质粒名称: | pIVT-dCas9-mCherry |
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出品公司: | Addgene |
目录编号: | 174443 |
存储实验室: | Michael Lampson |
载体骨架基本信息 |
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载体名称: | pIVT |
载体类型: | 体外转录载体 |
载体抗性: | 氨苄霉素100 μg/mL |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 低拷贝 |
插入基因信息 |
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插入基因名称: | Cas9 Endonuclease Dead,dCas9 |
物种来源: | Streptococcus pyogenes |
插入大小: | 4104 bp |
启动子: | T7 SP6 |
标签/融合蛋白: | mCherry(插入件上的C端子) |
克隆方法: | Ligation Independent克隆 |
5’测序引物 | CAGGAAACAGCTATGAC |
3’测序引物 | GTAAAACGACGGCCAGT |
文献引用方法 |
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材料和方法部分: | pIVT-dCas9-mCherry was a gift from Michael Lampson (Addgene plasmid # 174443 ; http://n2t.net/addgene:174443 ; RRID:Addgene_174443) |
参考文献部分: | Parallel pathways for recruiting effector proteins determine centromere drive and suppression. Kumon T, Ma J, Akins RB, Stefanik D, Nordgren CE, Kim J, Levine MT, Lampson MA. Cell. 2021 Sep 16;184(19):4904-4918.e11. doi: 10.1016/j.cell.2021.07.037. Epub 2021 Aug 24. 10.1016/j.cell.2021.07.037 PubMed 34433012 |
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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 pIVT-dCas9-mCherry 7882 bp ds-DNA circular SYN 30-SEP-2021 DEFINITION in vitro transcription template for dCas9-mCherry. ACCESSION . VERSION . KEYWORDS pIVT-dCas9-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7882) TITLE Parallel pathways for recruiting effector proteins determine centromere drive and suppression JOURNAL Unpublished REFERENCE 2 (bases 1 to 7882) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 30, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7882 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 96..200 /gene="bla" /label=AmpR promoter CDS 201..1061 /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 complement(419..438) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1232..1820 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1721..1740 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1974..1991 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2108..2129 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2144..2174 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2182..2198 /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)." primer_bind 2187..2209 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2206..2222 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2206..2222 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 2246..2264 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 2246..2263 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 2276..2295 /label=T7 /note="T7 promoter, forward primer" promoter 2276..2294 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" 5'UTR 2337..2379 /label=Xenopus globin 5'-UTR /note="translational enhancer from the 5'-UTR of the major beta-globin gene of Xenopus laevis" regulatory 2437..2446 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2443..6546 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6562..7272 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(6707..6725) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 6975..6994 /label=mCherry-F /note="mCherry, forward primer" 3'UTR 7300..7425 /label=Xenopus globin 3'-UTR /note="3'-UTR of the major beta-globin gene of Xenopus laevis" primer_bind complement(7488..7505) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7488..7504) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7497..7519) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(7713..7732) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7832..7854 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741 atagtcctat cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221 accatgatta cgccaagctc caacgattta ggtgacacta tagaagagaa ggaattaata 2281 cgactcacta tagggagaga gagagaatta ccctcactaa agggaggaga agcttgcttg 2341 ttctttttgc agaagctcag aataaacgct caactttggc agatcaagct tgcatgcctg 2401 caggtcgact atagggagac ccaagcttgg cgcgccgcca ccatggacaa gaagtactcc 2461 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag 2521 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac 2581 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga 2641 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt 2701 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg 2761 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg 2821 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact 2881 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga 2941 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc 3001 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt 3061 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc 3121 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc 3181 gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg 3241 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac 3301 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg 3361 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat 3421 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag 3481 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga 3541 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc 3601 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac 3661 aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa 3721 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt 3781 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact 3841 cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc 3901 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag 3961 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag 4021 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa 4081 gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa 4141 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc 4201 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc 4261 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt 4321 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa 4381 gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg 4441 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat 4501 ggatttgcca acaggaactt catgcagttg atccatgatg actctctcac ctttaaggag 4561 gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat 4621 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa 4681 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag 4741 aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag 4801 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt 4861 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag 4921 gaactggaca tcaatcggct ctccgactac gacgtggatg ctatcgtgcc ccagtctttt 4981 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag 5041 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg 5101 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt 5161 ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag 5221 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat 5281 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc 5341 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat 5401 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct 5461 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag 5521 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc 5581 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac 5641 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc 5701 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc 5761 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg 5821 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg 5881 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc 5941 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga 6001 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt 6061 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg 6121 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa 6181 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt 6241 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac 6301 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca 6361 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac 6421 ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca 6481 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt 6541 ggagacgcta gcggctctag aatggtgagc aagggcgagg aggataacat ggccatcatc 6601 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag 6661 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg 6721 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc 6781 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc 6841 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc 6901 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac 6961 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag 7021 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag 7081 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag 7141 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac 7201 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag 7261 ctgtacaagt agtgactgac taggatctgg ttaccactaa accagcctca agaacacccg 7321 aatggagtct ctaagctaca taataccaac ttacacttta caaaatgttg tcccccaaaa 7381 tgtagccatt cgtatctgct cctaataaaa agaaagtttc ttcacattct aaaaaaaaaa 7441 aaaaaaaaaa aaaaaaaaaa aanccccccc cccccaaggg cgaattcact ggccgtcgtt 7501 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 7561 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 7621 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 7681 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 7741 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 7801 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 7861 ccgtcatcac cgaaacgcgc ga //