LOCUS Exported 10266 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of human codon-optimized dCas9-DNMT3A fusion with T2A-EGFP for targeted DNA methylation in mammalian cells; cloning backbone for sgRNA. ACCESSION . VERSION . KEYWORDS pdCas9-DNMT3A-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10266) AUTHORS Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V TITLE Repurposing the CRISPR-Cas9 system for targeted DNA methylation. JOURNAL Nucleic Acids Res. 2016 Mar 11. pii: gkw159. PUBMED 26969735 REFERENCE 2 (bases 1 to 10266) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10266 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 440..725 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 727..1004 /label=chicken beta-actin promoter intron 1005..1233 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1245..1254 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1254..1319 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1326..1346 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1371..5471 /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5472..5519 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5547..6479 /codon_start=1 /product="catalytic domain of DNA methyltransferase 3A" /label=DNMT3A catalytic domain /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR LARQRLLGRSWSVPVIRHLFAPLKEYFACV" CDS 6507..6560 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 6561..7274 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 6561..7274 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(6603..6624) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6864..6883) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7211..7232 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(7307..7324) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7313..7520 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 7529..7669 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7529..7658 /label=AAV2 ITR rep_origin 7744..8199 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7831..7850) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8041..8062 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8216..8235) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8335..8357 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8395..8413) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8481..8585 /gene="bla" /label=AmpR promoter CDS 8586..9446 /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(8804..8823) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9617..10205 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10106..10125 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1381 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga 2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga 3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg acaggccaaa aagaaaaagc 5521 tcgagggcgg aggcgggagc ggatccccct cccggctcca gatgttcttc gctaataacc 5581 acgaccagga atttgaccct ccaaaggttt acccacctgt cccagctgag aagaggaagc 5641 ccatccgggt gctgtctctc tttgatggaa tcgctacagg gctcctggtg ctgaaggact 5701 tgggcattca ggtggaccgc tacattgcct cggaggtgtg tgaggactcc atcacggtgg 5761 gcatggtgcg gcaccagggg aagatcatgt acgtcgggga cgtccgcagc gtcacacaga 5821 agcatatcca ggagtggggc ccattcgatc tggtgattgg gggcagtccc tgcaatgacc 5881 tctccatcgt caaccctgct cgcaagggcc tctacgaggg cactggccgg ctcttctttg 5941 agttctaccg cctcctgcat gatgcgcggc ccaaggaggg agatgatcgc cccttcttct 6001 ggctctttga gaatgtggtg gccatgggcg ttagtgacaa gagggacatc tcgcgatttc 6061 tcgagtccaa ccctgtgatg attgatgcca aagaagtgtc agctgcacac agggcccgct 6121 acttctgggg taaccttccc ggtatgaaca ggccgttggc atccactgtg aatgataagc 6181 tggagctgca ggagtgtctg gagcatggca ggatagccaa gttcagcaaa gtgaggacca 6241 ttactacgag gtcaaactcc ataaagcagg gcaaagacca gcattttcct gtgttcatga 6301 atgagaaaga ggacatctta tggtgcactg aaatggaaag ggtatttggt ttcccagtcc 6361 actatactga cgtctccaac atgagccgct tggcgaggca gagactgctg ggccggtcat 6421 ggagcgtgcc agtcatccgc cacctcttcg ctccgctgaa ggagtatttt gcgtgtgtgt 6481 ccggccggcc cgaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg 6541 tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 6601 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 6661 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 6721 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 6781 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 6841 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 6901 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 6961 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 7021 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 7081 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 7141 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 7201 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 7261 acgagctgta caaggaattc aattctaact agagctcgct gatcagcctc gactgtgcct 7321 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 7381 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 7441 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag 7501 aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc 7561 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc 7621 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg 7681 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat 7741 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 7801 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 7861 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 7921 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg 7981 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 8041 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt 8101 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 8161 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa 8221 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 8281 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 8341 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 8401 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 8461 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 8521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 8581 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 8641 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 8701 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 8761 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 8821 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 8881 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 8941 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 9001 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 9061 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 9121 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 9181 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 9241 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 9301 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 9361 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 9421 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 9481 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 9541 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 9601 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 9661 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 9721 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 9781 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 9841 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 9901 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 9961 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 10021 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 10081 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 10141 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 10201 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 10261 acatgt //