质粒名称: | pML104 |
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出品公司: | 爱迪基因 |
目录编号: | 67638 |
存储实验室: | John Wyrick |
载体骨架基本信息 |
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载体名称: | pRSII426 |
载体抗性: | 氨苄霉素 |
空载体大小: | 5718 bp |
完整质粒大小: | 11251 bp |
载体类型: | Yeast Expression, CRISPR |
筛选标记: | URA3 |
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 |
启动子 | pTDH3 |
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 |
文献引用方法 |
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材料和方法部分: | pML104 was a gift from John Wyrick (爱迪基因 plasmid # 67638) |
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 |
买家导航
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 pML104 11240 bp ds-DNA circular SYN 10-JAN-2018 DEFINITION Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains URA3 marker for yeast transformation. ACCESSION . VERSION . KEYWORDS pML104 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11240) 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 COMMENT This file is created by Vector NTI http://www.biofeng.com/ COMMENT ORIGDB|GenBank COMMENT VNTAUTHORNAME|biofeng.com| COMMENT VNTNAME|pML104| FEATURES Location/Qualifiers source 1..11240 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1..1719 /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 4114..4134 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 4162..4349 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind complement(4217..4241) /label=pHybLex-R /note="ADH terminator, reverse primer" terminator 4467..4486 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter complement(4568..4836) /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" primer_bind complement(4867..4887) /label=T3 /note="T3 promoter, forward primer" promoter complement(4867..4885) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4906..4922) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4906..4922) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4919..4941) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4930..4946 /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(4954..4984) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4999..5020 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5137..5154) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5308..5896) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5388..5407) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6067..6927) /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 6690..6709 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6928..7032) /gene="bla" /label=AmpR promoter rep_origin 7059..8401 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind complement(8022..8069) /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." primer_bind 8442..8460 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(8498..8520) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 8620..8639 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 8663..8878 /gene="S. cerevisiae URA3" /label=URA3 promoter CDS 8879..9682 /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" rep_origin complement(9813..10268) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9950..9971) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 10162..10181 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10394..10416 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 10408..10425 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 10409..10425 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 10435..10454 /label=T7 /note="T7 promoter, forward primer" promoter 10435..10453 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 10478..10494 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 10479..10495 /label=KS primer /note="common sequencing primer, one of multiple similar variants" promoter 10540..11206 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 11076..11099 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" regulatory 11235..4 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 atggacaaga agtattctat cggactggac atcgggacta atagcgtcgg gtgggccgtc 61 atcactgacg agtacaaggt gccctctaag aagttcaagg tgctcgggaa caccgaccgg 121 cattccatca agaaaaatct gatcggagct ctcctctttg attcagggga gaccgctgaa 181 gcaacccgcc tcaagcggac tgctagacgg cggtacacca ggaggaagaa ccggatttgt 241 taccttcaag agatattctc caacgaaatg gcaaaggtcg acgacagctt cttccatagg 301 ctggaagaat cattcctcgt ggaagaggat aagaagcatg aacggcatcc catcttcggt 361 aatatcgtcg acgaggtggc ctatcacgag aaatacccaa ccatctacca tcttcgcaaa 421 aagctggtgg actcaaccga caaggcagac ctccggctta tctacctggc cctggcccac 481 atgattaagt tcagaggcca cttcctgatc gagggcgacc tcaatcctga caatagcgat 541 gtggataaac tgttcatcca gctggtgcag acttacaacc agctctttga agagaacccc 601 atcaatgcaa gcggagtcga tgccaaggcc attctgtcag cccggctgtc aaagagccgc 661 agacttgaga atcttatcgc tcagctgccg ggtgaaaaga aaaatggact gttcgggaac 721 ctgattgctc tttcacttgg gctgactccc aatttcaagt ctaatttcga cctggcagag 781 gatgccaagc tgcaactgtc caaggacacc tatgatgacg atctcgacaa cctcctggcc 841 cagatcggtg accaatacgc cgaccttttc cttgctgcta agaatctttc tgacgccatc 901 ctgctgtctg acattctccg cgtgaacact gaaatcacca aggcccctct ttcagcttca 961 atgattaagc ggtatgatga gcaccaccag gacctgaccc tgcttaaggc actcgtccgg 1021 cagcagcttc cggagaagta caaggaaatc ttctttgacc agtcaaagaa tggatacgcc 1081 ggctacatcg acggaggtgc ctcccaagag gaattttata agtttatcaa acctatcctt 1141 gagaagatgg acggcaccga agagctcctc gtgaaactga atcgggagga tctgctgcgg 1201 aagcagcgca ctttcgacaa tgggagcatt ccccaccaga tccatcttgg ggagcttcac 1261 gccatccttc ggcgccaaga ggacttctac ccctttctta aggacaacag ggagaagatt 1321 gagaaaattc tcactttccg catcccctac tacgtgggac ccctcgccag aggaaatagc 1381 cggtttgctt ggatgaccag aaagtcagaa gaaactatca ctccctggaa cttcgaagag 1441 gtggtggaca agggagccag cgctcagtca ttcatcgaac ggatgactaa cttcgataag 1501 aacctcccca atgagaaggt cctgccgaaa cattccctgc tctacgagta ctttaccgtg 1561 tacaacgagc tgaccaaggt gaaatatgtc accgaaggga tgaggaagcc cgcattcctg 1621 tcaggcgaac aaaagaaggc aattgtggac cttctgttca agaccaatag aaaggtgacc 1681 gtgaagcagc tgaaggagga ctatttcaag aaaattgaat gcttcgactc tgtggagatt 1741 agcggggtcg aagatcggtt caacgcaagc ctgggtacct accatgatct gcttaagatc 1801 atcaaggaca aggattttct ggacaatgag gagaaagagg acatccttga ggacattgtc 1861 ctgactctca ctctgttcga ggaccgggaa atgatcgagg agaggcttaa gacctacgcc 1921 catctgttcg acgataaagt gatgaagcaa cttaaacgga gaagatatac cggatgggga 1981 cgccttagcc gcaaactcat caacggaatc cgggacaaac agagcggaaa gaccattctt 2041 gatttcctta agagcgacgg attcgctaat cgcaacttca tgcaacttat ccatgatgat 2101 tccctgacct ttaaggagga catccagaag gcccaagtgt ctggacaagg tgactcactg 2161 cacgagcata tcgcaaatct ggctggttca cccgctatta agaagggtat tctccagacc 2221 gtgaaagtcg tggacgagct ggtcaaggtg atgggtcgcc ataaaccaga gaacattgtc 2281 atcgagatgg ccagggaaaa ccagactacc cagaagggac agaagaacag cagggagcgg 2341 atgaaaagaa ttgaggaagg gattaaggag ctcgggtcac agatccttaa agagcacccg 2401 gtggaaaaca cccagcttca gaatgagaag ctctatctgt actaccttca aaatggacgc 2461 gatatgtatg tggaccaaga gcttgatatc aacaggctct cagactacga cgtggaccac 2521 atcgtccctc agagcttcct caaagacgac tcaattgaca ataaggtgct gactcgctca 2581 gacaagaacc ggggaaagtc agataacgtg ccctcagagg aagtcgtgaa aaagatgaag 2641 aactattggc gccagcttct gaacgcaaag ctaatcactc agcggaagtt cgacaatctc 2701 actaaggctg agaggggcgg actgagcgaa ctggacaaag caggattcat taaacggcaa 2761 cttgtggaga ctcggcagat tactaaacat gtagcccaaa tccttgactc acgcatgaat 2821 accaagtacg acgaaaacga caaacttatc cgcgaggtga aggtgattac cctgaagtcc 2881 aagctggtca gcgatttcag aaaggacttt caattctaca aagtgcggga gatcaataac 2941 tatcatcatg ctcatgacgc atatctgaat gccgtggtgg gaaccgccct aatcaagaag 3001 tacccaaagc tggaaagcga gttcgtgtac ggagactaca aggtctacga cgtgcgcaag 3061 atgattgcca aatctgagca ggagatcgga aaggccaccg caaagtactt cttctacagc 3121 aacatcatga atttcttcaa gaccgaaatc acccttgcaa acggtgagat ccggaagagg 3181 ccgctcatcg agactaatgg ggagactggc gaaatcgtgt gggacaaggg cagagatttc 3241 gctaccgtgc gcaaagtgct ttctatgcct caagtgaaca tcgtgaagaa aaccgaggtg 3301 caaaccggag gcttttctaa ggaatcaatc ctccccaagc gcaactccga caagctcatt 3361 gcaaggaaga aggattggga ccctaagaag tacggcggat tcgattcacc aactgtggct 3421 tattctgtcc tggtcgtggc taaggtggaa aaaggaaagt ctaagaagct caagagcgtg 3481 aaggaactgc tgggtatcac cattatggag cgcagctcct tcgagaagaa cccaattgac 3541 tttctcgaag ccaaaggtta caaggaagtc aagaaggacc ttatcatcaa gctcccaaag 3601 tatagcctgt tcgaactgga gaatgggcgg aagcggatgc tcgcctccgc tggcgaactt 3661 cagaagggta atgagctggc tctcccctcc aagtacgtga atttcctcta ccttgcaagc 3721 cattacgaga agctgaaggg gagccccgag gacaacgagc aaaagcaact gtttgtggag 3781 cagcataagc attatctgga cgagatcatt gagcagattt ccgagttttc taaacgcgtc 3841 attctcgctg atgccaacct cgataaagtc cttagcgcat acaataagca cagagacaaa 3901 ccaattcggg agcaggctga gaatatcatc cacctgttca ccctcaccaa tcttggtgcc 3961 cctgccgcat tcaagtactt cgacaccacc atcgaccgga aacgctatac ctccaccaaa 4021 gaagtgctgg acgccaccct catccaccag agcatcaccg gactttacga aactcggatt 4081 gacctctcac agctcggagg ggatgaggga gctcccaaga aaaagcgcaa ggtaggtagt 4141 tccggatcct agactctcga ggcgaatttc ttatgattta tgatttttat tattaaataa 4201 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa 4261 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag 4321 gtcgctctta ttgaccacac ctctaccggc atgccgagca aatgcctgca aatcgctccc 4381 catttctcta gagcggccgt ggtatcgttt agattggcaa ttacagtgtc ttagctcaca 4441 tgcttataac taattacatg actcgaagac ataaaaaaca aaaaaagcac caccgactcg 4501 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat ttaaatgcat 4561 cgattctgat catttatctt tcactgcgga gaagtttcga acgccgaaac atgcgcacca 4621 actttcactt ctacagcgtt tgaccaaaat cttttgaaca gaacattgta gggtgtgaaa 4681 aaatgcgcac ctttaccgct agcccaagag ggcactacaa aatctagagt tgtacttcaa 4741 acgtacatgt aatcaccttg tatatactcg aaagaaaaca tcaagtttct gtataaatat 4801 gagtgaaagc ataatcatac attatctttt caaagattgt ccgcggtgga gctccagctt 4861 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc 4921 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 4981 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 5041 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 5101 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 5161 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 5221 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 5281 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 5341 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 5401 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 5461 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 5521 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 5581 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 5641 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 5701 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 5761 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 5821 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 5881 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 5941 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 6001 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 6061 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 6121 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 6181 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 6241 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 6301 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 6361 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 6421 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 6481 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 6541 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 6601 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 6661 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 6721 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 6781 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 6841 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 6901 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 6961 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 7021 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt 7081 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca 7141 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt 7201 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag 7261 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta 7321 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc 7381 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc 7441 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa 7501 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt 7561 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga 7621 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct 7681 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca 7741 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat 7801 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt 7861 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg 7921 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga 7981 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta 8041 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa 8101 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt 8161 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta 8221 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc 8281 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct 8341 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat 8401 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 8461 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 8521 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 8581 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 8641 tgagagtgca ccataccacc ttttcaattc atcatttttt ttttattctt ttttttgatt 8701 tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa 8761 ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg 8821 cccagtattc ttaacccaac tgcacagaac aaaaacctgc aggaaacgaa gataaatcat 8881 gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct 8941 atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac 9001 caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca 9061 tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc 9121 cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg gtaatacagt 9181 caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc 9241 acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac 9301 aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac 9361 tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg 9421 ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac 9481 acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata gaaccgtgga 9541 tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg 9601 aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag 9661 aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta tactaaactc 9721 acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt gaaataccgc 9781 acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa 9841 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 9901 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 9961 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 10021 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg 10081 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 10141 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 10201 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca 10261 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 10321 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 10381 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac 10441 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcgataag 10501 cttgatatcg aattcagtct actagtcagt tcgagtttat cattatcaat actgccattt 10561 caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt 10621 agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat 10681 atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg 10741 agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt 10801 tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc 10861 acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc tggagtaaat 10921 gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta 10981 ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa 11041 attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt 11101 aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta 11161 aaacaccaag aacttagttt cgaataaaca cacataaaca aacaaaagat ctgatatgga 11221 tcgaattaga tctcgccacc //
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