LOCUS Exported 10724 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial vector for expression of Snap-tagged Streptococcus pyogenes Cas9. ACCESSION . VERSION . KEYWORDS pNS20-SpCas9-SNAP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10724) AUTHORS Savic N, Ringnalda FC, Lindsay H, Berk C, Bargsten K, Li Y, Neri D, Robinson MD, Ciaudo C, Hall J, Jinek M, Schwank G TITLE Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. JOURNAL Elife. 2018 May 29;7. pii: 33761. doi: 10.7554/eLife.33761. PUBMED 29809142 REFERENCE 2 (bases 1 to 10724) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10724 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(18..36) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 104..208 /gene="bla" /label=AmpR promoter CDS 209..1069 /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(427..446) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1240..1828 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1729..1748 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1982..1999 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2014..2156 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2082..2101) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2242..2264 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2258..2449) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 3907..3926 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4010..4029 /label=T7 /note="T7 promoter, forward primer" promoter 4010..4028 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 4117..4139 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4159..4176 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4186..5286 /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 5260..5283 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 5341..5361 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 5368..9471 /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 9475..9501 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 9520..9540 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9547..9567 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9607..10149 /codon_start=1 /product="human O6-alkylguanine-DNA-alkyltransferase variant that forms covalent bonds with benzylguanine derivatives" /label=SNAP-tag(R) /note="optimized for bacterial expression" /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEV PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV VKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAV KEWLLAHEGHRLGKPGLG" CDS 10165..10185 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(10260..10278) /label=T7 Term /note="T7 terminator, reverse primer" terminator 10274..10321 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(10687..10715) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" ORIGIN 1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc 3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc 3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc 3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc 3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc 4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa 4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat 4141 atacatatga aatcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa 4201 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt 4261 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa 4321 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac 4381 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg 4441 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt 4501 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac 4561 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag 4621 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac 4681 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat 4741 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg 4801 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg 4861 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta 4921 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca 4981 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg 5041 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg 5101 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc 5161 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt 5221 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg 5281 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag 5341 gaaaacctgt acttccaatc caatgcaatg gacaagaagt acagcatcgg cctggacatc 5401 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa 5461 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggcgccctg 5521 ctgttcgaca gcggagaaac agccgaggcc acccggctga agagaaccgc cagaagaaga 5581 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc 5641 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag 5701 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag 5761 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg 5821 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag 5881 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc 5941 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc 6001 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc 6061 gagaagaaga atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac 6121 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac 6181 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg 6241 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag 6301 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac 6361 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc 6421 ttcgaccaga gcaagaacgg ctacgccggc tacatcgatg gcggagccag ccaggaagag 6481 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 6541 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc 6601 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca 6661 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac 6721 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 6781 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc 6841 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 6901 agcctgctgt acgagtactt caccgtgtac aacgagctga ccaaagtgaa atacgtgacc 6961 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaagccat cgtggacctg 7021 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 7081 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg 7141 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa 7201 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg 7261 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg 7321 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg 7381 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga 7441 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc 7501 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc 7561 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 7621 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag 7681 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg 7741 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 7801 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac 7861 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc 7921 atcgataaca aagtgctgac tcggagcgac aagaaccggg gcaagagcga caacgtgccc 7981 tccgaagagg tcgtgaagaa gatgaagaac tactggcgcc agctgctgaa tgccaagctg 8041 attacccaga ggaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg 8101 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg 8161 gcacagatcc tggactcccg gatgaacact aagtacgacg agaacgacaa actgatccgg 8221 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 8281 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc 8341 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc 8401 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag 8461 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc 8521 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aacaggcgag 8581 atcgtgtggg ataagggccg ggactttgcc accgtgcgga aagtgctgtc tatgccccaa 8641 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 8701 cccaagagga acagcgacaa gctgatcgcc agaaagaagg actgggaccc taagaagtac 8761 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag 8821 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga 8881 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa 8941 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag 9001 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa 9061 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat 9121 aatgagcaga aacagctgtt tgtggaacag cacaaacact acctggacga gatcatcgag 9181 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaggtgctg 9241 agcgcctaca acaagcacag agacaagcct atcagagagc aggccgagaa tatcatccac 9301 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc 9361 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc 9421 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga cgcctatccc 9481 tatgacgtgc ccgattatgc cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg 9541 gaagatccta agaaaaagcg gaaagtggac ggcattggta gtgggagcaa cggcagcagc 9601 ggatccgaca aagattgcga aatgaaacgt accaccctgg atagcccgct gggcaaactg 9661 gaactgagcg gctgcgaaca gggcctgcat gaaattaaac tgctgggtaa aggcaccagc 9721 gcggccgatg cggttgaagt tccggccccg gccgccgtgc tgggtggtcc ggaaccgctg 9781 atgcaggcga ccgcgtggct gaacgcgtat tttcatcagc cggaagcgat tgaagaattt 9841 ccggttccgg cgctgcatca tccggtgttt cagcaggaga gctttacccg tcaggtgctg 9901 tggaaactgc tgaaagtggt taaatttggc gaagtgatta gctatcagca gctggcggcc 9961 ctggcgggta atccggcggc caccgccgcc gttaaaaccg cgctgagcgg taacccggtg 10021 ccgattctga ttccgtgcca tcgtgtggtt agctctagcg gtgcggttgg cggttatgaa 10081 ggtggtctgg cggtgaaaga gtggctgctg gcccatgaag gtcatcgtct gggtaaaccg 10141 ggtctgggac ctgcaggcgg gtcacctaag aaaaaacgaa aagttgagta aggtaccacg 10201 cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac 10261 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 10321 gctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc atgatcgcgt 10381 agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca aagcggtcgg 10441 acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc gctagcagca 10501 cgccatagtg actggcgatg ctgtcggaat ggacgatatc ccgcaagagg cccggcagta 10561 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga 10621 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac 10681 taccgcatta aagcttatcg atgataagct gtcaaacatg agaa //