LOCUS Exported 13908 bp ds-DNA circular SYN 08-SEP-2023 DEFINITION Cre-dependet Cas9-GFP expression. ACCESSION . VERSION . KEYWORDS pTol2 hsp70l loxP-DsRed-loxP-nCas9n-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13908) AUTHORS Hans S, Zoller D, Hammer J, Stucke J, Spiess S, Kesavan G, Kroehne V, Eguiguren JS, Ezhkova D, Petzold A, Dahl A, Brand M TITLE Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish. JOURNAL Nat Commun. 2021 Feb 18;12(1):1125. doi: 10.1038/s41467-021-21427-6. PUBMED 33602923 REFERENCE 2 (bases 1 to 13908) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 8, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13908 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(7..462) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 356..375 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 589..611 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 603..620 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 604..620 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 630..649 /label=T7 /note="T7 promoter, forward primer" promoter 630..648 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4494..4527 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." regulatory 4534..4543 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4540..5217 /codon_start=1 /product="improved tetrameric variant of DsRed fluorescent protein" /label=DsRed2 /note="mammalian codon-optimized" /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH LFL" primer_bind complement(4720..4740) /label=DsRed1-N /note="DsRed1, reverse primer" primer_bind 5132..5155 /label=DsRed1-C /note="DsRed1, forward primer" polyA_signal 5338..5459 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind 5713..5746 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 5765..5785 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5792..9892 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes MGAS15252 Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" RBS 9805..9813 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 9908..9928 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9929..10645 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 9929..10642 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(9971..9992) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(10232..10251) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 10579..10600 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 10768..10849 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11671..11687) /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11671..11687) /label=pBluescriptSK /note="For pBluescript vector" primer_bind complement(11724..11744) /label=T3 /note="T3 promoter, forward primer" promoter complement(11724..11742) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(11763..11779) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11763..11779) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11776..11798) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11787..11803 /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(11811..11841) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11856..11877 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11994..12011) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12165..12753) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12245..12264) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12924..13784) /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 13547..13566 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13785..13889) /gene="bla" /label=AmpR promoter ORIGIN 1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc 481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 661 ccaaatagta ggaattaccc acctgtacaa gtgctgaaaa cttggatgaa taagcccgtt 721 tgccattttc tactgctatt ttaaatcttt tctgtatttg tctgcatttg tctttaccct 781 tgaataaatg ttttagttgt tttttttcaa ttatgactgt gtttaacaga cattattaca 841 ataatatgta atagtagtac acactattat tatgtaatag tacttgttga ctgtatttga 901 gaactggacc gagtgagtgt tacgtcaccc attcaaaatg acttacttct ggctccaaca 961 aaatgaagtt aattcagttg ccatttttca ctgtatggac atcgccgtgt tggagctaga 1021 cgttgccaat aagcaagaaa gagccgagat gcgtcgagtc tgagtcaccg ttcctatggc 1081 aacccctcta accaatcaga agtaagcttg ttggaagtcc acagcctacc acttgaaagc 1141 gggctgcaca aaatctgtca aacgttttga acgttggatg tgagagcaca tacttttatt 1201 aaggcatctg gttggtcagt ttataatatc aacaacttgg gctacagaaa agaaaagtta 1261 ttacagaaat tatgattaac aagtacatgt taaataaaga ttttaatatg aatgccacca 1321 ctggagcatt catgccattt ggagcttctt cctgtttgga tcactagaag gaggaggtca 1381 ctcattacag ttctcatata cagtcgttgg ttggttggtt ggttggtaga ttgattgatt 1441 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt 1501 gattggtagt caaaataaga aataatttcc acagattcat tacagaaatg attaaatgca 1561 tacataaaaa actggggggg gggggggata caacaacaca cttaagtcac atttgcctac 1621 gtaaagaaaa gtaaagaaaa tcaatagcta tattttacat ctcctttttt tgctgtcttt 1681 taattagcct tgttttgctg ttatcttgat tgaaactgta acttcttcac ctgcttcttt 1741 tctttgtagg ttttgccagc ccagaaacag gcatggctgt gcaaggccag agcaccatgc 1801 acaatgcgct gcatgtcttc atgaacggct caatgtcctc agtccagggc tcagccaacg 1861 accccatctt cctccttcac catgctttca ttgacaggta acaaacacgt catgacatta 1921 gactgcacag tttttgacaa agttcataca atctgttgtt tatagctgct acaattagtg 1981 aagtttgtga atgtacttgg atgagcagcg aaagatcaat tgagatcaat tgttagagtt 2041 tggttgccct gcagagcaaa gaacaaaaaa taatctggtg gctttactgc gtgaggttat 2101 tattggtgga atagaaacac aaaacataat tgcatttatt tgtttaattt tttatcttat 2161 cttaactttc atcttgcata tttgtttctt acatcatttc tagcatcttt gagcgctggc 2221 taagaactca tcagcctccc cggtccatct acccacgtac caatgcacca attggccaca 2281 atgacggcta ctacatggtg ccattccttc ctctttatag gaatggagac tacctcctgt 2341 ccaacaatgc tcttggatac gagtacgcct acctgttgga cccaggtcat tgcacaacac 2401 cagaaatgcc ctctgatctg caaaagacgt gaatatctgt tcagacaccc atatccactc 2461 tgttccacac aggtcagagg tttgtccagg agttcttgac agaggtgtaa aaagtactca 2521 aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa 2581 aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac 2641 ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagattc ttgtttaagc 2701 ttttaatctc aaaaaacatt aaatgaaatg catacaaggt tttatcctgc tttagaactg 2761 tttgtattta attatcaaac tataagacag acaatctaat gccagtacac gctactcaaa 2821 gttgtaaaac ctcagattta acttcagtag aagctgattc tcaaaattgt tagtgtcaag 2881 cctagctctt ttggggctga aaagcaatcc tgcagtgctg aaaagcctct cacaggcagc 2941 cgatgcggga agaggtgtat tagtcttgat agagaggctg caaatagcag gaaacgtgag 3001 cagagactcc ctggtgtctg aaacacaggc cagatgggcc cttcaggggt gtcgcttggt 3061 tatttccaaa aatcaaatta attttattaa actattagaa cgagcatgtt ttgtctatat 3121 gctacagaag ataaaaaata ataggagtta acagttataa aacaacacac tttgtttcta 3181 ttgattgttg accacactgg ggtctcatta agttagatta aagacacact aactgggtca 3241 aaagcagcag attgatttca tagcaccagg gtaaactttc taacactttt acggcaatca 3301 tatacattaa aattaaatac agaccacgac tgaacaagga ggatgatctc caatattaaa 3361 caaagagact tgtgcctatt tctctgaggg taaacatgac ctctcaagtt agcaagttgt 3421 ttttaacact acaaaaatag ttaagactgc aatcccagaa taaagtattg gttttaacca 3481 atcaatatag tacagtaaac atccatttgt tttgttgaaa cgttaaacaa atctgaccaa 3541 agctattagc ttatataaaa caggtttgcc ttctatgtag ctgaaaacac cacaggcccg 3601 attttgctac tgtgtaaaac atttcagcaa gattttttta ttgcattttt ttttactgaa 3661 tcgttcaaac attttatcat tttagtttgt tcattcattg caactggaaa aacaacacat 3721 cacacaaccg cacatatttc agcaataagt acaataaaac actcaaataa aaaaaacatt 3781 ttaaatctct ttgtattttt gaccgctgtt tcgcgtaatt tcacggtaaa actctggaaa 3841 tctccactac attcctctca gcggctcctc tcaatgacag ctgaagaagt gacgcggctg 3901 cctgctgtgt tttgattggt cgaattcact ggaggcttcc agaacagtgt agagtctgaa 3961 cgggtgcgcg ctctgctgta tttaaagggc gaaagagaga ccgcagagaa actcaaccga 4021 agagaagcga cttgacaaag aagaaaagag cagcctgaca ggactttttc cccgacgagg 4081 tgtttattcg ctctatttaa gaatctactg taaggtaagt ctcaatatat tgtactctat 4141 tggctaatca gaattatata gagattatat gtacttaatg tcaaaaaatc aactttgtat 4201 atgtaatctt tttacatgtg gactgcctat gttcatctta ttttaggtct actagaaaat 4261 tatatttccc gttttcacaa taaggatttt taaaaaaagc aatgaacaga cgggcattta 4321 ctttatgttg ctgacattat tttatatgag cataataacc ataaatacta gcaaatgtcc 4381 taaatgaatt tgtgttaatg ttgtctacaa aagaaaatta gcgttttact tgtacaacta 4441 ataataactt ggttattaag agaatttcac ttgttgacta gaaaaggccg gccataactt 4501 cgtatagcat acattatacg aagttatccg gtcgccacca tggcctcctc cgagaacgtc 4561 atcaccgagt tcatgcgctt caaggtgcgc atggagggca ccgtgaacgg ccacgagttc 4621 gagatcgagg gcgagggcga gggccgcccc tacgagggcc acaacaccgt gaagctgaag 4681 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac 4741 ggctccaagg tgtacgtgaa gcaccccgcc gacatccccg actacaagaa gctgtccttc 4801 cccgagggct tcaagtggga gcgcgtgatg aactttgagg acggcggcgt ggcgaccgtg 4861 acccaggact cctccctgca ggacggctgc ttcatctaca aggtgaagtt catcggcgtg 4921 aacttcccct ccgacggccc cgtgatgcag aagaagacca tgggctggga ggcctccacc 4981 gagcgcctgt acccccgcga cggcgtgctg aagggcgaga cccacaaggc cctgaagctg 5041 aaggacggcg gccactacct ggtggagttc aagtccatct acatggccaa gaagcccgtg 5101 cagctgcccg gctactacta cgtggacgcc aagctggaca tcacctccca caacgaggac 5161 tacaccatcg tggagcagta cgagcgcacc gagggccgcc accacctgtt cctgtagcgg 5221 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa 5281 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac 5341 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 5401 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa 5461 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 5521 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 5581 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 5641 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 5701 accctaatca agataacttc gtatagcata cattatacga agttatcccg tcatggcttc 5761 tccacctaag aagaagagaa aggtgggaag catggataag aagtatagca tcggcctgga 5821 tattggaact aactccgtgg gttgggcagt gattacagac gactacaagg tccctagcaa 5881 gaaatttaag gtgctgggta acaccgacag gcacagcatc aagaaaaatc tgattggagc 5941 cctgctgttc ggttctggag agactgccga agcaacacgc ctgaaaagaa cagcaagaag 6001 gcgctatacc agaaggaaga atagaatctg ttacctgcag gagattttct ctaacgaaat 6061 ggctaaggtg gacgattcat tctttcatag gctggaggaa agtttcctgg tcgaggaaga 6121 taagaaacac gagcgccatc ctatctttgg aaacattgtg gacgaggtcg cctatcacga 6181 aaaataccca accatctatc atctgcgcaa gaaactggct gactctactg ataaagccga 6241 cctgagactg atctatctgg ctctggccca catgattaag ttcaggggtc attttctgat 6301 cgagggcgat ctgaaccccg acaattccga tgtggacaag ctgttcatcc agctggtcca 6361 gatttacaat cagctgtttg aggaaaaccc tattaatgct tccagagtgg acgcaaaagc 6421 tatcctgtca gccaggctgt ccaagtcacg cagactggag aacctgattg cacagctgcc 6481 cggagaaaag aggaacggtc tgtttggaaa tctgatcgct ctgagtctgg gcctgactcc 6541 taacttcaaa agcaattttg atctggctga ggacgccaaa ctgcagctgt caaaggacac 6601 atatgacgat gacctggata acctgctggc acagatcgga gatcagtacg ctgacctgtt 6661 cctggctgcc aaaaatctgt ccgacgcaat cctgctgtca gatattctga gagtgaacag 6721 cgagattaca aaagctcctc tgagtgccag catgatcaag agatatgacg agcaccatca 6781 ggatctgacc ctgctgaagg ctctggtcag gcagcagctg ccagagaagt acaaggaaat 6841 tttctttgat cagtccaaga acggctacgc cggttatatc gacggaggcg catcacagga 6901 ggaattctac aagtttatca aacctattct ggagaagatg gacggaactg aggaactgct 6961 ggtgaaactg aatagagagg acctgctgag gaagcagcgc acatttgata acggttccat 7021 cccacaccag attcatctgg gagagctgca cgctatcctg aggcgccagg aagacttcta 7081 cccctttctg aaagataacc gcgagaagat cgaaaaaatt ctgaccttca gaatccctta 7141 ctatgtgggt ccactggctc gcggaaacag cagatttgcc tggatgactc gcaaatccga 7201 ggaaaccatt actccttgga acttcgagga agtggtcgat aagggcgcct ctgcacagtc 7261 cttcatcgag agaatgacta attttgacaa aaacctgccc aatgagaaag tgctgcctaa 7321 gcactccctg ctgtacgagt atttcactgt ctataacgaa ctgacaaagg tgaaatacgt 7381 caccgagggc atgagaaagc cagccttcct gtcaggagag cagaagaaag caatcgtgga 7441 tctgctgttt aaaaccaaca ggaaagtgac tgtcaagcag ctgaaggagg actacttcaa 7501 gaaaattgaa tgcttcgatt ccgtggagat cagcggagtc gaagacagat ttaacgcaag 7561 cctgggcgct taccacgatc tgctgaagat cattaaggat aaagacttcc tggacaacga 7621 ggaaaatgag gatatcctgg aagacattgt gctgacactg accctgtttg aggacagagg 7681 aatgatcgag gaaagactga aaacctatgc tcatctgttc gatgacaagg tgatgaaaca 7741 gctgaagaga aggcgctaca ctggctgggg tagactgagc aggaagctga tcaacggcat 7801 tagggataaa cagtcaggaa agacaatcct ggactttctg aaaagtgatg gcttcgccaa 7861 ccgcaatttt atgcagctga ttcacgatga cagtctgacc ttcaaagagg acatccagaa 7921 ggctcaggtg tctggacagg gccactccct gcatgagcag attgcaaacc tggctggaag 7981 cccagccatc aagaaaggca ttctgcagac agtgaaaatc gtcgatgagc tggtgaaagt 8041 catgggccat aagcccgaaa acatcgtgat tgagatggct cgcgaaaatc agacaaccca 8101 gaagggtcag aagaacagta gagagaggat gaaaagaatc gaggaaggca ttaaggagct 8161 gggtagccag atcctgaaag agcacccagt ggaaaacaca cagctgcaga atgagaagct 8221 gtatctgtac tatctgcaga atggaagaga tatgtacgtg gaccaggagc tggatattaa 8281 caggctgtct gattacgacg tggatcatat cgtcccccag agtttcatca aagatgacag 8341 cattgacaac aaggtgctga ccaggtccga caaaaacaga ggaaaatcag ataatgtccc 8401 tagtgaggaa gtggtcaaga aaatgaagaa ctactggaga cagctgctga atgccaaact 8461 gatcactcag aggaagtttg ataacctgac aaaagcagag cgcggtggac tgtcagaact 8521 ggacaaagct ggattcatca agaggcagct ggtggaaaca cgccagatca ctaaacacgt 8581 cgcacagatt ctggatagtc gcatgaacac aaagtacgat gagaatgaca aactgatcag 8641 agaagtgaag gtcattaccc tgaagagtaa actggtcagc gactttagga aagatttcca 8701 gttttataag gtccgcgaga ttaacaatta tcaccatgcc catgacgcat acctgaacgc 8761 cgtggtcggt accgcactga tcaagaaata cccaaaactg gagagcgaat tcgtgtacgg 8821 agactataag gtgtacgatg tcagaaaaat gatcgccaag tccgagcagg aaattggaaa 8881 agctactgcc aagtatttct tttactcaaa catcatgaat ttctttaaga cagagatcac 8941 cctggccaat ggagaaatcc gcaaaaggcc cctgattgag acaaacggag agacaggcga 9001 aatcgtgtgg gacaaaggca gagattttgc aaccgtgagg aaggtcctga gcatgcctca 9061 agtgaatatc gtcaagaaaa ctgaggtgca gacaggcggt ttctcaaaag aaagtattct 9121 gccaaaacgc aactctgata agctgatcgc tagaaagaaa gactgggacc ctaagaagta 9181 tggaggcttt gactctccca ctgtggcata ctccgtcctg gtggtcgcta aggtggagaa 9241 gggcaaaagc aagaaactga aatctgtcaa ggagctgctg ggtatcacaa ttatggagag 9301 aagctctttc gagaagaacc caatcgattt tctggaggcc aaaggttata aggaagtgaa 9361 gaaagacctg atcattaaac tgcccaagta cagtctgttt gagctggaaa acggcaggaa 9421 acgcatgctg gcaagcgctg gagagctgca gaaaggcaat gaactggccc tgccttctaa 9481 gtacgtgaac ttcctgtatc tggcaagcca ctacgagaag ctgaaaggat ctccagagga 9541 taacgaacag aaacagctgt ttgtggagca gcacaagcat tatctggacg agatcattga 9601 acagattagc gagttctcta aaagagtgat cctggccgac gcaaatctgg ataaggtcct 9661 gtctgcttac aacaaacaca gagataagcc catcagggag caggccgaaa atatcattca 9721 tctgttcact ctgacaaacc tgggcgcacc tgcagctttc aagtacttcg acactacaat 9781 cgatagaaag aggtacacct ccactaagga ggtgctggac gctacactga tccatcagag 9841 tattaccggc ctgtacgaaa caaggattga cctgtctcag ctgggtggcg actcaccggt 9901 gagatctcct aagaagaaga gaaaggtggt gagcaagggc gaggagctgt tcaccggggt 9961 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg 10021 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg 10081 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt 10141 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg 10201 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga 10261 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa 10321 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta 10381 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat 10441 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg 10501 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc 10561 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct 10621 cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc 10681 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 10741 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 10801 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 10861 ggtttgtcca aactcatcaa tgtatcgcta gcatatatgg cgcgccatca acgtcaaaac 10921 atgcacaccc aaaaaaaaaa aaaaaaaaaa aaaaaaaaan nctatcagta aaacaaaata 10981 cttaatgtac ttacctcaat atgcttcctt aggtttgatg gcgaactttt gaaggccgat 11041 atttctttat taagcgggag acagaggaca catttcatct tgaatgaatc tttatttaca 11101 ccacttaaag aaaagaattc gcgaagatac ggccacgggt gctcttgatc ctgtggctga 11161 ttttggactg tgctgctcgc agctgctgat gaatcacata cttcctccat tttcttccac 11221 tgattgactg ttataatttc cctaatttcc aggtcaaggt gctgtgcatt gtggtaatag 11281 atgtgacatg acgtcacttc caaaggacca atgaacatgt ctgaccaatt tcatataatg 11341 tgaaaacgat tttcataggc agaataaata acatttaaat taaactgggc atcagcgcaa 11401 ttcaattggt ttggtaatag caagggaaaa tagaatgaag tgatctccaa aaaataagta 11461 ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt 11521 aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca 11581 aaaataatac ttaagtacag taatcaagta aaattactca agtactttac acctctggtt 11641 cttgaccccc taccttcagc aagcccagca gatccactag ttctagagcg gccgccaccg 11701 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca 11761 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 11821 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 11881 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 11941 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 12001 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 12061 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 12121 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 12181 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 12241 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 12301 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 12361 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 12421 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 12481 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 12541 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 12601 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 12661 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 12721 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 12781 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 12841 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 12901 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 12961 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 13021 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 13081 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 13141 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 13201 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 13261 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 13321 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 13381 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 13441 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 13501 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 13561 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 13621 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 13681 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 13741 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 13801 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 13861 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtg //