质粒名称: | pML107 |
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出品公司: | 爱迪基因 |
目录编号: | 67639 |
存储实验室: | John Wyrick |
载体骨架基本信息 |
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载体名称: | pRSII425 |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 6841 bp |
完整质粒大小: | 12366 bp |
载体修饰: | -- |
载体类型: | 酵母表达,CRISPR |
筛选标记: | LEU2 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Cas9 |
物种来源: | S. cerevisiae (budding yeast); Streptococcus pyogenes |
插入大小: | 4151 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | pTDH3 (aka GAP promoter) |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | EcoRI (未破坏) |
3' 克隆位点: | XhoI(未破坏) |
5' 测序引物 | T7 |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pML107 was a gift from John Wyrick (Addgene plasmid # 67639 ; http://n2t.net/addgene:67639 ; RRID:Addgene_67639) |
参考文献部分: | New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T, Wyrick JJ. Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21. 10.1002/yea.3098 PubMed 26305040 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pML107 12367 bp ds-DNA circular SYN 28-AUG-2018 DEFINITION Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains LEU2 marker for yeast transformation. ACCESSION . VERSION . KEYWORDS pML107 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12367) AUTHORS Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T, Wyrick JJ TITLE New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. JOURNAL Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21. PUBMED 26305040 REFERENCE 2 (bases 1 to 12367) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 28, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12367 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..17 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 27..46 /label=T7 /note="T7 promoter, forward primer" promoter 27..45 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 70..86 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 71..87 /label=KS primer /note="common sequencing primer, one of multiple similar variants" promoter 132..798 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 668..691 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" regulatory 827..836 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 833..2551 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 4946..4966 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 4994..5181 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind complement(5049..5073) /label=pHybLex-R /note="ADH terminator, reverse primer" terminator 5299..5318 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter complement(5400..5668) /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" primer_bind complement(5699..5719) /label=T3 /note="T3 promoter, forward primer" promoter complement(5699..5717) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5738..5754) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5738..5754) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5751..5773) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5762..5778 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5786..5816) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5831..5852 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5969..5986) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6140..6728) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6220..6239) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6899..7759) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7522..7541 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7760..7864) /gene="bla" /label=AmpR promoter rep_origin 7891..9237 /label=2u ori /note="yeast 2u plasmid origin of replication" primer_bind 9278..9296 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(9334..9356) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 9456..9475 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" CDS complement(9969..11063) /codon_start=1 /gene="S. cerevisiae LEU2" /product="3-isopropylmalate dehydrogenase, required for leucine biosynthesis" /label=LEU2 /note="yeast auxotrophic marker" /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG DLGGSNSTTEVGDAVAEEVKKILA" promoter complement(11064..11471) /gene="S. cerevisiae LEU2" /label=LEU2 promoter rep_origin complement(11772..12227) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11909..11930) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 12121..12140 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 12353..8 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 12367..17 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" ORIGIN 1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg 61 ggccccccct cgaggtcgac ggtatcgata agcttgatat cgaattcagt ctactagtca 121 gttcgagttt atcattatca atactgccat ttcaaagaat acgtaaataa ttaatagtag 181 tgattttcct aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac 241 atgcccaaaa tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac 301 agtttattcc tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa 361 aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc 421 attctcttag cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct 481 caatggagtg atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat 541 gtatctatct cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag 601 ctgaaaaaaa aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat 661 ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc 721 tacttttata gttagtcttt tttttagttt taaaacacca agaacttagt ttcgaataaa 781 cacacataaa caaacaaaag atctgatatg gatcgaatta gatctcgcca ccatggacaa 841 gaagtattct atcggactgg acatcgggac taatagcgtc gggtgggccg tcatcactga 901 cgagtacaag gtgccctcta agaagttcaa ggtgctcggg aacaccgacc ggcattccat 961 caagaaaaat ctgatcggag ctctcctctt tgattcaggg gagaccgctg aagcaacccg 1021 cctcaagcgg actgctagac ggcggtacac caggaggaag aaccggattt gttaccttca 1081 agagatattc tccaacgaaa tggcaaaggt cgacgacagc ttcttccata ggctggaaga 1141 atcattcctc gtggaagagg ataagaagca tgaacggcat cccatcttcg gtaatatcgt 1201 cgacgaggtg gcctatcacg agaaataccc aaccatctac catcttcgca aaaagctggt 1261 ggactcaacc gacaaggcag acctccggct tatctacctg gccctggccc acatgattaa 1321 gttcagaggc cacttcctga tcgagggcga cctcaatcct gacaatagcg atgtggataa 1381 actgttcatc cagctggtgc agacttacaa ccagctcttt gaagagaacc ccatcaatgc 1441 aagcggagtc gatgccaagg ccattctgtc agcccggctg tcaaagagcc gcagacttga 1501 gaatcttatc gctcagctgc cgggtgaaaa gaaaaatgga ctgttcggga acctgattgc 1561 tctttcactt gggctgactc ccaatttcaa gtctaatttc gacctggcag aggatgccaa 1621 gctgcaactg tccaaggaca cctatgatga cgatctcgac aacctcctgg cccagatcgg 1681 tgaccaatac gccgaccttt tccttgctgc taagaatctt tctgacgcca tcctgctgtc 1741 tgacattctc cgcgtgaaca ctgaaatcac caaggcccct ctttcagctt caatgattaa 1801 gcggtatgat gagcaccacc aggacctgac cctgcttaag gcactcgtcc ggcagcagct 1861 tccggagaag tacaaggaaa tcttctttga ccagtcaaag aatggatacg ccggctacat 1921 cgacggaggt gcctcccaag aggaatttta taagtttatc aaacctatcc ttgagaagat 1981 ggacggcacc gaagagctcc tcgtgaaact gaatcgggag gatctgctgc ggaagcagcg 2041 cactttcgac aatgggagca ttccccacca gatccatctt ggggagcttc acgccatcct 2101 tcggcgccaa gaggacttct acccctttct taaggacaac agggagaaga ttgagaaaat 2161 tctcactttc cgcatcccct actacgtggg acccctcgcc agaggaaata gccggtttgc 2221 ttggatgacc agaaagtcag aagaaactat cactccctgg aacttcgaag aggtggtgga 2281 caagggagcc agcgctcagt cattcatcga acggatgact aacttcgata agaacctccc 2341 caatgagaag gtcctgccga aacattccct gctctacgag tactttaccg tgtacaacga 2401 gctgaccaag gtgaaatatg tcaccgaagg gatgaggaag cccgcattcc tgtcaggcga 2461 acaaaagaag gcaattgtgg accttctgtt caagaccaat agaaaggtga ccgtgaagca 2521 gctgaaggag gactatttca agaaaattga atgcttcgac tctgtggaga ttagcggggt 2581 cgaagatcgg ttcaacgcaa gcctgggtac ctaccatgat ctgcttaaga tcatcaagga 2641 caaggatttt ctggacaatg aggagaaaga ggacatcctt gaggacattg tcctgactct 2701 cactctgttc gaggaccggg aaatgatcga ggagaggctt aagacctacg cccatctgtt 2761 cgacgataaa gtgatgaagc aacttaaacg gagaagatat accggatggg gacgccttag 2821 ccgcaaactc atcaacggaa tccgggacaa acagagcgga aagaccattc ttgatttcct 2881 taagagcgac ggattcgcta atcgcaactt catgcaactt atccatgatg attccctgac 2941 ctttaaggag gacatccaga aggcccaagt gtctggacaa ggtgactcac tgcacgagca 3001 tatcgcaaat ctggctggtt cacccgctat taagaagggt attctccaga ccgtgaaagt 3061 cgtggacgag ctggtcaagg tgatgggtcg ccataaacca gagaacattg tcatcgagat 3121 ggccagggaa aaccagacta cccagaaggg acagaagaac agcagggagc ggatgaaaag 3181 aattgaggaa gggattaagg agctcgggtc acagatcctt aaagagcacc cggtggaaaa 3241 cacccagctt cagaatgaga agctctatct gtactacctt caaaatggac gcgatatgta 3301 tgtggaccaa gagcttgata tcaacaggct ctcagactac gacgtggacc acatcgtccc 3361 tcagagcttc ctcaaagacg actcaattga caataaggtg ctgactcgct cagacaagaa 3421 ccggggaaag tcagataacg tgccctcaga ggaagtcgtg aaaaagatga agaactattg 3481 gcgccagctt ctgaacgcaa agctaatcac tcagcggaag ttcgacaatc tcactaaggc 3541 tgagaggggc ggactgagcg aactggacaa agcaggattc attaaacggc aacttgtgga 3601 gactcggcag attactaaac atgtagccca aatccttgac tcacgcatga ataccaagta 3661 cgacgaaaac gacaaactta tccgcgaggt gaaggtgatt accctgaagt ccaagctggt 3721 cagcgatttc agaaaggact ttcaattcta caaagtgcgg gagatcaata actatcatca 3781 tgctcatgac gcatatctga atgccgtggt gggaaccgcc ctaatcaaga agtacccaaa 3841 gctggaaagc gagttcgtgt acggagacta caaggtctac gacgtgcgca agatgattgc 3901 caaatctgag caggagatcg gaaaggccac cgcaaagtac ttcttctaca gcaacatcat 3961 gaatttcttc aagaccgaaa tcacccttgc aaacggtgag atccggaaga ggccgctcat 4021 cgagactaat ggggagactg gcgaaatcgt gtgggacaag ggcagagatt tcgctaccgt 4081 gcgcaaagtg ctttctatgc ctcaagtgaa catcgtgaag aaaaccgagg tgcaaaccgg 4141 aggcttttct aaggaatcaa tcctccccaa gcgcaactcc gacaagctca ttgcaaggaa 4201 gaaggattgg gaccctaaga agtacggcgg attcgattca ccaactgtgg cttattctgt 4261 cctggtcgtg gctaaggtgg aaaaaggaaa gtctaagaag ctcaagagcg tgaaggaact 4321 gctgggtatc accattatgg agcgcagctc cttcgagaag aacccaattg actttctcga 4381 agccaaaggt tacaaggaag tcaagaagga ccttatcatc aagctcccaa agtatagcct 4441 gttcgaactg gagaatgggc ggaagcggat gctcgcctcc gctggcgaac ttcagaaggg 4501 taatgagctg gctctcccct ccaagtacgt gaatttcctc taccttgcaa gccattacga 4561 gaagctgaag gggagccccg aggacaacga gcaaaagcaa ctgtttgtgg agcagcataa 4621 gcattatctg gacgagatca ttgagcagat ttccgagttt tctaaacgcg tcattctcgc 4681 tgatgccaac ctcgataaag tccttagcgc atacaataag cacagagaca aaccaattcg 4741 ggagcaggct gagaatatca tccacctgtt caccctcacc aatcttggtg cccctgccgc 4801 attcaagtac ttcgacacca ccatcgaccg gaaacgctat acctccacca aagaagtgct 4861 ggacgccacc ctcatccacc agagcatcac cggactttac gaaactcgga ttgacctctc 4921 acagctcgga ggggatgagg gagctcccaa gaaaaagcgc aaggtaggta gttccggatc 4981 ctagactctc gaggcgaatt tcttatgatt tatgattttt attattaaat aagttataaa 5041 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt 5101 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct 5161 tattgaccac acctctaccg gcatgccgag caaatgcctg caaatcgctc cccatttctc 5221 tagagcggcc gtggtatcgt ttagattggc aattacagtg tcttagctca catgcttata 5281 actaattaca tgactcgaag acataaaaaa caaaaaaagc accaccgact cggtgccact 5341 ttttcaagtt gataacggac tagccttatt ttaacttgct atttaaatgc atcgattctg 5401 atcatttatc tttcactgcg gagaagtttc gaacgccgaa acatgcgcac caactttcac 5461 ttctacagcg tttgaccaaa atcttttgaa cagaacattg tagggtgtga aaaaatgcgc 5521 acctttaccg ctagcccaag agggcactac aaaatctaga gttgtacttc aaacgtacat 5581 gtaatcacct tgtatatact cgaaagaaaa catcaagttt ctgtataaat atgagtgaaa 5641 gcataatcat acattatctt ttcaaagatt gtccgcggtg gagctccagc ttttgttccc 5701 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5761 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5821 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 5881 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5941 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 6001 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 6061 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6121 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 6181 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6241 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6301 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 6361 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6421 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6481 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6541 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6601 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6661 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6721 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6781 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6841 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6901 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6961 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 7021 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 7081 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 7141 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 7201 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 7261 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 7321 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 7381 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 7441 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7501 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7561 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7621 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7681 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7741 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7801 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7861 cgcgcacatt tccccgaaaa gtgccacctg aacgaagcat ctgtgcttca ttttgtagaa 7921 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca 7981 gaacagaaat gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc ttcatttttg 8041 taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt 8101 ttacagaaca gaaatgcaac gcgagagcgc tattttacca acaaagaatc tatacttctt 8161 ttttgttcta caaaaatgca tcccgagagc gctatttttc taacaaagca tcttagatta 8221 ctttttttct cctttgtgcg ctctataatg cagtctcttg ataacttttt gcactgtagg 8281 tccgttaagg ttagaagaag gctactttgg tgtctatttt ctcttccata aaaaaagcct 8341 gactccactt cccgcgttta ctgattacta gcgaagctgc gggtgcattt tttcaagata 8401 aaggcatccc cgattatatt ctataccgat gtggattgcg catactttgt gaacagaaag 8461 tgatagcgtt gatgattctt cattggtcag aaaattatga acggtttctt ctattttgtc 8521 tctatatact acgtatagga aatgtttaca ttttcgtatt gttttcgatt cactctatga 8581 atagttctta ctacaatttt tttgtctaaa gagtaatact agagataaac ataaaaaatg 8641 tagaggtcga gtttagatgc aagttcaagg agcgaaaggt ggatgggtag gttatatagg 8701 gatatagcac agagatatat agcaaagaga tacttttgag caatgtttgt ggaagcggta 8761 ttcgcaatat tttagtagct cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt 8821 cttcagagcg cttttggttt tcaaaagcgc tctgaagttc ctatactttc tagctagaga 8881 ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc 8941 aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct 9001 gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta 9061 tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt 9121 ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca 9181 ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata 9241 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 9301 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 9361 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 9421 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 9481 agtgcaccat atcgactacg tcgttaaggc cgtttctgac agagtaaaat tcttgaggga 9541 actttcacca ttatgggaaa tggttcaaga aggtattgac ttaaactcca tcaaatggtc 9601 aggtcattga gtgtttttta tttgttgtat tttttttttt ttagagaaaa tcctccaata 9661 tataaattag gaatcatagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc 9721 ctcctccttg tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat 9781 tagtatcaat ttgcttacct gtattccttt acatcctcct ttttctcctt cttgataaat 9841 gtatgtagat tgcgtatata gtttcgtcta ccctatgaac atattccatt ttgtaatttc 9901 gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga 9961 gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg 10021 gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact 10081 gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca 10141 gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg 10201 tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca 10261 gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca 10321 aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg 10381 gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt cttgatggtt 10441 tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac 10501 caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt 10561 tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc 10621 tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct 10681 ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat 10741 ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt 10801 ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc 10861 ttcttggagg cttccagcgc ctcatctgga agtggaacac ctgtagcatc tatagcagca 10921 ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct 10981 ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg 11041 acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata tatatatatt 11101 gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc acctattgga 11161 aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc aagcatttag 11221 tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcgctctcac ctttcctttt 11281 tctcccaatt tttcagttga aaaaggtata tgcgtcaggc gacctctgaa attaacaaaa 11341 aatttccagt catcgaattt gattctgtgc gatagcgccc ctgtgtgttc tcgttatgtt 11401 gaggaaaaaa ataatggttg ctaagagatt cgaactcttg catcttacga tacctgagta 11461 ttcccacagt taactgcggt caagatattt cttgaatcag gcgccttaga ccgctcggcc 11521 aaacaaccaa ttacttgttg agaaatagag tataattatc ctataaatat aacgtttttg 11581 aacacacatg aacaaggaag tacaggacaa ttgattttga agagaatgtg gattttgatg 11641 taattgttgg gattccattt ttaataaggc aataatatta ggtatgtaga tatactagaa 11701 gttctcctcg accggtcgat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 11761 accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 11821 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 11881 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 11941 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 12001 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 12061 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 12121 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 12181 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc 12241 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 12301 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 12361 cgacgtt //