LOCUS Exported 10073 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression plasmid for SpCas9-HF1 variant. ACCESSION . VERSION . KEYWORDS pJSC111 - Bacterial expression plasmid for SpCas9-HF1 variant SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10073) AUTHORS Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA TITLE Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. JOURNAL Nature. 2017 Sep 20. doi: 10.1038/nature24268. PUBMED 28931002 REFERENCE 2 (bases 1 to 10073) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10073 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 167..186 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 270..289 /label=T7 /note="T7 promoter, forward primer" promoter 270..288 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 377..399 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 419..448 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 458..1558 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 1532..1555 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1613..1633 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1640..5743 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Kleinstiver et al., 2016)" /label=SpCas9-HF1 /note="carries the mutations N497A, R661A, Q695A, and Q926A" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" primer_bind complement(5872..5890) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5886..5933 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(6299..6327) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(6354..6372) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6440..6544 /gene="bla" /label=AmpR promoter CDS 6545..7405 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6763..6782) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7576..8164 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8065..8084 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8318..8335 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8350..8489 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8415..8434) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8575..8597 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8591..8782) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 61 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 121 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 181 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 241 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg 301 gtttccctct agtgccggct ccggagagct ctttaattaa gcggccgccc tgcaggactc 361 gagttctaga aataattttg tttaacttta agaaggagat atacatatga aatcttctca 421 ccatcaccat caccatcacc atcaccatgg ttcttctatg aaaatcgaag aaggtaaact 481 ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt 541 cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt 601 cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt 661 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga 721 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc 781 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa 841 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct 901 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta 961 tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg 1021 cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga 1081 caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa 1141 cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact 1201 gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa 1261 cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga 1321 tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta 1381 cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg 1441 tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt 1501 gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa 1561 ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg aggaaaacct 1621 gtacttccaa tccaatgcaa tggataagaa atactcaata ggcttagata tcggcacaaa 1681 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt 1741 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga 1801 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg 1861 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga 1921 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga 1981 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac 2041 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat 2101 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt 2161 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca 2221 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc 2281 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa 2341 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc 2401 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga 2461 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa 2521 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa 2581 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct 2641 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca 2701 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa 2761 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa 2821 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat 2881 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa 2941 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc 3001 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac 3061 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg 3121 catgacagcg tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct 3181 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat 3241 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa 3301 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg 3361 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta 3421 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga 3481 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga 3541 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg 3601 ccgttatact ggttggggag cgttgtctcg aaaattgatt aatggtatta gggataagca 3661 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat 3721 ggccctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc 3781 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa 3841 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca 3901 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca 3961 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca 4021 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta 4081 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag 4141 tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa 4201 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga 4261 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca 4321 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc 4381 tggttttatc aaacgccaat tggttgaaac tcgcgccatc actaagcatg tggcacaaat 4441 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa 4501 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa 4561 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg 4621 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa 4681 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc 4741 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa 4801 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg 4861 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat 4921 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag 4981 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt 5041 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc 5101 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt 5161 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt 5221 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct 5281 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa 5341 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca 5401 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag 5461 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata 5521 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac 5581 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa 5641 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg 5701 tctttatgaa acacgcattg atttgagtca gctaggaggt gactaataac attggaagtg 5761 gataacggat ccgcgatcgc ggcgcgccac ctggtggccg gccggtacca cgcgtgcgcg 5821 ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 5881 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 5941 gaggaactat atccggatat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata 6001 gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct 6061 ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag 6121 tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata 6181 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg 6241 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat 6301 taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt 6361 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 6421 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 6481 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataacatt gaaaaaggaa 6541 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 6601 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 6661 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 6721 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 6781 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 6841 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 6901 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 6961 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 7021 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 7081 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 7141 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 7201 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 7261 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 7321 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 7381 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 7441 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 7501 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 7561 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 7621 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 7681 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 7741 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 7801 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 7861 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 7921 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 7981 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 8041 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 8101 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 8161 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 8221 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 8281 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 8341 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa 8401 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc 8461 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc 8521 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 8581 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa 8641 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct 8701 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg 8761 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg 8821 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg 8881 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga 8941 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg 9001 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc 9061 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg 9121 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct 9181 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc 9241 gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg 9301 cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga 9361 attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat 9421 tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta 9481 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat 9541 cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc 9601 ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg 9661 catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt 9721 cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg 9781 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa 9841 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc 9901 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac 9961 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt 10021 gaaggctctc aagggcatcg gtcgacgctc tcccttatgc gactcctgca tta //