCG CGATGGACGA GTAGACGACG AACGCGGCGA 36601 GTCCGTCGCC CAGCACCTCG TCCAGGATGA CGGCACCGGT GACCTTCGCG TCGACGACGG 36661 CGGCGAATTC GGTCGCGTCG AGATCAGCCA GCGGATGTTC GGCCGCCACG CCCGCCGTGT 36721 GCACGACCGC ACCGACGGGG GCACCGCGCC CGGCCAGGTC GGCGGCGAGC GCCGCGACCT 36781 CGTCGCGGCT GGTCACGTCG CAGGACACCA GGTCCACCGT GGCACCGTGG GCGGCCAGTT 36841'CGGCCCGCAG GTCCGCGGCG CCGGGGGCGT CGGGCCCCTG GCGGCTGGCG AGGACGAGGT 36901 GCGGGGCACC CTGCTCGGCG AGCCGACGCG CCGTGTGCGC GCCGAGGGCG CCGGTGCCTC 36961 CCGTGATGAG GACCGAGCCG TGCGACCACC AGGGTTCGCG CGCGGCGGGC GGCTGCGGAT 37021 CACCGGTCCG CCCGGTGGCG TCGGCCCCCT CCGGGGCGAC GAGGGATTCC GGGGCGACGG 37081. GGCGTACGGG CTCGGGTGCG CCGTCCGGGC CTGCGGGCCG CAGGCGGCGC ACCCGTGCTC 37141 CGTCGGCACG CAGCGCGACC TGGTCCTCAC CACTGGATCC GGCCAGCAGC GCCGCCAGGC 37201 CGAAGGAGGC CTCGGCCGTC GCGGCGAGGG CGTGCCCGTC TGCCGCGGAC AGGTCGGGCG 37261 CGGGCAGGTC GACCAGGCCG CCCCACAGGG TGGGGTGTTC GAGGGCCGCG ACCCGTCCGA 37321 GGCCCCAGAC CTGGGCCTGC CACGGGTCGG GAGCGTCGTC GGACGCCGTC GCGCGGACCG 37381 CTCCGCGGGT GAGCGTCCAC AACCGGGTCG CGCTCCAGCC CGTGTCGAGC AGCGCTTGGA 37441 GAAGGCACAC GGAGGCCCAG GCGCCGGAGC CGACGCCGCG CGGCCCGGTG TGCTCGCGGC 37501 CGGACAGGGC GAGCAGCGAG ACCACTCCGG CGGGAGTGTC GTCGAGCCCG TTCAGCAGCT 37561 TGGCGATGGT CTGGCGGTCG ATGTCCTCGG GCGCGAGGGA CAGCGACTTC ACCTCGGCGC 37621 CGGCATCGGT CAGCACCCGA CGCACCTCGC CGTGCAGCCC GTCGTGCAGC CCGTCGTTGT 37681 CGAGCAGGTG GCCCGCGCGC AGGTCGCCTT CGGGTACGAC GATCAGCCAG GTGCCGTGCA 37741 GGGTGGCGGG CCCCTCGGGG GCGTGCTGCG CGGTCGGCCG CTCCCAGGCG ACGCGATAGC 37801 GCCATCCGTC CGTCTCGGAG GCCTCGATGT GCGTCTGGTG CCAGTCGCCG AGCGCGGGCA 37861 GGACGGTGTG CAGCGGAGCG TCGGGGTCCA CGCCGAGGTC GCTCGCCAGC CGCTGGAGGT 37921 CCTGTTCCTG GACGACCTTC CAGAACGCGC CGTCGCTCCC GGCCGTCCGG GATGCCGCCG 37981 ATCCGGGCCG GACGGAGGCG CCCTTGAGCC AGTGGTGTTC GTGCTGGAAG GCGTAGGTGG 38041 GGAGTTCACG GGCGAGGTCG TCGGCCCGGC CGAGAGCGGT CCAATCGACC TGGTGACCAC 38101 GCGCGTGCAC CCGGGCCAGC ATGCCGAGGA ACGCCCGGGT GTCCGTGGAA CGCCGGCTCA 38161 GCGTGGGCAC GAACGCCACG TCCCGCGCCG CCGAGTCCCG CTCGGCGGAC GCGGCGCGCA 38221 CGCGCTCGCC CAGCGCCGTC AGGACGGGGT CGGGCCCGAG TTCGACGACG GTCGCGACGC 38281 CCTGGGCCAG GACCGCGCCG ACTCCGTCCC CGAACCGCAC CGCTTCGCGC ACGTGCCGCA 38341 CCCAGTACTC CGGCGAGCAC AGCTCCTCGG CGTCCGCGAT CGTGCCGGTC ACGTTGGACA 38401 CGACCGGAAT CGACGGGGCA CGGAACTCCA CCTGCGCCAG CACGTCCGCG AACTCGGCGA 38461 GCATCGGCTC CATCAACGGC GAATGGAACG CGTGGCTGAC CGCCAACGCC CGTGTGCGCC 38521 GCCCCCGTCC GGCAAAGATG TCCGCGATCT GGTCCACCGC GGCGTCCTGA CCGGACACGA 38581 CCACAGCCCC TGGAGCGTTC ACGGCTGCCA GCGACACCAT GCCCCCGGCA GCCGCCACAT 38641 CCGCGACGAG CGGCGCGACC TCCTCCTCGG TGGCCTCCAC CGCCACCATC CGCCCACCCG 38701 ACGGCAACGA ACCCATCAAC CGGGCCCGGG CCACCACCAC CCGCACCGCA TCCGCCAACG 38761 ACCACACACC CGCCACATAC GCGGCGGACA ACTCCCCCAG CGAATGCCCG ATCAACACAT 38821 CCGCACGCAC ACCGAAAGAC TCCGCCAGCC GATACAACGC CACCTCGACC GCGAACAACG 38881 CAGGCTGAGC AACCCCCGTA TCCTCCAAAA CCCCCGCATC ATCACCGAAG ACCACCCCAA 38941 GCAGCTCTGC TCCCGTCTGC GCCTCGACCT CCGCACACAC CTCGTCCAAC GCAGCCGCGA 39001 AGACCGGGAA CCGCCCATAC AACTCACGCC CCATCCCCGG ACGCTGCGAG CCCTGACCCG 39061 AGAACGCCAC ACCCACACCA CCAGCGACAC GACGCTCAAA CACCACACCA CCGGCAGCGG 39121 AACCGTCACC CCGCGCAACC CCACCCACAC CGGCCAACAA CTCGTCCAAC GACCCACCAC 39181 TGACCACAGC ACTGTGATCG AACACCGAAC GCGACGACAC CAACGCCAGA CCCACACCCC 39241 CCACATCCAG CGCACCACCC CCGCGTCCCG CCACGAACGC CGCAAGCCGC GCCGCCTGAG 39301 CCCGCACCGC ACCCTCAGTA CGACCCGACA CAACCCACGG CAACTCCCCA GCAACCACCA 39361 GCGCTTGAGT GGACTCCACC GGAACCTGAG CGGACCCCAC CGGAGCTTCA GTGGATTCCA 39421 CGGGCTCGTG CTCCAGGATC ACGTGCGCGT TCGTCCCGCT GATACCGAAC GACGACACAC 39481 CCGCCCGCCG CGCACGACCC GTCTGCGGCC ACTGCCGAGC CCGCGTCAAC AACTCCACCG 39541 CACCCGCAGA CCAATCCACA TGCGGCGACG GCTGCGACAC ATGCAACGTC CGCGGCAACA 39601 CCCCGTGCCG CATCGCCATC ACCATCTTGA TCACGCCACC GACACCGGCA GCCGCCTGCG 39661 TATGACCGAT GTTCGACTTC AACGACCCCA GCCACAACGG ACGGCCCTCC GCCCGGCCCT 39721 GCCCGTACGT CGCGATCAAC GCCTGCGCCT CGATCGGATC ACCCAGCCTC GTCCCCGTCC . 39781 CGTGCGCCTC CATCACATCC ACGTCCGACG TCGACAACCC CGCACCCGCC AACGCCCGCA 39841 CGATCACCCG CTGCTGCGAC GGACCGTTCG GCGCCGTCAA CCCGTTCGAC GCACCGTCCT 39901 GGTTCACCGC ACTGCCCCGC ACCACCGCCA ACACCTCGTG CCCGTTGCGC CGCGCGTCCG 39961 ACAAACGCTC CAGCACCACC ACACCCACAC CCTCGGACCA GCCCGTCCCC TCCGCATCCG 40021 CGGAAAAAGA ACGACACCGG CCGTCCGCCG ACAGACCACC CTGACGACCG AACTCCACGA 40081 ACGCGTACGG CGTCGCCATC ACCGTCACAC CACCCGCGAG CGCCAACGAA CACTCCCCCG 40141 CACGCAACGA CTGCACCGCC AGATGCAACG CCACCAACGA CGACGAACAC GCCGTGTCCA 40201 CCGTCACCGC AGGACCCTCG AACCCGAACG AATACGAGAC CCGGCCCGAG ATGACGGAGC 40261 TGGCCGATCC CGTTCCGCCG ACGCCTTCGG GCGCGTCGAC GATCTCCGTG CCGACCAGGC 40321 CGTAGCCCTG GACGGCGCCG CCCATGAAGA CGCCAACCGG CTTGCCGCGC AACGAGTCGG 40381 CGCTGATGCC GGACCGCTCC ACCGCCTCCC AGCAGGTCTC CAGGGCGATG CGCTGCTGGG 40441 GGTCCATGGC GGCGGCATCG CGCGGCGAGA TGCCGAAGAA GCCCGCGTCG AACTCCGCGG 40501 CGTCGTGCAG GAACCCGCCG CCCGCGGCAG GCAGTCGTCC GAGGTCCCAG CCGCGGTCGG 40561 CCGGGAACGG CGAGATCGCG TCCCGCCCCT CGGCCACCAG TCGCCACAGG TCCTCGGGCG 40621 AGGCAACCCC GCCCGGATAC TTGCAGGCCA TGCCGACGAG CGCGATCGGC TCGTTCCCGG 40681 CTGCCTCCAG TTCCCGCAGC CGGCCGCGGG TACGCAGCAG ATCGCCGGTC AGTTCCTTGA 40741 C. GTAGTGGCG AAGCTTGTCT TCGTTGGTGG ACACGGTGCG CCAGCTCCTT GTTCGTGCTG 40801 AGGTTTGCGA ACGCCGGCGT CAGGAGATGC CGAATTCCTT CTCGATCAGA TCGAAGAGCT 40861 GATCGTCGGT TGCCGAGTCG AGTTGCTCGG CAGCTGTTTC CGCCGCGCCG GACGCGGCGG 40921 TGGGCTCGTC GTCGGCGTTC TGGAACCTGG TCAACAGGTT GGAAAGCCGC AGGGTGATAC 40981 GGCCGCGCGC GGCCGGGTCG GACCCGGCGT CGAGCGCGGC GAGGGCCGCT TCGAGCCGGT 41041 CCAGCTCACC GAGGACCGCC GACGCGCCCG ACGCGCCGCC GACCCGCTCC GCGAGGGCGC 41101 TCGCGACGAC CTGTGCCAGG GCCGCGGGCG TGGGGTGGTC GAAGACGAGC GTGGCGGGCA 41161 GCTTGAGGCC GGTGAGCTTT TCGAGCCGCT GCCGCAGACC GACTGCAGCC AGCGAGTCGA 41221 ACCCGAGTTC CTGGAAAGGG CGTTCCGGCT CGATCGTGCC GCCCGAGGCG TGCCCGAGTT 41281 CCGCGGCGGC CTGCGTGCAG ACGGTCTCCA CCAGCACCCG TCGGCGTTCC CCGCCGGACA 41341 GCGCGGTCCA GCGGGCGACG AAGGGGGTGG CCCCTGCGGT ACCGGCGTCG CTGTCGCCCG 41401 GGCCGGACAC GTCGTCCGCA CCGGCCGTGC CGGCCGCCGT CCCGTCCGCG CCGACGCCGC 41461 CGCGTTCCGT TTCCACGGTG CGGAGCGGGT CGAACAGGGG GCTCGGACGG TTGACGGTGA 41521 AGATGTCGGC CAGGCGTGAC CAGTCGATGT CGGCGAGGAC GACGGTGCCG TAGTCCGCCC 41581 TCACGACGCG GCCGAACGCG GCGACGGCCT CCTCCGGGTC GAGCGCGCTG ACGCCGCGGG 41641 CCTGCATCTC CCGTGTGAGG CGTTCGTCGG CCATGCCGCC CCCGCCCCAG GGGCCCCAGG 41701 CGAGGGCGGT GCCGGGACGT CCCTGGGCGC GCCGCCGCTC GATCAGCGCG TCGAGATGCG 41761 CGTTTCCTGC GGCGTAGGCA CCGGCACGAG CGCTGCCCCA GACCCCGGCG ATGGAGGAGT 41821 AGACGACGAA CGCGGCGAGT CCGTCGCCCA AGACCTCATC GAGGACCTGC GCGCCGACGA 41881 CCTTCGCGCG TACGACGGCG GCGTAGCCGT CCTCGTCGAG CTCCGCGAGC GGCAGTTCCG 41941 AGGCGACGCC CGCGGTGTGG ACCACGGTGC TGACCGGCGT TCCCGCGTCG GCAAGCCTGT 42001 CCCGGAGGGC GGCGAGGGCC ACGGCGTCGG TGACGTCGCA GGACTCGACG ACGACGTCCG 42061 CACCCCGCTC CTCGAGTTCC GTGCGCAGCG CGGCCACCGC GGGTGCGGCA GGGCCCTGAC 42121 GGCTGGTGAG GACGAGGGTC CGCGCGCCGT TCCGGGCGAG CCAGCGCGCC GTCTGCGCGC 42. 181 CGAGGGCGCC GGTGCCGCCG GTGATCAGGA CGGAGCCGTC GGTCCAGGGC GCCGTGGAGC 42241 CGGTCTCCGG CTCCGGCACG CTCGTGGGTA CCGTGCCGCG GATCAGCCGA CGACCGAGCA 42301 ACAGCTCGCC TCGCAGGGCG ATTTGGTCGT CACCGGTGGT GTTGGCAAGG GCAGCGGCAA 42361 GGCCGGTGAG CTGCGCTGCC GGACTCGTAC CGGAGACGTC GATCAGACCG CCCCAGAGGG 42421 TGGGGTGTTC GAGGGCCGCG ACCCGTCCGA GGCCCCAGAC CTGGGCCTGC CACGGGTCCG 42481 GCGCCGCGTC GTCGGCCTGC GCACACACCG CGTCGCACGT GAGGGCCCAT ACGCGGGTGT 42541 CGACTCCCGC GTCCTGCACG GTGTGCAGGA GATCGAGCAC GGCCAGGGCG CCGGTCGCGA 42601 TGCCGCGCTC CCGGTCGCGG TCGCGCTGGG CGCCGACCGC GGGCAGGCAG AGCACGCCGC 42661 GGGGCGCCAC TGCGGCCAGC CGTGCGCGCA GCTCGGCCGT CTCGCACCGC TCCACGCGCG 42721 CCCCCGCGTC GACGAGCGCC TTTTCGACCG CAATTACGAG GTCCGGTTCG ACGGGCTCGC 42781 CGGGCACCGC GACCAGCCAC GTGCCGTCGA GCGGCACCGC GTCCGTCGGC GACGGAAGCT 42841 CCGTCCAGGA GACGCGGTAG CGCAGGGCGT CGGCCGCTGC GAGCCGGGCC TGTTCCCTGG 42901 ACCAGGTCTG GAGCGCGGGC AGCACCGCGG TGAGCGGCGC GTCCGGCGCG AGGCCGAGGG 42961 TATGGGCGAG GCCGTCGACG TCCTGCTGCG CGACCGCGTT CAGGAGTACG GCCTGCTCCG 43021 GCACGTGCTC GACGACGCCG GGCTCGGGCG CGGGGGCGTC GAGCCAGTAA CGCTGATGCT 43081 GGAAGGCGTA GGTGGGGAGT TCACGGGCGA GGTCGTTCGC CCGGCCGAGA GCGGTCCAGT 43141 CGACCTGGTG ACCACGCGCG TGCACCCGGG CCAGAGCCGT CAGGAAACCG TTCACATCAC 43201 CCGTCCGCCG CCCCAGGGTG GGCAGGAACA CGGCACCGTT CTCGACGACT CCCGGATGCG 43261 AGGCACCCAT CGCCGTCAAC ACCGCCTCGG GCCCCAGCTC GACAACGGTC GCGACGCCCT 43321 GCGCCAGGAC CGCACCGACC CCGTCCCCGA ACCGCACCGC TTCCCGCACG TGCCGCACCC 43381 AGTACTCCGG CGAGCACAAC TCCGCAGCGG ATGCGACCTC ACCCGTCACG TTCGACACGA '43441 CCGGAATCGA CGGGGCACGG AACTCCACCC GCGCCAGCAC CTGCGCGAAC CCGGCGAGCA 43501 TCGGCTCCAT CAACGGCGAA TGGAACGCGT GCGAGACACG AAGACGAGTC GCACGACGCC 43561 CTCCCCCGCG CGCCCGCTCC ACCACCGCCT CAACGGCACC CTCCACACCC GAAACAACCA '43621 CCGCCGCCGG CCCGTTGACA GCAGCGATCA CCGCACCGTC CACCAGCCAG CCCGACACCT 43681 CCTCCTCGGT GGCCTCCACC GCCACCATCC GCCCACCCGA CGGCAACGAA CCCATCAACC 43741 GGCCCCGGGC CACCACCACC CGCACCGCAT CCGCCAACGA CCACACACCC GCCACATACG 43801 CGGCGGACAA CTCCCCCAGC GAATGCCCGA TCAACACATC CGCACGCACA CCGAAAGACT 43861 CCGCCAGCCG ATACAACGCC ACCTCGACCG CGAACAACGC AGGCTGAGCA ACCCCCGTAT 43921 CCTCCAAAAC CCCCGCATCA TCACCGAAGA CCACCGAAAG CAGCTCTGCT CCCGTCTGCG 43981 CCTCGACCTC CGCACACACC TCGTCCAACG CAGCCGCGAA GACCGGGAAC CGCCCATACA 44041 ACTCACGCCC CATCCCCGGA CGCTGCGAGC CCTGACCCGA GAACGCCACA CCCACACCAC 44101 CCGCGACACG ACGCTCAAGC ACCACACCAC CGGCAGCGGA ACCGTCACCC CGCGCAACCC 44161 CACCCACACC GGCCAACAAC TCGTCCAACG ACCCACCACT GACCACAGCA CTGTGATCGA 44221 ACACCGACCG CGACGACACC AGCGCCAGAC CCACACCCCC CACATCCAGC GCCCCCGCAC 44281 CGCCCCCGCG TCCCGCCACG AACGCCGCAA GCCGCGCCGC CTGAGCCCGC ACCGCACCCT 44341 CAGTACGACC CGACACAACC CACGGCAACT CCCCAGCAAC CAACGGAGCT TCAGTGGACT 44401 CCACCCGAGC CTGCACAGAC CCCACCGGAA CCTGAGCGGA CCCCACCGGA GCTTCAGTGG 44461 ATTCCACGGG CTCGTGCTCC AGGATCACGT GCGCGTTCGT CCCGCTGATA CCGAACGACG 44521 ACACCCCCGC CCGCCGCGCA CGACCCGTCT CCGGCCACTG CCGAGCCCGC GTCAACAACT 44581 CCACCGCACC CGCAGACCAA TCCACATGCG GCGACGGCTG CGACACATGC AACGTCCGCG 44641 GCAACACCCC GTGCCGCATC GCCATCACCA TCTTGATCAC ACCACCCACA CCGGCAGCCG 44701 CCTGCGTATG ACCGATGTTC GACTTCAACG ATCCCAGCCA CAACGGACGG CCCTCCGCCC 44761 GGCCCTGCCC GTACGTCGCG ATCAACGCCT GCGCCTCGAT CGGATCACCC AGCCTCGTCC 44821 CCGTCCCGTG CGCCTCGACC GCGTCCACAT CCGCCACGGA AAGTCCCGCG CCCGCCAGTG 44881 CCTGGCGAAT CACGCGCTGC TGGGACGGGC CGTTCGGCGC CGTGAGCCCG TTCGACGCAC 44941 CGTCCTGGTT CACCGCACTA CCCCGCACCA CCGCCAACAC CTCGTGCCCG TTGCGCCGCG 45001 CGTCCGACAA ACGCTCCAGC ACCACCACAC CCACACCCTC GGACCAACCG GTGCCCGATG 45061 CGTCGGCGGA GAACGAACGG CACCGGCCGT CGACGGCCAG TCCGCCGTGG CGTCCGAACT 45121 CCACGAACGC GTACGGCGTC GCCATCACCG TCACGCCACC GGCGAGCGCC ATCGAGCACT 45181 CCCCCGCACG CAACGACTGT GCCGCCAGAT GCATCGCGAC CAGCGACGAC GAACACGCCG 45241 TGTCCACCGT CACCGCAGGA CCCTCGAACC CGAACGAGTA CGAGACCCGT CCCGACGCGA 45301 TGCTGCCGGA GCTTCCGTTG CTGATGTAGC CCTCGTACCC CTGCGGCGAG CGGTTCAGGT 45361 GGCGGGCGCC GTAGTCGTTG TACATGACGC CCATGAACAC GCCGGTGCGG CTGCCGGTGA 45421 GCGTCTCCGG CCGGGTGCCG GCCGACTCCA GGGCCTCCCA GGAGGTCTCC AGGAGCAGTC 45481 GCTGCTGCGG GTCGGTCGCG GTCGCCTCGC GTGGCGAGAT GCCGAAGAAC TCCGCGTCGA 45541 ACTGGGCCGC GTCGTGCAGG AACCCGCCCT CACGGGTGTA CGTCTTGCCG GGCTGCTGCG 45601 GGTCCGGGTC GTAGATGCCG TCGAGGTCCC AGCCGCGGTC GGCCGGGAAC GGCGAGATGG 45661 CGTCCCGCCC CTCGGCCACC AGCCGCCACA GGTCCTCGGG CGAGGCAACC CCGCCCGGAT 45721 ACTTGCAGGC CATGCCGACG ATGACGATCG GGTCGTCGCC CGCATCCTGC GGATGGCTCG 45781 CCGACGCCCG CGCCGCGGGC TCGGCGACCG CGAGCGCGGT GGACCCGGCC GGCTCCCCGA 45841 GGACTCCGCG AGCGAGTTCG TCGTACAGGA ATTCCGCGAC CGCGAGCGGA GTCGGGTGGT 45901 CGAAGACCAG CGTCGCCGGA AGGCGCACGC CGGTGGCGGC GCCGAGCTGG TTGCGCAGTT 45961 CGACGGCGGT. GAGGGAGTCG AGGCCGAGCC GGTTGAACGG CTGGGCGCGG TCGACGGCCT 46021 CGCGGTCGGC GTGTCCGAGC ACGTACGCGA CCTTCTCCGC GACGAGGCCG CCGAGGATCT 46081 GAAGGCGCTC CTCGCGGTCG GCCACGCGAA GCTCGGCGAG CAGCGGCGCG CTCGCGCTGC 46141 CGGCGGTCGC CGCGGTGCCG CCGGCCACAC GGGAAGAGGG TCGGGGCCTG GTGCTGACGA 46201 CCGCTTGGAA GACGGCGGGC AGCGATCCGG CCGCGGCCTG CTCGTCGAGG ACGGGGGCGT 46261 TCAGCCGGGC GGGCACGAGC AGGCCGTCGG CGTGCGTTCC GACGGTCTCC GGGCCGGCCT 46321 CCGAAGCGCC GAAGGCTGCG CCGGCTGCTG CGAGAGCGGC GTCGAACAGC GTGACGCCCT 46381 GTTCGCGGCT GATCTCCAGG AGGCCGGTGC GCTTCAGCCG GGCCACGTTG GCCCGGTCGA 46441 GCTCCGCGGT CATCCCGCCC TCGGTGCTCC ACAGGCCCCA CGCGAGCGAG ACGCCCGGCA 46501 GCCCGAGCGC CCGGCGCCGA CGCGCCAGTG CGTCGAGGAA GGCGTTGGCT GCGGCGTAGT 46561 TGGCCTGTCC GGCCCCGCCG AAGACACCGG CGACCGAGGA GAACAGGACG AACGCGGAGA 46621 GCGGGGCCCG CGACGTCAGC TCGTGGAGGT GCAGCGCGGC GTCGGCCTTC GCGCGCAGCA 46681 CCTTCGTCAG CTGCGCGGGA GTGAGGGATT CGAGGAGCCC GTCGTCGAGT ACGCCCGCGG 46741 TGTGGACCAC GCCGGTGAGC GGATGGTCGC CCGGTACGCC GGCGAGCAGT TCGGCGACGG 46801 CGGAGCGGTC GGACATGTCG CAGGCGGCGA GCGTCACGTC GGCCCCGAGT GCTTCCAGTT 46861 CCGCGATGAG CTCGGCGGCG CCCGGGGCGT CCGGACCACG GCGGCTGGTC AGCAGCAGGT 46921 GCCGCACTCC GTGCACGGTG ACGAGGTGGC GGGCGAAGAG CGAGCCGAGG TCGCCGGTGC 46981 CACCGGTGAT CAGGACCGTT CCCTCGCCGG AGAAGGCGGG GGCGTCGGCG GAGCCATCGG 47041 CGTCGGTCGT CTCGGGGGCC GCGATCGCGC GGAGTCGCGG GACGGAGGGG ACCCCGGCAC 47101 GGAGCGCGAT CTGCGGCTCC GCGACGACGC CGGTGCCCGT GCCGATGTCT GTGCCGTTGC 47161 CGGTGCCGAG CAGCGTCGGC AGGGCGCGCA GCGAGGCCTC CTCCCCGTCG GTGTCGATCA 47221 GGCGGAACCG GCCCGGGTGC TCAAGCTGGG CGCTGCGTAT CAGGCCGCCC ACGGCGGCGG 47281 ACGCCAGGTC GACGGCTGCG GCCTCGGCCG CGTCGACCGC GAGCGCACCG CGGGTGAGCA 47341 GCGTCGCGGT GACGGATGCG AAGCGCGGCT GCTTCAGCCA GTCCTGGAGG AGATGGAGGA 47401 CCGTCTGCGT CCGCTGATGC GTGGCCGCGG CGATGTCGCC GTCCACTCCG CCCAGGGCGG 47461 GCAGCGCTAT CAGGACGTCA CGAGGCGCCT CGGCCGGATC GGCGTCGATG GCGGCGGCGA 47521 GGGCTGCGAG GTCGGCGTGG CGGCGCGCCG GAACGGATTC GACGGACCAG TCGGCGCCGA 47581 GGTCGAGGAG CGCCAATCCG GTCCCGGACG CGGACACGGC CGCAGACACA GCGGAACCGG 47641 CGCTTTCCAG CGGCGACCAC GCGATGCCGT ACAGCGATTC GACGCTCGAC ACGCGGGCCG 47701 AGCGAAGCTG CTCCAGCGTC ACGGGACGCA GCGCCAGCGA CCGCACGGTG GCGAGTGCCG 47761 CCCCCGCCTC GTCCAGCAGC TCCACGGAGG TCGCGCCCTC ACCGAGGCGG CGTATGCGGA 47821 CCCGGGCCGA ACTCACGGCG GTGGCGTGCA GCGTGACGCC GCTCCAGGAG AACGGCAGAT 47881 GGCACTCGTC CTGCCCGGAC AGCAGGTCGG GCAGCGCGAG CGAGTGCAGG GCGGTGTCCA 47941 GGAGCGCCGG ATGGATGCTG AAACGCCCGG CGTCGTCCGT CTGCCGGGTG GGCAGACGGA 48001 CTTCGGCGTA GACGGTGTCG CCGTCCCGCC ACGCCGTGTG CAGCCCGCGG AACGCGGGAC 48061 CGTAGTCGAA TCCGAGGCCG ATCAACCGCT CGTAGACGGC ATCGAGGTCG ACCGGTGTGG 48121 CCCCGCGCGG AGGCCATGCC TGCACCTGCG ACGGCTCCGG GGCGAGCACC GGGGCCGGCT 48181 CCGCCGCGTC GGAACGGAGG GTGCCGGTGG CGTGCCGGGT CCAGGGGGCG TCCTCGGCGG 48241 CGTGGACAGG CCGGGAGTGC ACGGTGATCG CACGGCCGCC GCCGGCCGCC TCGGCCTCGC 48301 CGACCAGGAC CTGCACGACG TGGGCCGACC CCCCGTCGAG GATGAGCGGC GCCTCCAGCG 48361 TGAGTTCCTC GACGCCCGCG CAGCCCACGC GGGCCGCGGC GGTGAAGGCG AGTTCGAGGA 48421 ACGCGGTCCC GGGCAGCAGC GTCGAGCCGA GCACGACGTG GTCGCCGAGC CACGGGTGGG 48481 TTCCCGGGGA GATCGTGCCG GTCAGCACTG TGCCACCG. GT GCCGGGCAAG GTGACCGCGG 48541 CGCCGAGGAA CGGGTGGTCG ACGGCGCGCA TACCGAGGTG CGCGGCGTCG ACGGAGGCGG 48601 CATTGCCGGC AAGCCAGTGG TGTTCGTGCT GGAAGGCGTA GGTGGGGAGT TCACGGGCGA 48661 GGTCGTTCGC CCGGCCGAGC GCGGTCCAGT CGACCTGGTG ACCACGCGCG TGCACCCGGG 48721 CCAGAGCCGT CAGGAAACCG TTCACATCAC CCGTCCGCCG CCCCAGGGTG GGCAGGAACA 48781 CGGCACCGTT CTCGACGACT CCCGGATGCG AGGCACCCAT CGCCGTCAAC ACCGCCTCGG 48841 GCCCCAGCTC GACAACGGTC GCGACGCCCT GCGCCAGGAC CGCACCGACC CCGTCCCCGA 48901 ACCGCACCGC TTCCCGCACG TGCCGCACCC AGTACTCCGG CGAGCACAAC TCCGCAGCGG 48961 ATGCGACCTC ACCCGTCACG TTCGACACGA CCGGAATCGA CGGGGCACGG AACTCCACCC 49021 GCGCCAGCAC CTGCGCGAAC CCGGCGAGCA TCGGCTCCAT CAACGGCGAA TGGAACGCGT 49081 GCGAGACACG. AAGACGAGTC GCACGACGCC CTCCCCCGCG CGCCCGCTCC ACCACCGCCT 49141 CAACGGCACC CTCCACACCC GAAACAACCA CCGCCGCCGG CCCGTTGACA GCAGCGATCA 49201 CCGCACCGTC CACCAGCCAG CCCGACACCT CCTCCTCGGT GGCCTCCACC GCCACCATCC 49261 GCCCACCCGA CGGCAACGAA CCCATCAACC GGCCCCGGGC CACCACCACC CGCACCGCAT 49321 CCGCCAACGA CCACACACCC GCCACATACG CGGCGGACAA CTCCCCCAGC GAATGCCCGA 49381 TCAACACATC CGCACGCACA CCGAAAGACT CCGCCAGCCG ATACAACGCC ACCTCGACCG 49441 CGAACAACGC AGGCTGAGCA ACCCCCGTAT CCTCCAAAAC CCCCGCATCA TCACCGAAGA 49501 CCACCGAAAG CAGCTCTGCT CCCGTCTGCG CCTCGACCTC CGCACACACC TCGTCCAACG 49561 CAGCCGCGAA GACCGGGAAC CGCCCATACA ACTCACGCCC CATCCCCGGA CGCTGCGAGC 49621 CCTGACCCGA GAACGCCACA CCCACACCAC CCGCGACACG ACGCTCAAGC ACCACACCAC 49681 CGGCAGCGGA ACCGTCACCC CGCGCAACCC CACCCACACC GGCCAACAAC TCGTCCAACG 49741 ACCCACCACT GACCACAGCA CTGTGATCGA ACACCGACCG CGACGACACC AGCGCCAGAC 49801 CCACACCCCC CACATCCAGC GCCCCCGCAC CGCCCCCGCG TCCCGCCACG AACGCCGCAA 49861 GCCGCGCCGC CTGAGCCCGC ACCGCACCCT CAGTACGACC CGACACAACC CACGGCAACT 49921 CCCCAGCAAC CAACGGAGCT TCAGTGGACT CCACCCGAGC CTGCACAGAC CCCACCGGAA 49981 CCTGAGCGGA CCCCACCGGA GCTTCAGTGG ATTCCACGGG CTCGTGCTCC AGGATCACGT 50041 GCGCGTTCGT CCCGCTGATA CCGAACGACG ACACACCCGC CCGCCGCGCA CGACCCGTCT 50101 CCGGCCACTG CCGAGCCCGC GTCAACAACT CCACCGCACC CGCAGACCAA TCCACATGCG 50161 GCGACGGCTG CGACACATGC AACGTCCGCG GCAACACCCC GTGCCGCATC GCCATCACCA 50221 TCTTGATCAC ACCACCCACA CCGGCAGCCG CCTGCGTATG ACCGATGTTC GACTTCAACG 50281 ACCCCAGCCA CAACGGACGG CCCTCCGCCC GGCCCTGCCC ATACGTCGCG ATCAACGCCT 50341 GCGCCTCGAT CGGATCACCC AGCCTCGTCC CCGTCCCGTG CGCCTCCATC ACATCCACGT 50401 CCGACGTCGA CAACCCCGCA CCCGCCAACG CCCGCACGAT CACCCGCTGC TGCGACGGAC 50461 CGTTCGGCGC CGTCAACCCG TTCGACGCAC CGTCCTGGTT CACCGCACTG CCCCGCACCA 50521 CCGCCAACAC CTCGTGCCCG TTGCGCCGCG CGTCCGACAA ACGCTCCAGC ACCACCACAC 50581 CCACACCCTC GGACCAGCCC GTCCCCTCCG CATCCGCGGA AAAAGAACGA CACCGGCCGT 50641 CCGCCGACAG ACCACCCTGA CGACCGAACT CCACGAACGC GTACGGCGTC GCCATCACCG 50701, TCACACCACC CGCGAGCGCC AACGAACACT CCCCCGCACG CAACGACTGC ACCGCCAGAT 50761 GCAACGCCAC CAACGACGAC GAACACGCCG TGTCCACCGT CACCGCAGGA CCCTCGAACC 50821 CGAACGAATA CGAAAGGCGT CCGGAGAGCA CGGAGCTGGC GTTGCCGGTG ATGAGGAATC 50881 CGGAGGCCTC GGTCGCACGG GACTCGCGCA GGTGTCCGAG GTAGTCCTGC ATGCCGGCGC 50941 CGATGAACAC GCCGGTGTCG CCGCCGCGCA GCGACTCGGG GACGATGCCG GTGCGCTCGA 51001 TCGCCTCCCA CGAGGTCTCC AGCAGCAGCC GCTGCTGGGG GTCCATGGCG GCGGCCTCGC 51061 GCGGCGAGAT GCCGAAGAAG CCCGCGTCGA ACTCCGCGGC GTCGTGCAGG AACCCACCGC 51121 CCGCGGTGAG CAGCCCGTCG GGAGCGGACG TCGAGCCGTC ACCGGCCACC CCGCCACTGG 51181 CCGGCCCGGC GCCGACGCCG TCGAGGTCCC AGCCGCGGTC GGCCGGGAAC GGCGAGATCG 51241 CGTCCCGCCC CTCGGCCACC AACTCCCACA GGTCCTCGGG CGAGGCCACC CCGCCCGGAT 51301 ACTTGCAGGC CATGCCGACG ACGGCGATGG GTTCCCGGGC CGCGTCTTCG ACGTCCTGGA 51361 GCCGACCACG CGTCTCGTGC AGCTCGGCGC TGACGCGCTT CAGGTAATCC AACAGCTGGT 51421 TCTCGGATGC CATTTCCCGC TCTCCCCATC AATTCCCGGA GGGTTCTCCA CTTGCCGCCG 51481 ACGACTCAGG ACTCGTCTAT CCCGGGCCCT CCAGCGGGGA GATGCCGAGC TGCCGGGTGA 51541 CGAAGTCGAG GACTTCGTCC TCGGTGGCCG ACTCCAGCAG CGCCCCCGTG TCCGGTGTGC 51601 CCCTCTCGGA CGAGGACCCG GAGGGCGAGG AGAACATGAC CGTCTCGGAA GCGGACCGTT 51661 TGCCTTCCCA GCCGTCCGCG AGCGCCCTGA GCCGGGACGC CGCGGCCTTC CGCAGGGCGG 51721 CGTCCAGCCG GAGTTCCTCG AGGCTCTGCG CCACTCGGTA CAGCTCGGCG AGGGCGGATT 51781 CCGCGGTGAC GGCCCGCTCC TCCGGCTGGA TCAGGCCGTG CAGTTGCGCG GCGAGTGCCT 51841 TGGGCGTCGG GTGGTCGAAG ACCATCGTGG CGGGCAGGTC CACGCCCGCG GCCCGCTGCA 51901 GTCTGTTCCG CAGCGCCATC GCGGTCATCG AGTCGATCCC CAGCTCCTTG AAGGGCTGGT 51961 CGACGGCGAG GGCGGCGGGG TCGGCGTAGC CGAGCACCGC CGCCGCGTGA TCACGGACGG 52021 TGTCCAGGAG CAGGCGGTCC GCGTCGGTCC GCGCCATGCC GGGCAGTCGC CGGGCGAGCG 52081 TGGGACCACC CGCGCCGCCC GCGCCGTCGG AACGCCCGGC GTCGGAGCCG TCGTCCGCGG 52141 CTTCCCCGGA TCGTGCGAAT GCGGCGAGCA GCGGGCTGGG CCGGGCCGCG GTGAAGCCGT 52201 CGGCGAACAG CGGCCATTCG ATCTCGGCCA GCACCTGGCT CGCGGGGCCG TCCTGGGCCA' 52261 GCGCGAGGTC GAGGGCCCGC ACGCCGAGTT CGGGCTCGAT GGGCGGCAGT CCGTTGCGCC 52321 GCATCCGCTG TTCGGTGGCC GCGTCGACGA GACCGCCGCC GCCCCAGGGC CCCCAGGCGA 52381 TCGAGAGGGC CGGGAGGCCC GCGGCGCGGC GGTGCTCGGC GAGGGCGTCG AGCACCGCGT 52441 TGGCCGCGGC GTAGTTGCCC TGGCCGATGC CGCCGACGGT ACCGACGAAG CCGGAGTAGA 52501 GCACGAACGA CGACAGGTTC AGGCCGGCGG TCAGTTCGTG CAGGTGCCAG GCGCCGAGCG 52561 CCTTGGGGCC GAGAACCGCG TCGAGCCGGT CGGCGTCCAG GTTCTCCAGT GCGGCGTCGT 52621 CGAGGACCGC GGCGGCGTGC ACCACGGAGA CGAGCGGCGC GTCGGCCGGT ACCGACTCCA 52681 GCAGGGCGCG TACGGCCTCG CGGTCGGCGA GATCGCAGGC GGCGATCGTC ACGCGCGCGC 52741 CCATGCTCTC GAGGTCGGCG CCAAGTCCGT CCGCGTCCAG CGCCTCAGGA CCGCGGCGGC 52801 TGGTGAGCAG CAGATGTTGC GCGCCCTGCG CCGCCAGCCG GCGGGCCAGG CGGCTGCCGA 52861 GGGCGCCGGT GCCGCCGGTG ATCAGGACCG TGCCCTCGCG CGGCCACACC GCATCCGCGC 52921 CCACGTTCCC GTCCGTGTCC GTGTCCGTGT CCGTGTCCGA CAGGGAAACC TGACCGCCGT 52981 CGCGTCCGCC GGTCGTGGGG TCCGGCAGCC GGAACGGGGT GCGGACGAGG CGTCGGGCGA 53041 GCACGCCCGT CGGGCGCAGC GCGAGTTGGT CCTCGTCGCC CGGGTCGGCC AGCACCGCGC 53101 AGAGCTGAGC GGCGGTGGCG GCGTCCGGCG CGGTCCGTCC GTCCTGCCCG TCGGCCTCAC 53161 GGGGCGCACG GGGCAGGTCG ATCAGACCGC CCCACAGCGT GGGGTGTTCG AGGGCGGCGA 53221 CGCGGCCGAG GCCCCAGACC GCGGCGGCGT GCGGTGCCGC CGGAGGGTCG GTCGGGTCGG 53281 TTCCCACGGC GCCGTGCGTG AGGCACCACA GCCGGCCCGG CAGGTTTGTC TGCGCGAGCG 53341 CCCGCACCAG. GAGCGTCGTC CCGGCCAGGC CGCGGCCGAC CGCGGGGCGT CCAGGTAGCG 53401 GCCGGTCGTC GAGCGCGAGC AGGGAGAGGA TCCCGGCCGG CCGGGCATCG GCCGCCGCGG 53461 CGTGCAGCGA CGCGGCCCAT GCGGCCGGGT CGGTGTCCTC GCGCGGGTCG AGTTCCACGA 53521 GCAGCGCCCG GCCGCCCGCG GCCTCGACGG CGTCGAGCAC CCACCCGACG AGCCGGTGGT 53581 CGGGGCCGAA TCCGTGCTCG GCGCCGGGTA TCGCGAGCAG CCAGGTGCCG GCGAGGGCGG 53641 GGACGGCGCC CGGCGCGTGC AGGACCGGCT CCCAGCGGAC GCCGTAGCGC CAACTGCCCA <BR> <BR> <BR> 53701 GCCGTGCCTG CTCCAGGTGG TCGCG CGCC ATGCCGCGAG GGCGGGCAGC AGGGCGTCCG<BR> <BR> <BR> 53761 CCGACTCGCC GTCCTTCAGC CCGAGGGCGT CCAGGAGCGC GGCGGGGTCG GCCTCCAGCG 53821 CCTCCCACAG ACCCGAGTCG GCCGCCTGCG CGGCATGCGG CGCGGACGCT CCGGCCAGGG 53881 CCGAGGGGCG CCCGCCCCGG CCGGCCTCGG ATCCGGCCCC GGAGCCATTC GTGGTGCGCG 53941 GCGTCGGCCA GAAGCGGCTG CGCTGGAAGG GATAGGTGGG CAGGTCGGTC ACCCGGCCGT 54001 CGCTGTCGCG CAGCAGGGCG GCCCAGTCCA CGTCGAGACC GTGCACCCAG CCCTCGGCAG 54061 CCGAGGTGAG GAAGCGGTCG GCTCCGCCCG CGTCGCGGCG CAGCGTCGGC ACGGTCGCCG 54121 GTCGGACGCC CGCCTCCTCC GCGGTGCGTT CCATCGCCCC GGTCAGCAGC GGGTGCGGGC 54181 TGATTTCGAC GAATACGGCG TCCCCGGACT CGGCGAGGCG GCGGACCGCG GCCTCGAACT 54241 CCACCGGGGC CCGCAGGTTG CGCACCCAGT ACTCCGCGGT CAGTTCGCCG TCGCGGACCG 54301 GCTCGCCGGT GACGGTGGAC AGCATCTCGA CCTCGGGGCG GCCCGGTGCG ACGTCCGCGA 54361 GCTCCGTCAG GATCCGCGGG CCGATCCGGT CGACGTGCGG CGAGTGCGAC GCGTAGTCGA.

54421 CCGCGATCCG CCGGACGCGC ACGTTGTCGG CCTCGCAGAC GGTGAAGAAC TCGTCCAGCG 54481 CGTCCGCGTC GCCGGACACC ACGGTGGCCT CGGGGCCGTT GGCGGCGGCG AGGAAGACGC 54541 GGCCTTCGAA GGGCGCGAGG CGGGCGGCGG TCTCGGCTCG GCCGAGCGCG ACGGACAGCA 54601 TGCCGCCGGC GCCCGCGATC CCGGTCAGCG TGCGGCTGCG CAACGCCACG ATCTTCGCGG 54661 CGTCTTCGAG GCTGAGGGCG CCCGCGACGC AGGCCGCCGC GACCTCGCCC TGCGAGTGGC 54721 CGACCACCGT GTGGGGGCGG ACGCCGACGG AGCGCCACAG CTCGGCCAGG GACACCATCA 54781 TCGCGAACAG CACGGGCTGG ACGACGTCGA CCCGGTCCAG GGACGGTTCG CCGGGCTCGC 54841 CGTTCAGCAC GTCGAGCAGC GACCAGTCGA CGTGCGGGGC GAGCGCGTCG GCGCACTCCC 54901 GCACCCGCGC GGCGAACACC GGCGAGCTGT GCAGCAGTTC GCGAGCCATG CCGGCCCACT 54961 GGGCGCCCTG TCCGGGGAAG ACGAACACCA CCCGGCGGCG AGGGGCTGCG GCGCCGTCAC 55021 CGTCCACCCG GTTCGCGCTC GGCACGCCCG CGGCCAGCGA CTCCAGTTCG GCGACGACCG 55081 TCGCACGGTC GCCGCCGAGC ACGACGGCCC GCTGCTCGAA GGCCGTCCTG GTCGTCGCGA 55141 GCGACAGCGC CACATCGGCC AGGGGCGACT CGTCGGCGGT CAGGCGCCTG CCGATCAGCC 55201 GGGCCTGGGC CGACAGGGCG TCGGGGGTGC GTGCGGAGAC CAGGAAGGGC AGAACTCCCG 55261 ATCCCTGAGC CGGAGTTCCG GTGCCCTTGG CCGTCGGCGC GGGTACGTCC GGTTCCGGGT 55321 CGGGGGCCTG TTCGAGGATC ACGTGTGCGT TGGTGCCGCT GATGCGGAAG. GACGAGATGC 55381 CGGCGCGGCG GGGCCGGTCG GTCCGCGGCC ACGGCTGTGC CTCGGTGAGC AGGCGGACGG 55441 CGCCCGCCGA CCAGTCGACG TGCGTGGCCG GCCGGTCGAC GTGCAGCGTG CGCGGCAGCA 55501 CGCCGTCGCG CATGGCCATC ACCATCTTGA TGATGCCGCC GACCCCGGCC GCGCCCTGCG 55561 TGTGACCCAG GTTCGACTTC AACGAACCAA GCCAGAGCGG GCGTTCGGCC GGGTGCTCGC 55621 GCCCGTAGGT GGCGAGCAGC GCCCGTGCCT CGATCGGGTC GCCGAGCGTC GTGCCGGTGC 55681 CGTGCGCCTC CACGACGTCG ACGTCTGCGG CCTGGACCTG CGCGTCCTGG AGGGCGCGCC 55741 AGATCACGCG CTCCTGCGCG GGGCCGCTCG GCGCGGCCAG ACCGTTGGAC GCGCCGTCCT 55801 GGTTGACCGC CGAACCGCGC ACGACGGCCA GCACGCGGTG CCCGTTGCGC TGCGCGTCCG 55861 AGAGGCGTTC GAGGACAAGC ATGCCGGCGC CCTCACCCCA GGAGGTGCCG TCGGCGGCGT 55921 CCGCGAAGGA CTTGCTGCGG CCGTCCGCGG CGAGGCCACG CTGCCGGGAG AACTCGGTGA 55981 AGGTGATGGG CGTCGGCATG ACTGTCACGC CGCCTGCGAG CGCCAGCGAG CATTCGCCGC 56041 GGCGCAGCGA CTGGGCGGCC AGATGCAGCG CCACCAGCGA CGACGAGCAG GCGGTGTCGA 56101 TCGTGACCGC GGGCCCGGAC AGCCCGAGGG CGTAGGAGAC ACGGCCCGAG ACGACGGCGC 56161 TGTTGTTGCC CGTCACGAGG TAGCCCTCGT ACTCCTCGGA CGAGGCCTGC AAGGGCACGT 56221 CGTAGAACGA GTAGTAGGCG CCGGTGAACA CGCCGGTCTC GCTCTCGCGC AGCGTGTCCG 56281 GGGCGATGCC GGCGCGCTCC AGGGACTCCC AGGCCACTTC GAGCATGAGG CGCTGCTGCG 56341 GGTCCATGGC GACGGCCTCG CGCGGCGAGA CGCCGAAGAA ACCTGCGTCG AACCAGGCGG 56401 CGCCGTGCAG GAACCCGCCC TCGCGGACGT ACGAGGTGCC CTCCTCGGTG CCGTCGCCGG 56461 CGAAGAGCGC GTCGAGGTCC CAGTCCCGGT CCTTGGGGAA GGCGCCGATC GCGTCGCGGC 56521 CCGTGCGCAC CAGGTCCCAC AGCGCCTCCG GGGAGTCGGA GCCGCCGGGG AAGCGGCAGC 56581 CCATGCCCAC GATCGCGATG GGCTCGGTGG CGCGGCGCTC GGTGTCCTTC AGGCGCCGGC 56641 GGGAGTCCTG GAGGTCGCCG GTGACCTTCC GCAGCGCCTG AAGAAGCTGT TCCTGGCTTA 56701 CGTCGTCAGA CATCCACGCC ACCGTTTTCC ACTCTCGGGC AAAGCACTGT CACTCGAAGC 56761 CCTTCTCGAT CAGCGCCAGC AGGTCGGAGG CGCTGGCGGT CGCGATGGCC GCGCCGCCGT 56821 CCGTGTCGTC GTCCGCGGCT TGTGCGGCCG GTTCGGTGTC GCGGGGTGTC TCGCGCAGCC 56881 GGGCGGCCAG CGCGCCGAGC CGGGTGGCGA GCCGGTCCCG GTCGGCGGCG GAGCCTTCGA 56941 GGTCCGCGAG CGACCGCTCC AGCGAGGCGA GGTCGGCCAG CGCGCGGGCG GCCTGTCCGC 57001 CGCCGTCCGG GACGGCTTGG CGGCAGAGGA ACGCGGCGAC GTCGGTCGGC GTCGGATGCT 57061 CGTAGACGAG CGTGGCGGGC AGGTCGAGAC CGGTGGACTC GCGCAGCCGG GTGCGCAGTT 57121 CGACGGCCAT CAGGGAGTCG AAGCCGAGGT CCTTGAAGGC GTGGTGCGGC GCGACGTCCC 57181 GGGCCGCGGC GTGACCGAGC ACGGCGGCGA TCTGGGCGCG CACTAGGTCG AGCGCGACGC 57241 GCTGCTGTTC GTCGGGACTG TGCCCGGCCA GCACTTTGGC GAAGTCCGGC GTGGTGCCGC 57301 CCGTCTCGTC GGTGCCCGGA CCCGCGCCGG TGACGCCGGC GAGGTCGGAG GCGGCACCGG 57361 GCACCGGAAC GAGTCCGGCG AGCAGCGGGC TGGGGCGGGC GGCGGTGAAG ACGGCGGTGA 57421 AGCGTCCCCA GTCGACGTCC GCGACGGTGA CCGTGGTGTC GCCGTGCCGC AGCGCCCGGT 57481 CCAGGGCGGT GACGGCGAGT TCCGGCGCCA TCGGGGCGAT GCCGAGTCGG CGCAGGTCGT 57541 CACCGAAGCT CCCGGCAGCC ATGCCGCCGC CGTCCCAGGG GCCCCAGGCG ATGGCCTTGC 57601 CTGCGGCGCC CCGGGCGCGG CGCCGCTCCA TCAGCGCGTC GACATGCGCG TTCGAGGCGG 57661 CGTAGGCGCC GCCGGACGCG TTGCCCCAGA CGCCTGCGAC GGAGGTGAAG GCGACGAACG 57721 CCGACAGGCC GTCGCCGAAG ACCTCGTCGA GGTTGCGCGC GCCGGTCACC TTCGCCGCCA 57781 TCACGGTCGC GAAGGTGTCG GCGTCGAGCG CGGCGACGGG GTTCTGGTCC CCGGTGCCCG 57841 CGGCGTGCAC CACCGCGCCG ACGGGCGTGT CCGCGGCGGC GAACCGGGCG GCCAGTTCCT 57901 CGACGTCGGC CCGGCGGGAG ACGTCGCAGT CCGCGAGGAC GAGTTCGGTG CCGTGCCGCG 57961 CCAGTTCGGC GCGGAGCCGG TCCGCGTCGG CGAAGCCACT GGCGCGGCGG CTCGCGAGCA 58021 CGACGATGGG CGCGCCGGAC TCGGCGAGCC GGCGGGCGGT GTGGACGGCG AGGGCGCCGG 58081 TGCCGGTGAT CAGCACGGGT CCCGAGGACC ACAGGTCCGC GTCCTCGGTG TCCTCGGCGG 58141 GCCGTTCGTG GGGGCTCACG GTCGGCTCGG CGCGGGTCAG GCGCCGGGCG TGGACGCTGT 58201 CGTCCCGCAG CGCGAGCTGG TCGTCGCCGC CGGATCGCCC GGCGAGGACG GCGGCCAGCC 58261 GGGCGAGCCC GCCGCAGCCG AGGGGATCGT CGTCCGTGCT CGCCGCGAGG TCGATCAACC 58321 CGCCCCAGAG TCCCGGGTGT TCGAGTCCGA CGACCCGGCC GAGGCCCCAG ACCTGGGCCT 58381 GCCACGGGTC CGGCGCCGCG TCACTCTCCA GCGCCCGCAC CGCGCCACGG GTCAGGGTCC 58441 ACAGCGGCGG GTCCGTGCGG CCCGTGTCGA GCAGAGCCTG TACGAGCAGC AGGGTGGCGT 58501 CCGCGGCGGC CGACACGCCG TCGCGGCGCG CGGTCTCACC GCTCGGCCAG GGCAGGGCGA 58561 GCACGCCCGC GAACCGCTCG TCCGACGCCG GGGTCCGGTC CAGTTCCCGG GCGAGTTCCT 58621 TCGCCAGGGT CGCGCGATCG GCGGTCGCCA CGTCGACCGT CAGGAGGCGG GTCGTCGCGC 58681 CCGCGTCGGC. CAGTGCGGCC GTGACGGCGT CGGCGAGCGC GGCCGACGCG CCGTCTTCGG 58741 CGGTGTCGGC GTCGGATTCG GGGGCGACGA TGAGCCAGGT GCCGTCAAGT GCCGCCGCCG 58801 GGGGCGCGGC GTCCGGAAGA CGTTCCCAGG TGACGTGGTA GCGCCACCTG TCGGTTCTGG 58861 CGATCATGTC CTGTCCGCGC CGCCAGTTGC CGAGCGCGGG CAGCACGGCG CTCAGCGGCG 58921 CGTCGGCGCC GAGGCCGAGC GTGCGGGCCA GGCCGTCGAG GTTCTGCTCC TCGACGACCT 58981 GCCAGAACGC CGTGTCCTCC GCGCTGTGCG TGCCGTCCGG CGCGGCCGGA ACGTCCGCCG 59041 TGTCCGGTGC GGCGTCCAGC CAGTAGCGCC GGTGCCGGAA GGCGTACGTC GGCAGTGCGA 59101 CACGCCGGGC GCCGGGCGTG AGCGCGGTCC AGTCGACCGG CACGCCGTTG ACGTACGTCC 59161 GGCTCAGCGC GGTCAGCACG GTCGCCGCCT CCGGCCGGCC GCGCCGGGAG GAGGCCGCGA 59221 CGGGCCGAGT CCCCTCGAAC AGCGGCGTCA GGGCGGCGTC CGGGCCGATC TCGATCAGCA 59281 CGTCCGCGTC CGGCAGGCCG CGCACCGCGT CGGCGAAGCG CACCGCGCGC CGCGCGTGCT 59341 CGATCCAGTA CTCGGGGGTG TCGAAGGCGT GCGTGGTGTC CGCGGCCGGG CTGACCTGGA 59401 TCTCGGCCGG GTGGAAGGTG AGGCCGTCGA CCACGGCGGC GAACTGCGCA AGCATCGGTT 59461 CCATCAGCGG GGAGTGGAAC GCATGGCTCA CCCGCAGCCG GGTCGTCCTG CGGCCCTCGC 59521 CGCGCCAGTG CTCCGCGATG CGCGTGACCG CGTCCTCGGC TCCCGAGACG ACGACCGCGG 59581 AAGGCCCGTT CACCGCCGCG ATCTCGGCTT CCGAAGTGTC CACGAGGTAG GCGAGGCTCG 59641 CGGCGACCTC GTCGTCGGTC GCCTGGATCG CGACCATCGC GCCGCCCTCG GGCAGCGCCT 59701 GCATCAGCCG TCCGCGGGCG ACCACGAGCC GGGCGGCGTC CTCGACGGAC AGCACCCCGG 59761 CGACCACGGC CGCCGCGATC TCCCCGACGG AGTGCCCGGC GAGGGCCGAG AACTCGACGC 59821 CCCACGACAT CCACAGTCGC GCCATGGCGA GTTCGAAGGC GAACAGGGCG GGCTGCGCGA 59881 ACTCGGTTCG CTCCAGCACC TCCGCGGGCT CCTGCCACAG TACGTCCCGC AGCGGACGTC 59941 CGAGCAGCGG GTCGATCACG TCGCAGATCT CGTCCAGGGC GGCGGCGAAC ACCGGGAAGT 60001 GCTCGGCGAG TTCACGCCCC ATCCCGGGCC GCTGTGCTCC CTGGCCGGAA AACAGCGCCG 60061 CGGCCCGTGT GCCGCCCGTG GTGCCGGCCT CGGGACCGGT GACCGCCTCG GCCGGGTGCA 60121 CCGCGTCCGG GGTACCGAGC GCCGCGAGCG CACCGAGCAG GCCGTCGCGG TCCTCCGCGA 66961 CGGTGATCGC GTCGCGTCCC TCGGCCACCA GCTCCCACAG CTCCTCGGGC GAGGTCACCC 67021 CGCCCGGGTA GCGGCAGCTC ATGCCGACGA TCGCGATCGG CTCGCGGGCC GCGGCCTCCG 67081 CATCGCGCAG CCGGGATCGC GTACGTTGCA GGTCGGCGGT CACCTTCCGC AGGTACTCGA 67141 CGAGCTGCTG TTCGTCAGTC ATGTTCCTCG CCCATCGGCG TACGGCGGGT TCGCTGGCTT 67201 CGCGAACCCG GCATCGAATG AACTGCACGA GCCCGCCGAC CGGATCGAAT CCGGCGGTCT 67261 TCGTCTCGGC TCTCAATGCG GCGCGGACTG CGGCGGCCGT GCCGAATCGG ATTTCTTCGA 67321 TCCAAGCACG GAAACAGCGG CGCCCCCTAC TCAGGCACCC CCCTAAAACA CCCGGCATGG 67381 CCTTCGGTTG GGGTTCGACC AGGGGTGATG CGGCAGCGCC GGATGGGCGG GCACGGCATG 67441 CAAGAGCCGG GCGCGGGCAG CGGCGATCGC GGCCCGCGCC CGGCGCGGTT CAGTCGTCCC 67501 CAGGGAACCG TGGATCAATG GCTCCACGAA CCACGGATCT ACGGATCTGG AGGGAACTGG 67561 AGGGGATACG GATCCGGAGG GGACACCAGA ATTCAGGATT CAGGAAGCCG GTGAGTCCGC 67621 ACGCTTCTCG GCGGCTCCCT CGGCACGCTC TTCGGCACAC CACCATGCCG GCTGTACGGT 67681 CGCCTGCGGG ACCGAGCCGA GAGCGGCCCG GATGGGCTGC GCGCCCGCCG AGACGGCCAG 67741 CACGCCGCGC ACCGGCTCCG GCGCGAACAG CGCCTCGCAG ATCGTCTCGG CGAGATCCGT 67801 CCTGGAGACC GTGCCCTCGG GCTGCGGGTC GGCGCCGCCG CGTACCGTGA CCAGGCCGGT 67861 GGCCGGGGAG TTGTCGAGCA TCCCCGGACG CACCACGCAC CAGTCCAGGT CGTGTCCGGC 67921 CAGCTTCCGC TCCACGTCCC GCTTCTCCGC GAGGTACGAC TCCAGCTCGT. CACCGAGCCT 67981 GGTGTGCAGC TCGTCGTCCG GCAGATATGC GCTGACCAGG ACGAAGCGCC GGATGCCGAC 68041 CAACTGCGCG ACCCCCATCA ACTCCCCTAC GAGCGAGGTG GACGAGGTGT CCGTGGCGTC 68101 CGGGCCCCAG CCGGTCCCGG TGGCCACGGC GATCCCGCCG CACTCGCCCA TCGCCCGTAG 68161 CGCCGCCGGG CTCGTCCTGT CCGCCTCGGA CGCGATCACC AGCGGCTCTG TCCCTGCGGC 68221 CCGCAACGAC TCGCTGTGCC CGGCCTCCCC GATGAGCCCC ACCGGGGTCA GGCCGCGGGC 68281 GAGGATCGAC TCGGCGAGCC GCCGGCCCAG CGCGCTCGTG ACGCCGAGGA CGACGACGTT 68341 CTTCTTCCCC GCCATCGGCC CCGCCTGCGC CGTCCGTGCG GCTTCCGCCC TCAGCGCGGC 68401 ATCGGCTGTC CGCGGGGCGT CCGCCGTCTC ATCGGCTTTC GATATCGGCA TGTTCGAGAT 68461 GTCTTCTTTC ATCGGCTCGG TCGCCATATC AGTCCGCTCA CGCCACGTCC TGGATTTCCG 68521 CGGGCGTGTG GTCCGGAGCA CCGCGCGATT CGACGATGGC GCCGATCTCG GCGCGGCGCC 68581 TGATATCAAA TTTTCGGTAA ATACGGGTCA AGTGCTGTTC AATGGTGCTC GCGGTGACGT 68641 AGAGCGACTC CGCGATTTCG CGGTTCGTGT ATCCCTGAGC CGCCATTCTG GCGACATTCC 68701 ACTCGGCCGG GGTGAGGCTG CGCCCGTCGC GTCGTGCCGC GGGCGGCGGC ACCTTGCGCC 68761 TCGCCTGCGA TCCGCCGGAC ATCTCCGCAA GCGCCCACTG CGCCCCGCAG CCGCGCGCCG 68821 TCTTCTCGGC CAGCCGCATC GTCTTACGAC CGCCGGCCAG GTCTCCGGTG TGCTTGTAGA 68881 TCTGCGCGAG CTGGCCGAGC GCGCGGGCGT ATTCGAGTTC GTCGCCGCAG GACTGGAGAA 68941 CGGCGATGGA TTTCCGGCAC GCCAGCGCCC GTTCCCCCGG CGGGACCGTC GCCACCCGCA 69001 GGCGCAGCCC GATGCCGCGC GCCCGGGTGT TCGAGTCCGG CGAGAACGCC AGCTGCTCGT 69061 CGATGAGCTC CGCCGCCTCG GCGTACTCCT CCAGCTTCAG CAGCGCCTCC GCGAG. GTCCA 69121 GGCGCCACGG CACCAGGCCC GGCATCTCCA TGCCCCAGGC CGTGAGGATG GCGCCGCAGC 69181 TGCGGAAATC CGCGACGGCG GCGCGCAGGG CGCCGGTCGC CAGCCGGAAA CGCCCGCGGG 69241 CGCGCAGGTA CACCAGGCCG TACGGGCTGG CCAGGTCCGC CTCGGTCATC GGGCGGGCGA 69301 GTTGCGCGGC TGCCTCCTCG AAGCGTCCTT GCTCGGTCAG CATCAGCGCG GACAGGCCGC 69361 GCGGCAGACC GGCGAGCACG CCCCACCGGT CCGCGTCCCA GCGCTCGAAG CCGCGGCGCA 69421 CGGCCGTCTG GGCCGCGACG AGGTCACCCT TCCACCAGGC AGCGACCGCC TCGGCCACGC 69481 TCAGCATGGA GGCCAGCGGC AGCGGCATAC GTTCGTTGTC GGCGAGGGCG GCGTTGTCGG 69541 CGAGGGCGGC GTACCAGGGG GCGCGCGGGT CGAGTCGTCC CGTCGCCATC AGGCAGAACA 69601 GCGCCACGAG CATCGGCTCC AGGCTCGAAT AGTCGAAGTT GGCCGACTGG AGGATCTCCT 69661 CCGCGCAGGT CACCGCGGCT TCCGCGTCTT TCACGGCCTT CGCGGAACCT GCGGAATCCG 69721 CGGAACCCGT GGGCCGTGGA CCGGAGTTCG GCGCATCGGA GCACTGGACG CCCGGCGCGA 69781 GCAGCCACGA GAGCCGTTGC GCGCTGGACA GCCACGAGAA TCCCTCGGCG ATCAGCTGGG 69841 GGTGCGGCTG GTCGTCGGGC GCGGGCGCGT CCGGGCAGGC GTCGGGGAAC AGATGACGCA 69901 ACCACAGCCC GCTGAGCGTT CTCTGGACCT CGGCACGGAC ATCCGCGGCG CCGAGGGCGT 69961 CGCCGGGACT GACAGCACCG ACAGCGTTGC CGCGTCCAGG CGCGGGTTCG GCCAGGCGCA 70021 GCATGTCGGA CGCCTGCGCC ATCTCGCCGT GTCTGGCCAG ATGCCGCGAG AGACGGGCTA 70081 ACGAACCCGG CTTGAAAGTG CCGTCGGACG CGGCAGCCGC CAGCCGCCGC AGCCGCGGTG 70141 CGGTGAGCGC CGGGTCCATC CACCACACCA TGTCGGTGAT CCGGACGCCG GCCTCCTCCC 70201 GCAGGTCGGG CCCGGAGCCC CAGAACGAGG CGGACTCCAG GAGTTGGACA GCCAGCCGGT 70261 GCCGGCCGAG ACGCGAGGCG TGCTCGGCGG CCTCATAGAG GACCCCACCC GCCCAGGGCT 70321 GCGGCGCGAT CCCGGCCTCG TGCAGATACG GAGCGATCTC CCAGGCGGGC ACGCCCTGTT TABLE 4 FOSTRIECIN SYNTHASE GENE CLUSTER ORF 8 ORF7 ORF6 ORF5 FosB (module 2)" FosA (modules 0-1) FosK FosJ FosI FosH FosG" FosF (module 8 and thioesterase) FosE (modules 6-7) FosD" (module 5) FosC (modules 3-4) ORF4 ORF3 ORF2 ORF1 t0094j All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

[00951 Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description are for purposes of illustration and not limitation of the following claims.