LOCUS Exported 8960 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Cas9 nuclease and gRNA. ACCESSION . VERSION . KEYWORDS pX330A-1x6 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8960) AUTHORS Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T TITLE Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. JOURNAL Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400. PUBMED 24954249 REFERENCE 2 (bases 1 to 8960) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8960 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 8..293 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 295..572 /label=chicken beta-actin promoter intron 573..801 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 813..822 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 822..887 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 894..914 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 939..5039 /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5040..5087 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5115..5132) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5121..5328 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5337..5477 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5337..5466 /label=AAV2 ITR rep_origin 5552..6007 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5639..5658) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5849..5870 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6024..6043) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6143..6165 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6203..6221) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6289..6393 /gene="bla" /label=AmpR promoter CDS 6394..7254 /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(6612..6631) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7425..8013 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7914..7933 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8075..8315 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8075..8095 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8246..8265 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8342..8417 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS complement(8503..8808) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL" primer_bind 8658..8680 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8672..8689 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8673..8689 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 8699..8718 /label=T7 /note="T7 promoter, forward primer" promoter 8699..8717 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(8765..8785) /label=T3 /note="T3 promoter, forward primer" promoter complement(8765..8783) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(8804..8820) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8804..8820) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8817..8839) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8828..8844 /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(8852..8882) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8897..8918 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat 241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc 601 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 661 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 721 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 781 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg 841 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga 901 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc 961 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca 1021 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg 1081 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca 1141 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg 1201 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag 1261 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc 1321 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg 1381 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc 1441 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg 1501 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca 1561 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc 1621 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga 1681 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg 1741 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc 1801 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga 1861 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc 1921 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag 1981 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc 2041 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac 2101 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca 2161 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt 2221 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc 2281 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga 2341 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc 2401 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc 2461 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat 2521 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg 2581 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact 2641 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg 2701 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca 2761 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca 2821 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga 2881 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg 2941 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg 3001 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc 3061 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg 3121 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga 3181 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga 3241 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca 3301 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg 3361 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg 3421 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg 3481 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca 3541 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg 3601 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga 3661 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa 3721 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc 3781 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg 3841 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc 3901 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg 3961 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa 4021 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg 4081 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa 4141 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca 4201 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt 4261 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta 4321 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag 4381 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca 4441 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag 4501 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg 4561 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc 4621 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc 4681 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga 4741 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca 4801 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata 4861 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca 4921 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc 4981 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca 5041 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag 5101 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 5161 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 5221 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 5281 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga 5341 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 5401 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 5461 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 5521 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 5581 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5641 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5701 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5761 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5821 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 5881 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 5941 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 6001 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 6061 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 6121 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 6181 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 6241 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 6301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 6361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 6481 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 6541 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 6781 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 7081 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca 7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 7261 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 7321 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7501 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7561 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7621 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7681 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7741 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7801 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7861 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 7921 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 7981 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 8041 ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc 8101 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa 8161 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc 8221 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt 8281 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc 8341 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 8401 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta 8461 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagagac cggcgccgct 8521 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 8581 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 8641 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta 8701 atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcc tccagctttt 8761 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg 8821 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 8881 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 8941 ctttccaccg gtggtctctg //