LOCUS Exported 10477 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of a catalytically inactive, human codon-optimized Cas9 under the control of Murine Stem Cell retroVirus LTR promoter for mammalian gene knockdown. ACCESSION . VERSION . KEYWORDS pdCas9-humanized SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10477) AUTHORS Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA TITLE Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. JOURNAL Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. PUBMED 23452860 REFERENCE 2 (bases 1 to 10477) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10477 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 579..920 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" CDS 987..1403 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 1333..1355 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 1371..1387 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" regulatory 1417..1426 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1423..5526 /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 5539..5559 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5563..5583 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5587..5607 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 5620..6119 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(5655..5678) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 6052..6071 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 6140..6739 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(6140..6159) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6636..6656 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 6839..7353 /label=3' LTR /note="3' long terminal repeat from murine embryonic stem cell virus" primer_bind complement(7522..7538) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7522..7538) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7535..7557) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7546..7562 /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(7570..7600) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7615..7636 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7753..7770) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7924..8512) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8004..8023) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8683..9543) /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 9306..9325 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9544..9648) /gene="bla" /label=AmpR promoter primer_bind 9716..9734 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(9772..9794) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 9894..9913 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 10107..10129 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 10257..10276 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" LTR 10476..515 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" ORIGIN 1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc 301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc 361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata 421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca 481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga 541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg 601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt 661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa 721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga 781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag 841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa 901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg 961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct 1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga 1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag 1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc 1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc 1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg 1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag 1381 ccctcactcc ttctctaggc gccggaatta gatctcgcca ccatggacaa gaagtattct 1441 atcggactgg ccatcgggac taatagcgtc gggtgggccg tgatcactga cgagtacaag 1501 gtgccctcta agaagttcaa ggtgctcggg aacaccgacc ggcattccat caagaaaaat 1561 ctgatcggag ctctcctctt tgattcaggg gagaccgctg aagcaacccg cctcaagcgg 1621 actgctagac ggcggtacac caggaggaag aaccggattt gttaccttca agagatattc 1681 tccaacgaaa tggcaaaggt cgacgacagc ttcttccata ggctggaaga atcattcctc 1741 gtggaagagg ataagaagca tgaacggcat cccatcttcg gtaatatcgt cgacgaggtg 1801 gcctatcacg agaaataccc aaccatctac catcttcgca aaaagctggt ggactcaacc 1861 gacaaggcag acctccggct tatctacctg gccctggccc acatgatcaa gttcagaggc 1921 cacttcctga tcgagggcga cctcaatcct gacaatagcg atgtggataa actgttcatc 1981 cagctggtgc agacttacaa ccagctcttt gaagagaacc ccatcaatgc aagcggagtc 2041 gatgccaagg ccattctgtc agcccggctg tcaaagagcc gcagacttga gaatcttatc 2101 gctcagctgc cgggtgaaaa gaaaaatgga ctgttcggga acctgattgc tctttcactt 2161 gggctgactc ccaatttcaa gtctaatttc gacctggcag aggatgccaa gctgcaactg 2221 tccaaggaca cctatgatga cgatctcgac aacctcctgg cccagatcgg tgaccaatac 2281 gccgaccttt tccttgctgc taagaatctt tctgacgcca tcctgctgtc tgacattctc 2341 cgcgtgaaca ctgaaatcac caaggcccct ctttcagctt caatgattaa gcggtatgat 2401 gagcaccacc aggacctgac cctgcttaag gcactcgtcc ggcagcagct tccggagaag 2461 tacaaggaaa tcttctttga ccagtcaaag aatggatacg ccggctacat cgacggaggt 2521 gcctcccaag aggaatttta taagtttatc aaacctatcc ttgagaagat ggacggcacc 2581 gaagagctcc tcgtgaaact gaatcgggag gatctgctgc ggaagcagcg cactttcgac 2641 aatgggagca ttccccacca gatccatctt ggggagcttc acgccatcct tcggcgccaa 2701 gaggacttct acccctttct taaggacaac agggagaaga ttgagaaaat tctcactttc 2761 cgcatcccct actacgtggg acccctcgcc agaggaaata gccggtttgc ttggatgacc 2821 agaaagtcag aagaaactat cactccctgg aacttcgaag aggtggtgga caagggagcc 2881 agcgctcagt cattcatcga acggatgact aacttcgata agaacctccc caatgagaag 2941 gtcctgccga aacattccct gctctacgag tactttaccg tgtacaacga gctgaccaag 3001 gtgaaatatg tcaccgaagg gatgaggaag cccgcattcc tgtcaggcga acaaaagaag 3061 gcaattgtgg accttctgtt caagaccaat agaaaggtga ccgtgaagca gctgaaggag 3121 gactatttca agaaaattga atgcttcgac tctgtggaga ttagcggggt cgaagatcgg 3181 ttcaacgcaa gcctgggtac ctaccatgat ctgcttaaga tcatcaagga caaggatttt 3241 ctggacaatg aggagaacga ggacatcctt gaggacattg tcctgactct cactctgttc 3301 gaggaccggg aaatgatcga ggagaggctt aagacctacg cccatctgtt cgacgataaa 3361 gtgatgaagc aacttaaacg gagaagatat accggatggg gacgccttag ccgcaaactc 3421 atcaacggaa tccgggacaa acagagcgga aagaccattc ttgatttcct taagagcgac 3481 ggattcgcta atcgcaactt catgcaactt atccatgatg attccctgac ctttaaggag 3541 gacatccaga aggcccaagt gtctggacaa ggtgactcac tgcacgagca tatcgcaaat 3601 ctggctggtt cacccgctat taagaagggt attctccaga ccgtgaaagt cgtggacgag 3661 ctggtcaagg tgatgggtcg ccataaacca gagaacattg tcatcgagat ggccagggaa 3721 aaccagacta cccagaaggg acagaagaac agcagggagc ggatgaaaag aattgaggaa 3781 gggattaagg agctcgggtc acagatcctt aaagagcacc cggtggaaaa cacccagctt 3841 cagaatgaga agctctatct gtactacctt caaaatggac gcgatatgta tgtggaccaa 3901 gagcttgata tcaacaggct ctcagactac gacgtggacg ccatcgtccc tcagagcttc 3961 ctcaaagacg actcaattga caataaggtg ctgactcgct cagacaagaa ccggggaaag 4021 tcagataacg tgccctcaga ggaagtcgtg aaaaagatga agaactattg gcgccagctt 4081 ctgaacgcaa agctgatcac tcagcggaag ttcgacaatc tcactaaggc tgagaggggc 4141 ggactgagcg aactggacaa agcaggattc attaaacggc aacttgtgga gactcggcag 4201 attactaaac atgtcgccca aatccttgac tcacgcatga ataccaagta cgacgaaaac 4261 gacaaactta tccgcgaggt gaaggtgatt accctgaagt ccaagctggt cagcgatttc 4321 agaaaggact ttcaattcta caaagtgcgg gagatcaata actatcatca tgctcatgac 4381 gcatatctga atgccgtggt gggaaccgcc ctgatcaaga agtacccaaa gctggaaagc 4441 gagttcgtgt acggagacta caaggtctac gacgtgcgca agatgattgc caaatctgag 4501 caggagatcg gaaaggccac cgcaaagtac ttcttctaca gcaacatcat gaatttcttc 4561 aagaccgaaa tcacccttgc aaacggtgag atccggaaga ggccgctcat cgagactaat 4621 ggggagactg gcgaaatcgt gtgggacaag ggcagagatt tcgctaccgt gcgcaaagtg 4681 ctttctatgc ctcaagtgaa catcgtgaag aaaaccgagg tgcaaaccgg aggcttttct 4741 aaggaatcaa tcctccccaa gcgcaactcc gacaagctca ttgcaaggaa gaaggattgg 4801 gaccctaaga agtacggcgg attcgattca ccaactgtgg cttattctgt cctggtcgtg 4861 gctaaggtgg aaaaaggaaa gtctaagaag ctcaagagcg tgaaggaact gctgggtatc 4921 accattatgg agcgcagctc cttcgagaag aacccaattg actttctcga agccaaaggt 4981 tacaaggaag tcaagaagga ccttatcatc aagctcccaa agtatagcct gttcgaactg 5041 gagaatgggc ggaagcggat gctcgcctcc gctggcgaac ttcagaaggg taatgagctg 5101 gctctcccct ccaagtacgt gaatttcctc taccttgcaa gccattacga gaagctgaag 5161 gggagccccg aggacaacga gcaaaagcaa ctgtttgtgg agcagcataa gcattatctg 5221 gacgagatca ttgagcagat ttccgagttt tctaaacgcg tcattctcgc tgatgccaac 5281 ctcgataaag tccttagcgc atacaataag cacagagaca aaccaattcg ggagcaggct 5341 gagaatatca tccacctgtt caccctcacc aatcttggtg cccctgccgc attcaagtac 5401 ttcgacacca ccatcgaccg gaaacgctat acctccacca aagaagtgct ggacgccacc 5461 ctcatccacc agagcatcac cggactttac gaaactcgga ttgacctctc acagctcgga 5521 ggggatgagg gagctgatcc aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag 5581 gtagatccaa aaaagaagag aaaggtatag aattctaccg ggtaggggag gcgcttttcc 5641 caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg 5701 gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg 5761 gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc 5821 agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga 5881 tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc 5941 tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca 6001 ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca 6061 cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga 6121 cctgcagccc aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga 6181 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg 6241 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct 6301 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc 6361 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg 6421 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc 6481 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca 6541 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc 6601 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct 6661 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac 6721 ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc 6781 acgaccccat gcatcgataa aataaaagat tttatttagt ctccagaaaa aggggggaat 6841 gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg 6901 gaaaatacat aactgagaat agagaagttc agatcaaggt taggaacaga gagacagcag 6961 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 7021 cagatggtcc ccagatgcgg tcccgccctc agcagtttct agagaaccat cagatgtttc 7081 cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg 7141 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc 7201 tcactcggcg cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg tatccaataa 7261 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga 7321 gtgattgact acccgtcagc gggggtcttt catgggtaac agtttcttga agttggagaa 7381 caacattctg agggtaggag tcgaatatta agtaatcctg actcaattag ccactgtttt 7441 gaatccacat actccaatac tcctgaaata gttcattatg gacagcgcag aagagctggg 7501 gagaattaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 7561 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 7621 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 7681 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 7741 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 7801 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 7861 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 7921 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 7981 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 8041 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 8101 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 8161 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 8221 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 8281 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 8341 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 8401 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 8461 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 8521 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 8581 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 8641 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 8701 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 8761 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 8821 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 8881 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 8941 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 9001 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 9061 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 9121 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 9181 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 9241 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 9301 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 9361 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 9421 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 9481 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 9541 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 9601 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 9661 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 9721 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca 9781 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 9841 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc 9901 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 9961 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg 10021 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 10081 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggcgcaagg 10141 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata 10201 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg 10261 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg 10321 cgtccggcgt agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct 10381 ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa 10441 taaaagattt tatttagtct ccagaaaaag gggggaa //