LOCUS Exported 9674 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Protein expression plasmid for 3xNLS SpCas9 in pET-21a backbone. ACCESSION . VERSION . KEYWORDS pET-21a_3xNLS_SpCas9_protein_expression SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9674) AUTHORS Wu Y, Zeng J, Roscoe BP, Liu P, Yao Q, Lazzarotto CR, Clement K, Cole MA, Luk K, Baricordi C, Shen AH, Ren C, Esrick EB, Manis JP, Dorfman DM, Williams DA, Biffi A, Brugnara C, Biasco L, Brendel C, Pinello L, Tsai SQ, Wolfe SA, Bauer DE TITLE Highly efficient therapeutic gene editing of human hematopoietic stem cells. JOURNAL Nat Med. 2019 May;25(5):776-783. doi: 10.1038/s41591-019-0401-y. Epub 2019 Mar 25. PUBMED 30911135 REFERENCE 2 (bases 1 to 9674) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9674 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 964..983 /label=LacI-R /note="LacI, reverse primer" promoter complement(1003..1080) /gene="lacI" /label=lacI promoter primer_bind 1286..1305 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1389..1408 /label=T7 /note="T7 promoter, forward primer" promoter 1389..1407 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1408..1432 /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)." RBS 1447..1469 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1477..1509 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 1519..1545 /codon_start=1 /product="nuclear localization signal (Makkerh et al., 1996)" /label=NLS /translation="PAAKKKKLD" CDS 1558..5661 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5707..5727 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5740..5787 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5794..5811 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5864..5882) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5878..5925 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 5962..6417 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6049..6068) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6259..6280 /label=F1ori-F /note="F1 origin, forward primer" promoter 6443..6547 /gene="bla" /label=AmpR promoter CDS 6548..7408 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6766..6785) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7579..8167 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8068..8087 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8321..8338 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8353..8492 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8418..8437) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8578..8600 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8594..8785) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(9594..1002) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc 61 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag 121 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc 181 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg 241 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc 301 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc 361 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg 421 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac 481 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg 541 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc 601 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag 661 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac 721 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc 781 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg 841 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg 901 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac 961 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg 1021 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc 1081 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt 1141 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca 1201 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga 1261 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac 1321 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc 1381 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga 1441 aataattttg tttaacttta agaaggagat atacatatgg ctagcatgac tggtggacag 1501 caaatgggtc gcgccgctcc ggcagctaag aaaaagaaac tggatggcag cgtcgacatg 1561 gataagaaat actcaatagg cttagatatc ggcacaaata gcgtcggatg ggcggtgatc 1621 actgatgaat ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac 1681 agtatcaaaa aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg 1741 actcgtctca aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat 1801 ctacaggaga ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt 1861 gaagagtctt ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat 1921 atagtagatg aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa 1981 ttggtagatt ctactgataa agcggatttg cgcttaatct atttggcctt agcgcatatg 2041 attaagtttc gtggtcattt tttgattgag ggagatttaa atcctgataa tagtgatgtg 2101 gacaaactat ttatccagtt ggtacaaacc tacaatcaat tatttgaaga aaaccctatt 2161 aacgcaagtg gagtagatgc taaagcgatt ctttctgcac gattgagtaa atcaagacga 2221 ttagaaaatc tcattgctca gctccccggt gagaagaaaa atggcttatt tgggaatctc 2281 attgctttgt cattgggttt gacccctaat tttaaatcaa attttgattt ggcagaagat 2341 gctaaattac agctttcaaa agatacttac gatgatgatt tagataattt attggcgcaa 2401 attggagatc aatatgctga tttgtttttg gcagctaaga atttatcaga tgctatttta 2461 ctttcagata tcctaagagt aaatactgaa ataactaagg ctcccctatc agcttcaatg 2521 attaaacgct acgatgaaca tcatcaagac ttgactcttt taaaagcttt agttcgacaa 2581 caacttccag aaaagtataa agaaatcttt tttgatcaat caaaaaacgg atatgcaggt 2641 tatattgatg ggggagctag ccaagaagaa ttttataaat ttatcaaacc aattttagaa 2701 aaaatggatg gtactgagga attattggtg aaactaaatc gtgaagattt gctgcgcaag 2761 caacggacct ttgacaacgg ctctattccc catcaaattc acttgggtga gctgcatgct 2821 attttgagaa gacaagaaga cttttatcca tttttaaaag acaatcgtga gaagattgaa 2881 aaaatcttga cttttcgaat tccttattat gttggtccat tggcgcgtgg caatagtcgt 2941 tttgcatgga tgactcggaa gtctgaagaa acaattaccc catggaattt tgaagaagtt 3001 gtcgataaag gtgcttcagc tcaatcattt attgaacgca tgacaaactt tgataaaaat 3061 cttccaaatg aaaaagtact accaaaacat agtttgcttt atgagtattt tacggtttat 3121 aacgaattga caaaggtcaa atatgttact gaaggaatgc gaaaaccagc atttctttca 3181 ggtgaacaga agaaagccat tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt 3241 aagcaattaa aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca 3301 ggagttgaag atagatttaa tgcttcatta ggtacctacc atgatttgct aaaaattatt 3361 aaagataaag attttttgga taatgaagaa aatgaagata tcttagagga tattgtttta 3421 acattgacct tatttgaaga tagggagatg attgaggaaa gacttaaaac atatgctcac 3481 ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc gttatactgg ttggggacgt 3541 ttgtctcgaa aattgattaa tggtattagg gataagcaat ctggcaaaac aatattagat 3601 tttttgaaat cagatggttt tgccaatcgc aattttatgc agctgatcca tgatgatagt 3661 ttgacattta aagaagacat tcaaaaagca caagtgtctg gacaaggcga tagtttacat 3721 gaacatattg caaatttagc tggtagccct gctattaaaa aaggtatttt acagactgta 3781 aaagttgttg atgaattggt caaagtaatg gggcggcata agccagaaaa tatcgttatt 3841 gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg 3901 aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt 3961 gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctccaaaa tggaagagac 4021 atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt cgatcacatt 4081 gttccacaaa gtttccttaa agacgattca atagacaata aggtcttaac gcgttctgat 4141 aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac 4201 tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg 4261 aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg 4321 gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact 4381 aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa 4441 ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac 4501 catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat 4561 ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg 4621 attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat 4681 atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct 4741 ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc 4801 acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag 4861 acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct 4921 cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat 4981 tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa 5041 gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt 5101 ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat 5161 agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa 5221 aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat 5281 tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag 5341 cataagcatt atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt 5401 ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca 5461 atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc 5521 gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa 5581 gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat 5641 ttgagtcagc taggaggtga caccggtggt ggtcccgggg gtggtgcggc cgcaggcagc 5701 ggaagcccta agaaaaaacg aaaagttggc agcggaagca aaaggccggc ggccacgaaa 5761 aaggccggcc aggcaaaaaa gaaaaagctc gagcaccacc accaccacca ctgagatccg 5821 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 5881 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 5941 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6001 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 6061 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6121 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 6181 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6241 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 6301 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 6361 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg 6421 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 6481 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 6541 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 6601 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 6661 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 6721 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 6781 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 6841 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 6901 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 6961 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 7021 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 7081 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 7141 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 7201 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 7261 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 7321 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 7381 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 7441 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 7501 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 7561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 7621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 7681 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 7741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 7801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 7861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 7921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 7981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 8041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 8101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 8161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 8221 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 8281 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 8341 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 8401 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc 8461 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg 8521 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 8581 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt 8641 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca 8701 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa 8761 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg 8821 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac 8881 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc 8941 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac 9001 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact 9061 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg 9121 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct 9181 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca 9241 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg 9301 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg 9361 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg 9421 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga 9481 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 9541 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 9601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 9661 ggagaggcgg tttg //