Abstract:
This invention relates generally to non-steroidal estrogen-receptor (ER) antagonists that are useful as therapeutics in the treatment of advanced stage breast cancer. More particularly, this invention relates to the cloning and heterologous expression of genes encoding ER antagonists. This invention also relates to the use of such expression to overproduce the ER antagonist and analogs thereof.

Description:
This application claims benefit of Provisional Ser. Nos. 60/243,458 filed Oct. 25, 2000 and 60/179,305 filed Jan. 31, 2000. 
    
    
     This invention was made in part using United States government funding through the National Institute of Health Grant No. CA 77248. 
    
    
     FIELD OF THE INVENTION 
     This invention relates generally to non-steroidal estrogen-receptor (ER) antagonists that are useful as therapeutics in the treatment of advanced stage breast cancer. More particularly, this invention relates to the cloning and heterologous expression of genes encoding ER antagonists. This invention also relates to the use of such expression to overproduce the ER antagonist and analogs thereof. 
     BACKGROUND OF THE INVENTION 
     Non-steroidal estrogen-receptor (ER) antagonists are useful in the treatment of advanced stage breast cancer. One class of ER antagonists that has been shown to be particularly useful are the R1128 substances (Y. Hori et al., The Journal of Antibiotics, 46(7): 1055-1062.) These substances are anthraquinone natural products with in vitro and in vivo potency approaching that of Tamoxifen. However, many of these types of ER antagonists exhibit variable effects in different target tissue. For example, tamoxifen acts as an ER antagonist in breast tissue, but an ER agonist in bone (Love, R. R. et al., New England Journal of Medicine 26: 852-856 (1992)) and uterine tissue (Kedar, R. P. et al., Lancet 28: 1318-1321 (1992)). Likewise, Raloxifene is an ER antagonist in breast tissue and an ER agonist in bone but not in uterine tissue (Black, L., et al., J. Clin. Invest. 93: 63-69 (1994)). 
     Estrogen receptors are transcription factors that bind to estrogen response elements in the promoter region of certain estrogen-related genes. Thus, estrogen-like ligands are useful to modulate transcription of these genes (Katzenellenbogen, J. A. and Katzenellenbogen B. S. (1996) Chem. Biol. 3: 529-536.) Recent studies have suggested that the tissue-dependent variability exhibited by these ligands may be due to differential ligand binding and/or activation of the two ER subtypes, ER-α and ER-β, which each have different tissue expression patterns and ligand binding characteristics (Mosselman, S. et al. (1996) FEBS Letters 392: 49-53.) It has also been reported that the ligand-bound ERs may modulate transcription activation by binding to different types of DNA enhancer elements, such as the AP1 transcription factors (Paech, K. et al. (1997) Science 277: 1508-1510). 
     Accordingly, there is thus a significant need for new ER antagonists which can inhibit ER function in a predictable way at both ER-dependent and AP 1-dependent DNA binding sites. 
     SUMMARY OF THE INVENTION 
     The present invention includes the estrogen receptor antagonist shown in FIG.  3 . Other aspects of the invention are found throughout the specification. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 depicts the structure of the R1128 compounds A (FIG.  1 A), B (FIG.  1 B), C (FIG. 1C) and D (FIG.  1 D). 
     FIG. 2 depicts the biosynthetic pathway leading to the R1128 compounds A-D. 
     FIG. 3 depicts the product, HU235. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to the cloning and expression of the R1128 gene cluster in a manner that favors production of alternatively cyclized derivatives of the R1128 anthraquinones. 
     The isolation, fermentation, structures and properties of R1128 A-D (shown in FIGS. 1A to D) were described in 1993. See Hori, Y. et al. (1993) J. Antibiot. 46: 1055-1062 (Part I); 1063-1068 (Part II); and 1069-1075 (Part III). In Part II, the authors compared their pharmacological properties to tamoxifen, the non-steroidal estrogen-receptor antagonist that is a leading choice for treatment of advanced breast cancer. These molecules, which were identified as potent ER antagonists, had IC 50  values in the range of 0.1-0.3 mM. Although these activities were approximately 7-17 fold less than that of tamoxifen, their selectivity (50-fold better binding to ER as compared to the androgen receptor) and low toxicity (significant inhibition of tumor volume in a subrenal capsule assay at 100 mg/kg; no acute toxicity in mice or rats at 500 mg/kg) profiles were promising. (Yori, H. et al., supra, Part III.) Accordingly, it appears that manipulating the structures of natural tamoxifen-like products offers an opportunity to optimize their pharmacological properties. 
     In order to further study the pharmacological properties of R1128 as a first step towards engineering the structure to enhance its pharmacological properties, the present invention provides for a cloning procedure that allows the capture of the entire R1128 biosynthetic gene cluster and its expression in a model heterologous host,  Streptomyces lividans  K4-114 (Ziermann, R., et al. (1999) BioTechniquies 20: 106-110. Although described herein as an exemplary cloning procedure, it should be understood by one of skill in the art that other known cloning procedures and hosts can also be utilized to clone the compounds describe herein using routine cloning and expression optimization methodologies. 
     Recent studies on bacterial aromatic polyketide biosynthesis have demonstrated considerable promise with respect to altering four aspects of product structure: primer unit incorporation, chain length, degree and regiochemistry of reduction, and regiochemistry of cyclization. (R. McDaniel, et al., Nature 375: 549-554 (1995); and L. Han, et al., J. Bacteriol. 180:4481-4486 (1988).) Most of these features are potentially attractive targets for manipulation in the R1128 family of ER antagonists. For example, the incorporation of an additional ketide unit in the polyketide backbone could lead to the introduction of an aliphatic ketone functionality in place of the variable alkyl group. Likewise, elimination of the C-9 hydroxyl has been accomplished in similar systems by co-expression of a suitable ketoreductase with regiospecificfty towards the C-9 carbonyl of the nascent polyketide backbone. Finally, of particular interest is the variable functional group in the R1128 anthraquinone series, which is presumably derived from alternative primer unit incorporation in the polyketide backbone. The recent identification of a single protein as the principal determinant of primer unit specificity in bacterial aromatic polyketide biosynthesis suggests that it should also be possible to predictively alter this functionality in the natural product. 
     A minimal prerequisite for biosynthetic engineering of natural products is the availability of cloned genes, which are typically clustered and coordinately regulated in microorganisms. An ideal strategy for cloning a desired gene cluster would involve its direct identification through heterologous expression in a biologically friendly host. Building on the pioneering studies of Hopwood and coworkers (Malpartida, F. et al. (1984) Nature 309: 462-464), recent studies have successfully demonstrated the ability of an organism, such as  S. lividans,  to produce heterologous aromatic polyketides (Hong, S. T., et al. (1997) J. Bacteriol. 179: 470-476; and Gould, S. J., et al. (1998) 51: 52-57.) However, due to the limitations of the integrative vectors used in such efforts, genomic libraries had to be pre-screened for candidate cosmids carrying putative polyketide genes prior to their transfer into  S. lividans.    
     Accordingly, other vector systems capable of exhibiting a larger DNA carrying capacity for DNA are more preferred for use in the present invention. The pHU204 vector, which is a 12.16 kb derivative of pRM5, has such properties (McDaniel, R., et al. (1993) Science 262: 1546-1550.) Among other features, it has the well-characterized SCP2* replicon. While its carrying capacity as a cosmid cloning vector in  E. coli  is limited to 40 kb inserts, SCP2* vectors have been used to clone significantly larger fragments in  S. lividans.    
     The organization of the R1128 gene cluster is shown below in Table I and the sequence is given below in Seq. ID No. 1 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Organization of the R1128 Gene Cluster 
               
             
          
           
               
                 Gene 
                   
                   
                   
                 Length 
                 Length 
                 Start 
                 Stop 
               
               
                 No. 
                 Gene Function 
                 Start 
                 Stop 
                 (bp) 
                 AA 
                 Codon 
                 Codon 
               
               
                   
               
             
          
           
               
                   
                   
                   
                   
                 0 
                   
                   
                   
               
               
                 1 
                 Chain Length factor 
                 1623 
                 376 
                 1248 
                 416 
                 ATG 
                 TAG 
               
               
                 2 
                 ketosynthase 
                 2873 
                 1620 
                 1254 
                 418 
                 ATG 
                 TGA 
               
               
                 3 
                 Acyltransferase 
                 3067 
                 4113 
                 1047 
                 349 
                 ATG 
                 TGA 
               
               
                 4 
                 Acetyl-CoA carboxylase subunit 3 
                 6207 
                 4822 
                 1386 
                 462 
                 ATG 
                 TGA 
               
               
                   
                 (Biotin carboxylase) 
               
               
                 5 
                 Acetyl-CoA carboxylase subunit 2 
                 6651 
                 6214 
                 438 
                 146 
                 ATG 
                 TAG 
               
               
                   
                 (Biotin carrier protein) 
               
               
                 6 
                 Acetyl-CoA carboxylase subunit 1 
                 8901 
                 7165 
                 1737 
                 579 
                 ATG 
                 TGA 
               
               
                 7 
                 Acyl Carrier Protein 1 
                 9182 
                 8922 
                 261 
                 87 
                 ATG 
                 TGA 
               
               
                 8 
                 Priming ketosynthase 
                 9448 
                 10467 
                 1020 
                 340 
                 ATG 
                 TGA 
               
               
                 9 
                 Aromatase/Cyclase 
                 10903 
                 11412 
                 510 
                 170 
                 ATG 
                 TGA 
               
               
                 10 
                 Cyclase 2 
                 11409 
                 12179 
                 771 
                 257 
                 GTG 
                 TAG 
               
               
                 11 
                 Oxygenase 1 
                 12183 
                 13757 
                 1575 
                 525 
                 ATG 
                 ? 
               
               
                 12 
                 Amidase 
                 13754 
                 15277 
                 1524 
                 508 
                 ATG 
                 TGA 
               
               
                 13 
                 Oxygenase 2 
                 15270 
                 16823 
                 1554 
                 518 
                 GTG 
                 TGA 
               
               
                 14 
                 Acyl Carrier Protein 2 
                 16861 
                 17109 
                 249 
                 83 
                 ATG 
                 TGA 
               
               
                 15 
                 Transcriptional regulator (has a 1 bp 
                 17249 
                 18060 
                 812 
                 271 
                 GTG 
                 TGA 
               
               
                   
                 frameshift) 
               
               
                 16 
                 Transporter (has a 1 bp frameshift) 
                 18154 
                 18923 
                 770 
                 257 
                 ATG 
                 TGA 
               
               
                 17 
                 Oxygenase/Transporter 
                 18920 
                 20122 
                 1203 
                 401 
                 GTG 
                 TAG 
               
               
                 18 
                 Activator 
                 21003 
                 20185 
                 819 
                 273 
                 ATG 
                 TGA 
               
               
                 19 
                 Repressor 
                 22124 
                 21357 
                 768 
                 256 
                 ATG 
                 TGA 
               
               
                 20 
                 Activator 
                 22255 
                 22992 
                 738 
                 246 
                 ATG 
                 TGA 
               
               
                   
               
             
          
         
       
     
     When this gene cluster was isolated and expressed as described in Example I, an alternatively cyclized, optically active product designated HU 235 (FIG. 2) was also produced. Because a mutation in the cloned gene structure abolishes production of HU 235, but still allows for production of R1128B and C, this suggests that the bioactive natural products might actually be side products, rather than the principal products, of the biosynthetic pathway shown in FIG.  3 . 
     EXAMPLES 
     Example 1 
     Cloning of the R1128 Gene Cluster 
     Anticipating that the R1128 gene cluster was under 40 kb in size, a cosmid library was constructed in pHU204 (McDaniel, R., et al. (1993) Science 262: 1546-1550) using genomic DNA derived from  Streptomyces sp.  R1128, the R1128 producer. The entire library was transformed without prior screening into  S. lividans  K4-114. Several pigmented colonies were identified; one of them, which was designated K4-114/pHU235, was cultivated on a larger scale (500 mL on semi-solid R5 agar medium). After neutral extraction and pre-purification using column chromatography, two major UV-active peaks were collected from reversed-phase HPLC, dried, and analyzed. Mass-, 1H and 13C NMR spectra revealed that one of the compounds was identical to R1128C (FIG.  1 C). The purified yield of the compound was 0.5 mg/L. The second compound, designated HU235 (FIG.  2 ), was produced in considerably greater quantifies (16 mg/L), and was structurally characterized by 1H, 13C, 1H, 1H-COSY, HMBC, HMQC, ROESY-NMR, and IR spectroscopy. Mass spectrometry was performed with the acetylated derivatives 6 and 7. The polyketide origin of HU235 was established by feeding of 1,2-13C sodium acetate, which gave the expected incorporation pattern as observed in the 13C NMR spectrum. It was initially suspected that HU235 was a shunt product resulting from low activity of a late-stage enzyme in the R1128 pathway. However, optical rotation analysis revealed that the compound HU235 was optically active ([a]D=33.0°), suggesting that it arose from stereocontrolled cyclization of a common biosynthetic intermediate (FIG. 1 a ). To test this hypothesis, we subjected cosmid pHU235 to in vitro transposon mutagenesis (R. McDaniel, et al., Nature 375: 549-554 (1995). Transformation of the resulting pool of mutagenized cosmids into  S. lividans  led to the identification of one clone, Tn164, that produced both R1128B and R1128C in 1.4 mg/L and 1.6 mg/L, respectively, while completely abolishing HU235 production. The result confirms that one or more enzymes encoded within the biosynthetic gene cluster are involved in the final stages of HU235 biosynthesis. If so, then the biologically active R1128 metabolites are probably side products of a biosynthetic pathway whose principal product is HU2353 
     Example 2 
     Isolation and Characterization of Products 
     To further study the products obtained in Example 1, the cultures were extracted with EtOAc. The extract was dried with MgSO4, evaporated, and subjected to column chromatography (SiO2, Hexane/EtOAc 1:1). The yellow fractions were dried and further separated on a preparative reverse phase HPLC column (25×150 mm, C18-IP column, Beckman, isocratic run with H2O (1% HOAc)/MeCN 55:45, flow 20 ml/min, UV-detection at I=250 and 350 nm. Three major UV-active peaks (tR=10.2, 22.5, and 28.3 min) were collected, dried, and analyzed. Spectral data of HU235 (5): [a]D (in CH3OH, c=0.63)+33.0°; 1H NMR (500 MHz, (CD 3 )2SO): d=16.10 (s, 1 H-O-C(13)), 10.23 (s, 1 H-O-C(9)), 9.81 (s, 1 H-O-C(11)), 4.84 (s, 1 H-O-C(3)), 2.95 (2 d, J=16.3, 2 H-C(4)), 2.58 (dd, J=8.1, 3.0, 1 H-C(16)), 1.75 (m, 1 H-C(2′)), 1.64 (m, 1 H-C(1′)), 1.57 (m, 1 H-C(1′)), 1.25 (s, 3 H-C(2)), 0.95 (d, J=6.4, 3 H-C(3′)), 0.91 (d, J=6.6, 3 H-C(3′)). 13C NMR (125 MHz, (CD3)2SO, (coupling constants of 13C-enriched material, 100 MHz, (CD3)2SO)): d=206.4 (C(15), J (C,C)=39.9), 165.3 (C(13), J (C,C)=63.0), 161.3 (C(9), J (C,C)=65.0), 159.1 (C(11) J (C,C)=62.5), 140.8 (C(7), J (C,C)=58.2), 136.2 (C(5), J (C,C)=64.9), 115.9 (C(6), J (C,C)=64.9), 106.9 (C(14), J (C,C)=63.0), 106.4 (C(12)), J (C,C)=62.5), 101.7 (C(8), J (C,C)=58.2), 101.1 (C(10), J (C,C)=65.0), 71.2 (C(3), J (C,C)=36.8), 55.1 (C(16), J (C,C)=39.9), 41.2 (C(4), J (C,C)=36.8), 34.4 (C(1′)), 28.0 (C(2)), 26.5 (C(2′)), 22.5 (C(3′)), 21.8 (C(3′)); IR (KBr):˜=3448m, 2956m, 2553w, 1638s, 1597s, 1458w, 1420w, 1384w, 1163m, 1095w, 917m, 848m. Acetylation of HU235: HU235 (5 mg) was stirred in 1 ml pyridine/Ac2O (3:2) for 2 h, the mixture was poured into 2 N HCl and extracted with ether. Two products were isolated after chromatographic separation (hexane-EtOAc 3:2). HU235Ac2 (6): 1H NMR (400 MHz, CDC13): d=7.33 (d, J=2.1, 1 H), 7.02 (s, 1 H), 6.87 (d, J=2.1, 1 H 3.21 (d, J=16.5, 1 H), 3.00 (d, J=16.5, 1 H), 2.60 (dd, J=8.3, 4.0, 1 H), 2.38 (s, 3 H), 2.33 (s, 3 H), 1.78 (m, 1 H), 1.66-1.53 (2 m, 2 H), 1.37 (s, 3 H), 0.99 (d, J=6.4, 3 H), 0.93 (d, J=6.5, 3 H); MS (FAB+): m/z (%): 459 (15) [M-H+2 Na+], 437 (100) [M+Na+]; 415 (20) [MH+]; HRMS (FAB+) calcd. for C23H26O7 Na+437.1576, found 437.1589. HU235Ac3 (7): 1H-NMR (400 MHz, CDC13): d=7.57 and 7.55 (2 s, 1 H), 7.48 and 7.45 (2 d, J=2.1, 1 H), 6.95 and 6.93 (2 d, J=2.1, 1 H), 3.31 (m, 1 H), 3.15 (m, 1 H), 2.58 (m, 1 H), 2.47 and 2.45 (2 s, 3 H), 2.40 and 2.38 (2 s, 3 H), 2.33 and 2.31 (2 d, 3 H), 1.65-1.51, 1.78 (m, 1 H), 1.66-1.53, 1.42-1.34 (2 m, 3 H), 1.36 and 1.34 (2 s, 3 H), 0.96-0.84 (m, 6 H); MS (FAB+): m/z (%): 479 (100) [M+Na+]; HRMS (FAB+): calcd. for C25H28O8 Na+479.1682, found 479.1695. 
     Numerous modifications may be made to the foregoing systems without departing from the basic teachings thereof. Although the present invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention as set forth in the claims which follow. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. 
     
       
         
           
             1 
           
           
             1 
             23673 
             DNA 
             Human 
             
               misc_feature 
               (1)...(23623) 
               n = A,T,C or G 
             
           
            1
ccaccggggc tcgtccccgt gccgcaccgg gtcggttgtt cgtgtcgcgt cgccctgcga     60
cgacgcggca gtcactccgt gcggcggcaa accgggtttc acacgtcggt ggttcgcacc    120
gacgggcgtg ctcccggtcg tgggtcgagc ccggccgggc gtcgggcggc ggcggttcgc    180
gccgtcgtcc gtcgctccgg ccacgccggc ctcccccgtt gccgcgcgga tgcgcgacga    240
cgggaggccg gccagggtcc cggcggcggt ggttcgcacc gtcgcccggc cctccgagat    300
cgaggtcccg gtggtcgtgg gccgccgacg tgcgccgcgc ggtcgcgtcg ggtcgcatgt    360
cggtcggctc ggcttctaca gttcggggtt catcgcgcgg accacgacgg ccgcgttgaa    420
cccgccgtgg ccgcgggcca gcaccagcac cgtccgcacg cgcgcgttgc gcggttcgga    480
cagcacgagg tcgagcccgt agtcggccgg cacgtcggtg gtgttgggcg tcggcgggac    540
gacgtcgtcg cggatcgaca acagcgcggc caccacgtcg agcggcggcc cgcccgagtt    600
gaggcgtccc accaggctct tgggcgccgt caccggcacc gcgccggccc cgaacacctc    660
gttgatcgcc tcggcttcgg cgcggtccag gtccggtacg ccggccgcgt ccgcgaacac    720
cgcgtccacg tcctcgggcc gcacgcccgc gtcgtcgagc gcgaggcggg ccgcgcgggc    780
cagccccggc ggccgttcgg agccgggcgg cgggtcgaag gtggccgcgt agcctgcgat    840
ctcgccgtag agcgtggccg cgccccgggc ccgggcggcg cgttcgtcct ccaggacgag    900
gatggccccg ccctcccccg gcacgtagcc gtcggcggcc gcgtcgaacg gccggtaggc    960
cagctcgggt tcggtcgcgc gggagatccg tccggtggtc aggtggctga cgtatcccca   1020
cgggtcgagc gccgagtcgc atccgccggt gaccacgagg tcgatgccgc gccgcaggtt   1080
gcggcgcgcg tggccgatgg cgtcgaggcc gccggcttgt tcgccgacca ggacgccgct   1140
gggcccgcgc atgccgtgcc ggatggacaa ctggccggtg ttgacggcgt agaaccacgc   1200
gaacgactcg tagacgctga cgtgttgcgg tcccttggtc cagagcttgt gtacctcgcc   1260
gtgggtgaac tcgaagccgc cgatggcgct ggaggtgacg acgcccatcg agtagtcgac   1320
gagtgcctgc gggtcgacgc cggcgtcgtc gagcgcgctc tggccggcga cgagggcgag   1380
tcgcgtgacg cggtcggtct gcggcaacag ccttcccgga atgtgctgtt cggcctcgaa   1440
gccgagtatc tggccggcga gtcgggacgg gaatccggac gcgtcgtagc ggtgcagcgg   1500
gtggatgccg gagcgtccgc gcagcgtggc cttccagtac tcctcggtgc cgaagccgtt   1560
gggggcaacg gcgccgaggc cggtgacgac ggcgcgccgc gtcgtggggg ctgcctgggt   1620
catcgcgcgt cctcctgaag gcggcgcagc accatcgcgc tctggaagcc accgaagccg   1680
ctgccgaccg acagcacggt gtcggtgcgg tgttcgcggg cggtcaccgg cacgtagtcc   1740
aggtcgcatt cggggtcgga ctgggtcagg ttcgcggtcg gcggcacgac gttgtgctgg   1800
atcgcgagga cgctggcggc gatctcgatg gagccgatcg cgccgagcga gtgcccgatc   1860
atcgacttga tggagctgac cgggacgtcg taggcgtggt cgccgagggc gaccttgaac   1920
gcgttggtct cgtgccggtc gttctgcttg gtcccggacc cgtgggcgtt gatgtagtcc   1980
accgccgtcg ggtcgatgcg cgattcgttc agcgcgtagg tgatggcgtc ggccatctcc   2040
cgtccgtcgg ccttgagtcc ggtcatgtgg tacgcgttgc agcgggtcgc gtagccggtg   2100
acctcggcgt agatgtgcgc gccgcgcgcc ttggcgtgct cgtactcctc gaggacgaac   2160
atcgccgccc cctcgccgat cacgaatccg ctgcgggtca ggtcgaacgg cctcgacgcg   2220
gactcgggtt cgtcgttgcg cggggtggtg gccttgatgg cgtcgaagca cgcgacggcg   2280
atcggcgaga tcggcgcgtc ggtggcgccg gtgaccatga tgtcggcggt gccctcgacg   2340
atgagctggt aggcgtagcc gaccgagtcc aggcccgagg tgcagccgga ggacacgatg   2400
gtcgcggggc cctccgcgcc gacgctccag gccacttcgg cggcgatcgc gccgggcgtg   2460
aagtagtcgt acatgtgcgg cgtggcgtag tcggcgtcga ccacccagtc gcgtccggag   2520
tcggagagca cgaggtattc gcgttcgagg ctcgtggtgg cgccgacggc gctgccgacg   2580
ctgactccga tccgggtcgg gtcgaggtcg gcgaacgcca gtccgctgtc ggcggcgcag   2640
tcccgcgcgc tggcgacggc gaacagcgac gcgcggtcca ggcggcggat ctcgcggtgc   2700
gtgaagccgg ccaggaccgg gtcgaagtcg cattccgcgg ccacctggga ccggaacggc   2760
gacgggtcga agtgggagat gcgccgggtg gcggtgcggc ctgaggtgag caggtcccag   2820
aaccccttgg ttccggtgcc gcccggggcc acgaccccca tcccggtgat caccgctcgt   2880
cgttccaaga caagcctcca gcggtggtgt cggttgttgc tgtgctcttc gagcgtgcgt   2940
ggcgcgactc gaaccgtgct caaggacgga tcgcgcggag tcgcgggccg ttgtcgagtg   3000
ggggtcgagt ggcgtgcgcg agcctcgtgg acccctgtag atgtactcgg gtcgaggaga   3060
cctccgatga cgatcagact ggttccgccg cgccgccgtg tcgccctgct gctgccgggg   3120
cagggctcgc aacaccccgg tatggggctg gagttgtacg gctacgacgg tgtgttcacc   3180
gagacgatgg acgagatgtt cgagctgctc gggccggtgg acggcgcgtc gctgcgcgcc   3240
gactggctgg ccggcgacgc gctcgcgccg gagctgctga acgacgcgag ccgcgcgcag   3300
ccgctgttgt tcgcggtcga gtacgcgctg ggccgcagcc tgttgtcgca gggcgtcgac   3360
gtggacgtgc tgctcgggca cagcgtcggc gagttgtcgg cggcggcgtt ggccggcgtc   3420
gtcgacctgc cgggcgcggc gcgcgtgatg gcggcccgga ccgcggcgtt ggccgcgctt   3480
cccccgggcg ggatgctcgc ggtcgcggcg accgccgagg agttggcgcc gttcgtggcc   3540
gccgagcacg tcgagatcgg cgtggtggtg ggcgcgttga acgcgccgcg gcagaccgta   3600
ctggcggggc ccgagccgcg gttgaccgag gtggagggcg aactgcgggc ggccggtgtg   3660
atgttccgtc gggtgccggc gcggcagccg ttccactccc ccgcgtcggc gccggccgcc   3720
gaggtggtgg agaaggtgtt gtccggggtg acgttgcgcg agccgacgat cccgatcatg   3780
tcgacgcact cgggcgccta cgtgacccgc gagcaggccc tcgacccggc cttctgggcc   3840
tggcacttcg tgcgcgtggt gttgttctgg ccggcgttgg acgcgctgct ctccgaggcc   3900
gaccacgcgc tcgtcgagct gggcccgggc caggagctga cggtgctggc gcgccggcac   3960
aaggcggtcc ggtcgggcgc cagcacggtg cgcgcggtgt tgccgcgcgg ggcgcgcggt   4020
acgcgggagg cgtgggacga cgcgctggcc gaactggccg agtacaccgg gccggccggg   4080
ggtgtgccgc tcgcctgcgc ggcggaacgg tgaacgtggc actccgccgc gggcggcgcc   4140
gccaacgccc ttgagaagcc cggaccgtgg cggtggtccc gtgtcctcgt caccttccgg   4200
tgggcgacgt cgcctcctac attggcgggt aaggcggcga ggacatggag gctcggatga   4260
gcggtgcctg ggtgcgcagt gccgacggac tctcgttgat ccgcgcggtg gagatcgtct   4320
ccctgagggt caacggcacc gccctcgaat gcgactcgcg cctggcccgc gccttcgtcc   4380
tggccaccgg cgccgaggag gatctggtcc ggggcgcacg ggagttgatc gagacgctgg   4440
cccgactgcc gcacgacgcc tacatcgtct cggccgatgt gaccgacggg cgtgtcgtgt   4500
gggcgctctc cgagatcggc ccggtcgagg acgacccgcc gtacgccgcg ggcgggcagg   4560
cttacggggc gaccggccag gtgtacggcg cgggcatgcc cggtaggccg atgccgggga   4620
tgccgatggg gggcgcgtgg ccgccctggg gcaacccgcc gccgcacagg tgagcctcgg   4680
gtccggcaca tcccgttccg ggcccggccc ccgcctcgcg gtacgcgaac acccccgccc   4740
ggcccgcggc gtcgtcgttc gcgggccggg cggaggcggg ggtcggcgtg cctgggagca   4800
ccgccgggca cgccgaccgc ttcacgcgga caggggcact cccgtccgcg cgttccgggc   4860
ctcggcggcg agcatctcgt cgacgaggga ggtgctgtgg cgtccggcgg cgaacttgtc   4920
gttgtcgaac acctcgcgca ggaacccgat ggtggtgcgc acccgttggc cctcgacctc   4980
gaactggtcg agcgcgcggc gcatgcgggc gagggcctgt tcgcggtccg gtgcccacac   5040
cacgagcttg gccagcagcg agtcgtacga cgcgggcacg gtgtacccgg cgtgcacgtg   5100
tgtgtcgacg cgcacgaacg ggccgccggc gggcgtgaac gtgtcgatca gccctggcgt   5160
cggcacgaat ccgcgcgtcg ggtcctcggc gttgatgcgg cactccatgg acacgccgtt   5220
gggtcggatg tcttcctggc gcaccgtcag cggacggccg gcggcgacca ggatctgctc   5280
gcgcaccagg tcgatgccgg tgaccatctc tgtgaccggg tgctcgacct ggatacggca   5340
gttgacctcc atgaaggaga acgttccgtc gggcgcgagc aggaactcga acgtgccggc   5400
gccgacgtag ccgaccgcgc gcgccccggc gacggccgcc tcgcccatcc gggcggcgag   5460
gccgtccgga agccggggcc cgggcgtctc ctcgacgagc ttctggtgcc ggcgctgcac   5520
ggagcagtcg cgttcgccca ggtggacgac gttgccgtag cggtcggcga gcacctggat   5580
ctcgacgtgc cgggcctcgg gcaggtagcg ctcgacgtac acccgcccgt cgccgaacac   5640
cgccaacgcg gtggcctggg tgcggcggaa cgtgtcgggg aagtcgtcgc tggagtggac   5700
caccgacatg ccgcgcccgc cgccgccggc cgccgccttg acgatgacgg ggtagccgat   5760
cgtgtcggcg agcgcgtgcg cctcgcggtc ggagagcggg tcgaggctgc cgggcagcag   5820
cggaaggccg gcctgcgaca tgatcgaccg ggcggacgcc ttgttcccga gccgcgacat   5880
cacctcggcg ggcgggccga tgagggtgat gccctcgtcc tcgcacgcct cggcgaagtc   5940
cgggtcctcg gacaggaatc cgtagccggg gtggaccgcc tcgacgccgg tctgcacggc   6000
ggcctggagc acggcggcgg cgttgaggta gctggcgcgc ggcggggcgg ggccgacgca   6060
cacggcctcg tccgcgaacc gcacggcggc ggagtggcgg tcggcatcgg agtgcacgac   6120
gacggtccgg atgccgagct cccgacaggt cgcggcgaca cgcagcgcga tctcgccgcg   6180
gttggcgatg agtaccttct cgaacatgtc gggctacgcg ccttccacgg ggaccagcgc   6240
gacgagcggc tcgccgtact cgacctggtc gccgttggcg cgcaggatcg cggacacccg   6300
gccgggacgg tcggcctcga cggggagcat gagcttcatc gcctcgacga tgccgatctg   6360
ttgaccgggg cggaccacgt cgccgacccg cacgaacggc gcggcgccgg gctcggtggc   6420
gtggtagaac acgccgacgc tgggcgcgca cacctggtgc gcggtcggct ccgccggggg   6480
cgcgggcacg ggcggcggcg cggcgacggc gggcacggtg gcgacgggaa cggcggacac   6540
gacgggctgg ggcaccgccg gggcctgcgg cgcctcccag gacacctcga tcccgacgcc   6600
gtccgcgcgc acgttgagcc gcgtgggccg ctcgggcaac gcttcgagca tctcgatgac   6660
ggagtgccgc agggtgttca acaaccgcgc ggcgtcctcg tcggcgccct cgccgccgcg   6720
taccgcgggg tcggcgatcg gggtgatggt cacggtgtct ccttctcctt gattcctgta   6780
cgtctacgcg gccgcgccac ggacggcccc tcgggacaca ccacctgcgc ggccgccccg   6840
accccgtgcg cgagcgccac ggcgagcccg cccgagagca cgagccgaaa cccgaccccg   6900
accacacccg cgacggtcga cggggagtcc tcccccggcc gcacgtggtg ggcgcgaagc   6960
aacgcccgcg aaacgggctg aacggcttcg acgaggggct gaggcgtcat gccgtcacca   7020
cacacgcgcg ggtcgcgcgc accaggccga agcagcgagg cggcggggcc gcggatgcga   7080
ggacgccgct gggcgacgcg gcgactcgag gcaccgacac cccgccgtca cccccacaac   7140
cgccgcgcgc agcgccggtg ggtctcacgt cgggtcgcct ttcatggcgg tgatctcggc   7200
gtcggtggtg cgttccgcgc catagcggcg gaagcgggcc cagcggtcgg cgaccaactg   7260
gtggccgtcg aggggggaca actccgcgag gccggccgcg atcgcgtcgc ggagggcgtc   7320
ggcggcgcgc agcgggtcgg ctccggtgcc gccttcgggt tccgggagga cgccgtcgac   7380
gatgcccagg ccgaggagtt cgcgggcggt gaccctcagc gcgcgggcgg cacgcccggc   7440
ggcggcgggg tcctgccaca ggatggccgc gcagccctcg gggctgatga ccgagtagat   7500
gccgttctcg aacatcagga cacggttggc gacggcgagc gcgagcgccc cgccgctgcc   7560
gccctcgccg gtgacgacgg tgacgaccgg cacgcgcagg cccgccatca ggcgcaggtt   7620
ctcggcgatg gcgacggcct ggccctgctc ctcggcgacc gcgccggggt aagcgccggg   7680
ggtgtcgacg aacgtgacga ccgggagacc gagtttgtcg gcgagccgca tgatccgggc   7740
ggccttgcgg tagcccgacg ggatcggcat gccgaagttg cggcggcgca gctcctgcgc   7800
gtcgtggccc ttctggtggc cgatgacggc gacgggtcgg ccgccgaagc gggccaggcc   7860
ggcgacgatg gcggggcagt cgcccgagat gcggtcgccg cgcagttcga cgaagtcctc   7920
gcacagcagc gcgatgtagt cgagcgtggt gggccggtcg atggcccgcg cggcgcgcac   7980
ctggcggctg gggtcgtgcg cggcgaggtc ggcggggtcg acgacgtacg gcgatgcccc   8040
cgcgtccagg ccgccgcgtc gggccggttc cccgtccccg cccttgggcc gcttcgcgcc   8100
ggcccgcagc aggcgggcca gttcgtcgcg cagcgcgcgg cgcggcacga tcaggtcgat   8160
caggccgtgt tcgaggaggt attcggcggt ctggaactcc ggcggcagtt cctcgcggat   8220
ggtctgcgcg atgacccgcc ggccggcgaa gccgagacgc gcgccgggtt cggcgacgat   8280
gacgtcgcac agggtcgcga aggacgccgc gacgccgccg taggtggggt cggtgaccag   8340
cgagatggtc aacacgccgg cctcgtccag ttgcccgagg gcggcgctgg tcttggccat   8400
ctgcatgagc gcgacggcgc cttcctgcat gcgcgcgccg ccggacgcgc tgacgatcag   8460
cagcggcagg tgttcggcga gcgcggtctc ggcggcgagg gtgatcagtt cgccgaccgc   8520
gccgcccagg ctgccgccga ggaacgcgaa gtccatcgcg gcgacgacga gcggctggcc   8580
gtggatccgg ccggtcacga tgacgacggc ctcgtcgagc ccggtggcgg cacgcgcggc   8640
ggcgagccgc gcggggtagg gcttggtgtc ggtgaagccg agcacgtcga aggtgcgcac   8700
gtcgttgcgg atcggctcgg cggagccggg gtcgaggagc tggtcgaggc gttcgcgggc   8760
gccgaccggg tggtgtttgg cgcattcggg gcagacccgc aggccgcggg cccagcgctt   8820
gccgtacacg gcggcgcggc agttgccgca ctgcacccag tcggcgacgc cggtcgtggg   8880
ccggatggcg gtttcggtca tgtcgttgtc tccctcggtt ctcagcccgc ggtctcgacg   8940
agcaggccgt tgacgaacgc gaccatgtcg cgcggtgtct ccaggccgtc gatgtcgtcg   9000
tcggagatgg cgacgccgcg ctcgtcctgg atccgggtga cgacctcgta gacggcgagc   9060
gagtcgaggc cgaggtccag gaacggggtg gccgcgccgg tctcgtcgag ctggaccgcg   9120
tcgtcggtgc cgacgcacgc gtcgatcagc cgcttgaggt cgtcgagcgt gaaggggtcc   9180
atgggttcgt cctcccggtg gtctggcttc ctgccgaacg cagacagcca ctgtcggccg   9240
gccggatgga ggtctggtcg agagccgctg gatgtgcgcc ggagggcgcg gcgtcggctt   9300
cgagtcggct tccggcgggc ttcgaggggg cgccgcgcat gctcggtgcg cgcccctctc   9360
ccgccggacg ccgccggacg cccggcgggt cccagacggg gttcaggcgg gtgtcaggcg   9420
agaccagaac cgcgaaaggt ggtcgcgatg cccggacttc gggttcccga gcggcgtttc   9480
agccgtgttc tcggggtcgg ttcgtaccgg ccccgccgtg aggtgagcaa caaggaggtg   9540
tgcacctgga tcgactcgac ggaggagtgg atcgagacgc gcaccgggat caggtcgcgg   9600
cggatcgccg agccggacga gacgatccag gtgatggggg tggccgcgag ccgccgcgcg   9660
ctggagcacg ccggcgtgga tccggccgag atcgacctgg tcgtggtctc cacgatgacc   9720
aacttcgtgc acacgccgcc gctttcggtc gcgatcgcgc acgagctggg tgccgacaac   9780
gccggcgggt tcgacctgtc ggcggcgtgc gcggggttct gccacgcgtt gtcgatcgcg   9840
gcggacgcgg tggagtccgg cggttcgcgg cacgtgctcg tggtggcgac ggagcggatg   9900
accgacgtga tcgacctggc ggaccggagc ctgtcgttcc tgttcggcga cggcgccggc   9960
gcggccgtgg tcggcccgtc cgacgtgccc ggcatcggtc cggtggtgcg cggcatcgac  10020
ggcaccggcc tgggctcgct gcacatgagc agttcgtggg accagtacgt cgaggacccc  10080
tcggtgggcc ggccggcgct ggtgatggac ggcaagcggg tgttccgctg ggcggtcgcg  10140
gacgtcgtcc cggcggcccg cgaggcgctc gaggtggccg gcctgaccgt cggggacctg  10200
gtggcgttcg tcccgcacca ggccaacctg cggatcatcg acgtgctggt ggaccggctc  10260
ggcgtgccgg agcacgtggt cgtctcgcgc gacgcggagg acaccggcaa cacctcgtcg  10320
gcgtcggtgg ccctggccct ggaccggctg gtgcgctccg gcgcggtgcc cggcggcggg  10380
cccgcgctga tgatcggttt cggcgccggg ctgagctacg cgggccaggc tctactgctt  10440
cccgacccgc cgtccactcc ggcttgagcg gcgttctagc cgccgcccgc aagctcggca  10500
tcaaccccag tcggccgcat cacccgtcaa ggagatccac cgtgatcgtg cacaccctga  10560
tctaccgttt cccggcctcc gtcccgcagg aggacctgga cgcgttcttc gacgcggcgc  10620
gcgagctggt gctgagcacc ggcctgatga acgcgttcga cgtcaagccg cacctgtggc  10680
tgcccgccga cgaccgcgcc cgcggcatga cggccggcta catcgtgcag ttcgtgtgcg  10740
acgacctgga cgcgctcgcg aagttctccg agctgcccac cgtctacgac ttcatcacgg  10800
actggaaggc ccgcctgcag ttcgaggcgg cgtacgccaa ccacgaggcg ctcgacctca  10860
cccccaccgc ctgagcccgc ccaaccaccg aggagcaccc tcatgcgtca cgtcgaacac  10920
accgtcaccg tagcggcccc ggccgacctg gtctgggagg tgctggccga tgtgctcggc  10980
tacgccgaca tcttcccgcc gaccgagaag gtcgagatcc tcgaggaggg tcagggctac  11040
caggtcgtcc gcctgcacgt ggacgtcgcc ggcgagatca acacctggac ctcgcgccgc  11100
gacctggacc cggcccgccg cgtcatcgcc taccggcagt tggagacggc cccgatcgtc  11160
gggcacatga gcggcgagtg gcgcgccttc acgctggacg ccgagcgcac ccaactggtg  11220
ctcacccacg acttcgtgac gcgcgcggcc ggcgacgacg gcctggtggc cggcaagctc  11280
acccccgacg aggcgcgcga gatgctggag gcggtcgtcg agcgcaacag cgtcgccgac  11340
ctgaacgcgg tgctcggcga ggccgagcgt cgggtgcgcg cggccggcgg cgtcgggacg  11400
gtgaccgcgt gagcggccgc aagacgttcc tcgacctgag cttcgcgacc cgggacaccc  11460
ccagcgaggc caccccggtc gtcgtcgacc tgctcgacca cgtgaccggc gcgacggtgc  11520
tcgggctgag ccccgaggac ttccccgacg gcatggccat ctcgaacgag acggtcaccc  11580
tcaccacgca caccggcacg cacatggacg cgccgctgca ctacggcccg ctcagcggcg  11640
gcgtgccggc caagtcgatc gaccaggtcc cgctggagtg gtgctacggc ccgggcgtgc  11700
gcctggacgt gcggcacgtg ccggccggcg acggcatcac cgtcgaccac ctcaacgccg  11760
cgctcgacgc cgccgagcac gacctggcgc ccggcgacat cgtcatgttg tggaccggcg  11820
cggacgccct gtggggcacc cgcgagtacc tgtccacctt ccccggactg accggcaagg  11880
gaacgcagtt cctggtcgag gcgggcgtga aggtgatcgg catcgacgcg tggggcctgg  11940
accggccgat ggccgcgatg atcgaggagt accggcgtac cggcgacaag ggcgccttgt  12000
ggccggcgca cgtctacggg cgcacccgcg aatacctcca gttggagaag ctcaacaacc  12060
tgggcgcgct gcccggcgcc accggatacg acatcagttg cttcccggtc gccgtcgcgg  12120
gcaccggtgc gggctggacc cgggtggtcg ccgtgttcga gcaggaagag gaggactagt  12180
ccatggacgc cgacgtcatc atcgccggtg ccggccccac cggactcatg ctggccgccg  12240
agctgcggct ggccggcgcc gacgtgctga tcctggagct gctgccggag cccaccggcc  12300
agtcgcgcgc gctcggcatc aacccgcgcg ccgtcgaact gctggacgcg cgcggcctga  12360
tgggccggtt cgccgaccag cacaccatca ccaactcgca ctacggcgcg ctgcaccgcc  12420
cgctggactt cagccggctc gacaccgcgc acggcgtcat gatggtgccg cagcgccgca  12480
ccgagaagga actgggcgag tgggcccggg ggttgggcgc acgaatactg cacgggcaca  12540
ccgtggtcgg actgacccag tacgccgacc gggtcgaggt cgaggccgac acgtccgagg  12600
gcacacgcac cttcaccgcg cggtacgtgg tcggcgccga cggcagccgc agcagtgtgc  12660
ggcagttgtc cggcatcggc tacccgggcg tgccgtcgac cgtcgacgcg ctcatgtgcg  12720
acgtgtcggg cctggacctg gccttcaagt tcttcgagcg caacgaccgt ggcttgtggg  12780
cggtgttccc ggtcggcgac ggcgtcttcc gggtcgtggt ctacgcgttc gaccgcccgc  12840
cggtgcgcgg cgcggacgcg ccctccttcg aggaggtcaa cgacgcggcc cgcaccattg  12900
cggggatcga cctgaccggc ggcacgccgc actggctgag ccggcacggc aacaccaccc  12960
ggatcgccga ccggttccgc gccgagcggg tgttcctggc cggcgacgcg gcgcacgtgg  13020
tcccgccggc gggcggccag ttgctgacca ccgggctgca cgacgcgacc aacctgggct  13080
ggaagctcgg cgcggcggtc gccggctggg ggtccgacga cctgctcgac agctaccagg  13140
acgagcggta cccggtcgcc gagcggatca tcctcaacgc gcgcgtgcag aacctgctca  13200
tgtcgggcgg cctggacatc gccgcgctgc gcgaactgtt caccgagctg gtcgagttcg  13260
acgaggtgaa cttctacctg tcgggtcagc tcagcgcggt gtcggtgcgc tacgacgtcg  13320
gcggcgacca cccgatgctg ggcctggggc tgccgcaccg cacgctggag accgacgcgg  13380
gcgaggtgac caccaccgag ctgctgcggt cggcgcgcgg gctgcttctg gaggtcgggg  13440
acgcgcctgg cttcgcggac ctggtcgcgc cgtacgcgga ccgggtggac gtccgggtcg  13500
cgcgcgtccc ggccggctcc cccgggttcg acggggtcac cgccgtgctg gtccgcccgg  13560
acacgcacgt cgcgtgggtc gccgagaccc ccgcgggcgc cgacgcggcg gacgcggacc  13620
gcgacgcgct cggcaccgcg ctgcgccgct ggttcggcgc gccggccgac acgaactccg  13680
gcgcggaatc cggtacggag tccggcacgc agaccgccgc gcccgtcggc gccgggaccg  13740
ccgggggcac gccatgaccc tcgacgacgg cgcggtcctc gcgcccgccg ccgtcctgac  13800
ggcggcgctg cgccgccggg agatctccag ccgcgaactg ctcgacctgt acctggcccg  13860
cgtcgaggcg gtgaacccgg cgctgaacgc cgtggtcacc ctcgacgtgg aacgggcccg  13920
ccgcgaggcg gccgaggcgg accgcgcgac ggcggccggc gcccgtaccg ggccgctgca  13980
cgggctgccg atgacggtca aggacaccct ggagaccgag ggtctgcgca ccacggccgg  14040
cgccgcggaa ctggccgagc acgtgccggc ccgcgacgcg gactcggtgg cgctgctgcg  14100
ccgcgcgggc gcggtggtgt tcggcaagac caacaccgcc acctacgcct ccgacgcgca  14160
gacctacaac ccggtgttcg gcaccaccaa caacccgtgg gacacctccc gcgcgccggg  14220
cgggtcctcg ggcggcgcgg cggccgcgct cgccgccggg ctgacgtcgc tggaactggg  14280
cggcgagctg tccggttcgg cgcgctaccc ggcgcactgc tgcggcgtgt tcgcgctgcg  14340
gccgtccttc gggatcgtgc cgatgcgcgg ccacatcccg cgccaaccgg ggtcgctgaa  14400
gaccaacgac atggtgacgc tggcgccgct cgcccgcagc gcggccgacc tcgacctgct  14460
gctcgacgtg ctggcccggc cggccgccga caaggcgccc gcgtggcgcc tggacctgcc  14520
cgcgccgcgc gccgaatcgc tgggcgggct gcgcgtgggg ctgtggctgg acgacccgct  14580
gtgcccggtc gacgccgagg tcggcgacgt gctggcggcg gcggtcgacg ccctcaaggg  14640
caccggcgtc gcacggctgg cggacgtcac gccggtcgac cgcgcgacgc acgaccggct  14700
ctacgagcgg ctgttgcagg gcgcggtcag tctcggactg cccgccccgg tgtacgcggc  14760
caaccaggcc gcggccgccg cgctcgcccc cgacgacgac tcgccgcgcg cgtcgttcct  14820
gcgcgccgcg acgctgtcgc accgcgactg gctggcggcc gacgaggagc gcgagcacca  14880
gcgggcccgt tgggccgagg tgttcgacga gttcgacgtc gtcctgtcgc ccgtcgcgcc  14940
ggtcgtcgcg acgccgcacg accagcgtcc cgacctgtcc gcgcggcgga tcaccgtcaa  15000
cggcgcccaa cggccctact gggacatcat ccgctggacc agcccggcca ccgcggccgg  15060
cctgccggcc gcgtccgtcc cggtgggggt ggcccgttcg ggcctgccgg tcgggctcca  15120
ggtggtcggc ccgcacctgc acgaccgcac cgtcacgtgg ttcgcccggc gtgtctcgga  15180
gctgctcggc ggcttccagg ccccgcccct gcgcgccgcc gggaacgacg gggcgtcccc  15240
cggatcatca cgaacggaag gagcggaccg tgcctgagtc cgacggcatc gacggcacct  15300
accccgtcgt catcgcgggc ggcggcccgg tcggcaccat gctggcgtgc gggctgcggc  15360
aggccggcgt cgacgttctg gtgctcgaac gccgcaccga gcccgacctg ttgccgcgcg  15420
ccggctcgat gggcccgctg gcgttcgagg cgctggaacg gctcggcctg cacgacgcgc  15480
tgctggccgc cgagcaggac acgctcgccg agtacggcaa gatgttcgcc gactgggcgg  15540
ccaagaaggg catcggccag ccggccaccc gctccgcgcc caaggagcac ttcgcgggcc  15600
tggagaagat cgacccggcc cgccgcaccg accccgagcg ccgccgggtg cgcgtcgagc  15660
agccggtgct cgaggagatc ctgcaccggc acgcggtgtc gctcggcgcg gtgatcctgc  15720
gcggccacga gatcgtcggc gtgttccagg acgaggagcg cgtcatcgtc gacgtgcgca  15780
ccgtcgacgg gaccgtcgcc caggtgacca cgcagtacct ggtcggcgcc gacggcgcgg  15840
acagcagcgt gcgcggcttc gtcggcttcg acttccccgg caccgacgcg accgtcaccg  15900
gccgcatggc ggtcgtggag ttggccgacg aggagaaact ctccccgggc ttccactaca  15960
cgccggtcgg cctgtacgtg cacggcctgg gcgtcaaccg gctgtcgacg gtcgagttcg  16020
acggtccgcc gaccgacgac gaggacatga ccgccgagga gctgcagggc agcatccgac  16080
gggtcagcgg cacggacgtg acgatcagcg agatgtcctc gggcacccgc tggatcgaca  16140
cggcgcggca ggcgtccgcg taccggctcg gccgggtgct gctggccggc gacgcggcgc  16200
acgtgtacgc gccggtcggc ggccagggcc tcaacgtcgg cctggtcgac gcggccaacc  16260
tcgtgtggaa gctggccgcg caggtacagc gctgggcgcc gcactatctc ctcgacacct  16320
acggttcgga gcgctacccg gtcgccgcgc ggctgttgca gaacacccgc gcgcagatcg  16380
cgctcatgca ccccgacccg cagaccaccg cgctgcgcga gatgttcgag gaactgctcg  16440
acatcgacga ggtgcaccgc acgatcgccg acatgatggc cggaatggac gtgcggtacg  16500
ccgtcgaggg cgagcatccg ctcctcggca cgctggtccc ggacgccaag ctcaccggcg  16560
gcaaggccgc cgacctgtgg gcggacgggc gcggcgtgct cgtcgacttc gccgaccgcg  16620
cggacgtgcg cgacgcggtc ggcgcgtggt cggcgcgggt caacgtcgtc acgttgcccg  16680
gcgcgcgcga ggacgtcgac gcgctgctgg tccggcccga cggctgcgtg gtgtgggcgc  16740
tgcccaccgg cgcggcgctc gccgacgcgc ccccgaccga ggccctgacc acgtggttcg  16800
gacgcgcccc gcgcgtgtcc tgagcaatcc caccccatcg cgaaggagat tccgaacacc  16860
atgacgatcg acgacctgcg caggatcctg accgagtgcg ccggcgagga cgagtccgtc  16920
gacctgggcg gcgacatcct ggacacgccg ttcaccgagc tgggctacga ctcgctcgcg  16980
ctgatggaga ccgccgcccg catcgagcag gagttcggtg tcgcgatacc ggacgacgag  17040
ttcgccgaac tggcgacgcc gcgtgcggtg ttggccgcgg tctccacggc ggtctcggcc  17100
gcggcctgag ccgcggcccg gcccccgggt tccgaggcgc ggtgcttgcg gtgcccgcgc  17160
tgccgcggtg ccgcagcccg gttccgcggc cggaatccgg ccaccgtctc ccgtgtcccg  17220
cccccatcgg ctcaggggcg ggacacgggt gtgttccgcg ccgcgactcg cccccgagcg  17280
tgccggtgtg aggtaatcgt cccgggaaag ggaaggcgtc gccatgtcgc tcggtcgggt  17340
gctcgtcgtc gacgacgaac ccaagatccg catgatcgtc cggggttacc tcgaggccga  17400
cggctacaag gtcgtcgagg cgtgccacgg tcgcnaccgg cccgacttgt tccggacggc  17460
ctgcgcgcca tcgtgcgcga ccggcccgac ctggtcgtcc tggacgtgat gttgcccggc  17520
ctggacggct tcgaggtgct gcgccggttg cgcgccaccg tcgaccggac cccggtcatc  17580
ctgctgaccg cgcgcgccga ggaggtggac cgggtcatcg ggttcaccgc cggcggcgac  17640
gactacgtca ccaagccctt cagcgcccgc gaactcgcct tgcgggtccg cgcggtgctg  17700
cgccgctgcc agccggacga gcgggccgcc gagcccgacg acacgctgcg cttcgccgac  17760
atcgagatcg acccgaccgc gcgggtcgtc accgtcgacg gcgcccccgt cgggccgctg  17820
accgcactgg acttcgacct gctgctcgca ctcgcccgcg cgcccggacg cgtcttcacc  17880
cgccaccggc tcctcgaaca cgtctggggc cgcgacgcgt tcggcgacga ccgggtggtg  17940
gacgtgcacg tgcgcacact gcgccgcgcg ctcggggacg acgccgccgc gccgcgattc  18000
gtcgggacgg tgcgcgcggt cggctaccgg ttcctggccg gcgccgccgc ccacgcctga  18060
gccggcgagt gtgagactcg ggcgagcccc gcttcacaca agcttcacgg agcgcaccgc  18120
cgtcgtcaca cgaactcccg gtgtgctgga ggcatgacaa ccaccgaagc ggcacctccc  18180
cccgcagcgt ccacgcgcgt ggccgcctcc gtcgtcgccg gcgtcaagtc ctacggttcg  18240
ggcgacgacg agatccgcgc gctcgacggc gtgaccatag agttcgcctc cggcgagttc  18300
accgccgtca tgggcccctc cggctcgggc aagtcgaccc tgatgcactg cgcggcgggc  18360
ctggaccggc tcacctcggg ccacgcgtcg cgtacatcgg cgacaccgac ctcggcaccc  18420
tgccggaccg cgagttgacg ttgctgcgcc gcagccgggt cggcttcgtc ttccaggcgt  18480
acaacctcat ctccacgctc aacgcgcggg agaacatcct gctgccgcag aacctcgccg  18540
gcgtgcgccc ggacctggag ttcctgggcg aggtggtgga ccggctcggg ctcggcgcgc  18600
gtctgcggca ccgccccggc gagttgtccg gcgggcagca gcagcgggtc gcggtggcgc  18660
gcgccctggt cagcaggccg gacatcgtct tcgccgacga gccgaccggc aacctggaca  18720
gcgccgcgag caccgaggtg ctggagttcc tgcgcggcac cgcgaacgag ctgtgccaga  18780
ccatcgtgat ggtcacccac gacccgcaca ccgccgccta cgccgaccgc gcggtgttcc  18840
tcgccgacgg ccgggtcgtc gacagcatcg ccgcccccac cgccgaagcg gtcctggagc  18900
gtatgaaggg cttccgaagg tgaacggttt ggtgcgcgcc gcgctcgccg gtgtgtgggt  18960
gcgccgcgct cggtatgcga tgaccgtcgc ggcggtcgtc ctgtcggtca tgttcatgac  19020
cgccaccctc atgctcacca gcggtctggg cgaccgcgag aaggaccaga tcacccgggc  19080
cggcgccggc gtcgacgcgg tggtgcgcgg cccggtggtc gccgacccgg agggcgggcc  19140
cggcgaggtg cagacgcccg cgcgggtgcc ggtcgacgac gcggtgctcg cggcggtacg  19200
ggcgaccccc ggcgtgagcg cggcggccgg cagcgtcaac ggctatgcgc gcctggcccg  19260
ttccgggtcg cggctgctcg gtttcgagga cgccacccgc gacgtgggct cggcgtggat  19320
cgacgacgac gcgctgaatc cgtatcggct cgccgagggc cacgcgccgc acggcgcggg  19380
cgaggtcgtc gtggacgcgg ccgccgcgcg ccgcgaccac atctccgtcg gcgaacggtt  19440
gtcgctgctg accccgagcg ggcgcggcga cgtgacggtc gtgggcgtgg cccgctacgt  19500
cggcgacgat gggccggcgg acaagagcgc tgtgctgctc gacgcggccg tcgcgcccgg  19560
cctgttcggg cacgcgggcg gctacgaggc catcctggtg cgcggcgccg gcgggcacga  19620
cgcggcggca cgcgaggcat tggcgacctc ggtggggcgg acggtcgcgg ccgtgccgcg  19680
cgccgggccg gtgccggcac cgggcgcggt cgccgggccg gtgtggacgg gcgagctgcg  19740
cgacgcggtc ggcaccgcgt acgacaccct cacgatcatc ctggacggcg cggcgttcat  19800
cgcgctgctg gtcggcgccg cgctggtgca caacacgttc accgccgtca ccgcgcaacg  19860
gctgcgcgaa ctggcgctga tgcgggcact gggcgccacc cggcggcagg tcaccgcgtc  19920
ggtgctggcc gaggcggcgg tggtcggcgc ggtggccggc gtgctcggga tagcgctggg  19980
cgcgctcgcc gcgcggatcc ggcgcatgcg cttggcgatc tgctcgactt cggtgacctg  20040
ggcccgttcg aagatcaggt tgagcagccg gtctttcatc ggttgcctcc tcgtgacgaa  20100
gggaaacggg agccccgggt agggccgggg cttccgtgct caccggacgg gcggtccgcc  20160
aaccgcgccg tccgtcggcc ggagtcaccc gaccagttgg tactcgcggc cttccagcgc  20220
gggatcggcg tcgaggatcg cgcgctgcaa ctggtgcagc cacggcgacg gttcgaggcc  20280
gagctcgtcg agcatcgtca ggcgcagccg ctggaacacg tccagcgcgt gggtgcgcct  20340
gccgcaccgg tacagggcca gcatgagctg gccgtgcagg ccctcgtgcg tccggtcgag  20400
cgcggcgagc ccggacagct cgtccacgat cgtgtagtgc cggccgagcg cgaggtcgat  20460
ctggatgcga cggtcgacca ccgcgcgacg ccggccttcg aggcggagga tctccgcttc  20520
gaggaccggg ccgacgtgca cgtcgaccaa ggcggggccg cgccacagcg cgacggcctg  20580
ccgtaagagt tccgaggagg tgcggtagtc gccctcctcg cacttgcgca tgcccaggtt  20640
cgcgaggcgg tcgaactcga ccacgtccga catgccgtcc gggctggcca acaggtagcc  20700
ggtcccacac gtcatgatga tttccttggc gcgcgccttg gagttctccg ccgacgttcc  20760
cgcgaccagt cgcttcgcga tacggttccg gatatgcaga atataagtct gcaacgtcgt  20820
cgaagcgctc ttgggaaggt ccacgggcca cagctcgtct cggattctcc gagtcgtgac  20880
cacgttgttc gaattgactg ccagcagcgt cagaaggcta cgcggcttgg gggcgctcgg  20940
gacgacggtc aagccgtcca gatcgacctg cagcggtccc agtatgccga tatccaccat  21000
catcccccga tgtgaataca tccttggagc ggctgtcatt gggtttttga cctgcgattt  21060
tgtggacggt aattcccctc ttcccctccc ttgcggagca ctttccggta ccgtcgcacg  21120
cgggtcgcca tcccgatctc ggacactatc acccggtgcg gaactgccgt gttgccggaa  21180
cgtaaaaatg gctgccaata ggacggttgg tgcactgccg ggctcattcg cgacggtccg  21240
acgaccggaa atggcggtta tcagggcagc agaaacgacc cgccagtgtg cgggtctgga  21300
atattctgtt ctggagggtc ctgcgggaac tccgtcgaaa atcggaggga aaaccctcag  21360
gaatcgacgg gggtcgcggt cagaccgttg atcaggacgt tgaccgcgaa atcgaagcgt  21420
tgttccgcgg tgccgtaggt caggtgttcc gagagtgccg ccagggtcgg atagcggccg  21480
gactccaggg cgtcgaggac gtcgacgatg cccgaggcgc gccggcgctc ggactgcgcc  21540
tcgtccaact ggtggtcggt cccgcccggg cttccgttcg aacccgccgc gccggccggg  21600
gcgccggtcg tcgggtcggc gggtcgggcg acgccttcca ggctggcctc gtacgcgacc  21660
gccgaggtgt agaggccgag caggtcgcac gcccacgccg cggtctgcgc cgagacgccg  21720
ccggccagca gcagcgccgt ggtgcgttcc gcgatcgcga tggacgccgg gccggtgggg  21780
atgcggccca tcgagacctg ggcgatgccc gggtgcgcgt tcatggcagc caccgtcaag  21840
cgcagcagtt cgaccaactg gctgcgccag cgcgccggtt cgatcgggtc gaggggaacc  21900
gtcgacaaca ccgcgtcgag cgcctggcgc agcaggtcgt cgcggttgtc gacgtagacg  21960
tacaacgacg ccggtccggt gtcgagctcg ttggcgaccc ggcgcatggt gaccgacgag  22020
agtccttcgc tctgcaggat gcgcagagct gtctcgacga ccacctcctg gctgaggggc  22080
gccttggcgg ggcgggctct ggtactgcgc gaagtgacgg ccatgcggcg agtgtagcag  22140
ttgacacgaa tgttgttcgt gacagacgat gttcgtaacg aacaacgttc gttacgaaca  22200
gcgtttgccg ctctcgggcc cctgtccggc ccactcttcc gccaaggaaa cgccatggat  22260
tccaacccga tcacccttcc cgcgaacgga ccggtggccg aaccattggg cgagtcgccg  22320
gccgagccac ccggcaccgg accggcgccg tacgcggctc gctggatggt cctcgccgtc  22380
gtcttggtcg ccgaggtcat ggacctgctc gacgcgacca tcaccagcgt cgccgcaccg  22440
tcgatgtcgg cgccgcgtac accctgccgt tcgccgtctt cctcatcacc gccggccgac  22500
tcggcgacat ctggggccgg cgccggctct tcgtcatcgg cgcgggcggc ttcacgatcg  22560
cgtccgcgtt gtgcgcgctg gcgaactcgc cggaaatgct gatcaccacc cgcgcgctgc  22620
agggcgcgct cggcgcgctg ctcatcccgc agggcttcgg gctgatcaag gaggtcttcc  22680
cggacaagga actgggcaag gcgttcgcgc tgttcggccc ggtcatgggc ctgtcggcgg  22740
tgctcggccc gatcatcggc ggcgcgctgg tggacggcga cctgttcggc accggctggc  22800
ggatgatctt cctgatcaac ctgccgctcg gtgtcctcgc cgtactcggc gcgttccgct  22860
acatgccgac cggcgcgcgg atccggtgtg ctcaacgccg tccagcagtt cgccaacgcg  22920
atcggcgtcg cggtcgtcgc gacgatcttc ttcgcctacc tcgagacggc cacacctcct  22980
ccgaggcgat gacccgcacc gccgtcgcca cctgcgggct gctcgcgctg gcgttcctcg  23040
ccggcttcgc gctgccgcgc acggcacggg aagaaggcca ctagccggcc gccccggccg  23100
cgtcgtcgcg gccacacggt ggccgcaccc tggccacacc cctggaaaag ccggtcggac  23160
cgggcacgca ccggcggcac tcgcggtgcg tcacgtcccc ggtccggccg gcccggccga  23220
ccgccctcgg ccctcaagcg cggtcccctc cgtgaccgtg accggtgacg cgcctcgcgt  23280
cccacctccg ccgccactcc ggcccgtccg ccccgggcat caccgcaccg acgaacgcgc  23340
caggagcaca tcacgtgtcc acgcaccgca cccccgagcc cgaacgcgcg gccgagcacc  23400
gcgaccgcga acgccttcgg ggccacgatc gagaccgaga ccgcgaccga gaccggatgc  23460
cacgccggat cggcgcgggt ttcgcgcgtg gtccccacgt ccacaccacg cacgtcctga  23520
agaccgagca gatcctcgac gtcgccgccg cgcacttcgg tgtcgagccg cccgagtgcc  23580
ggctcgtcga cgaggccgtc gaacacaccg ggaaacgcct cgcgttgaag gaggtgtacg  23640
cgatctggga ctagggtccg gattttttct aga                               23673