LOCUS Exported 10582 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid encoding Cas9-53BP1-DN1S fusion with BbsI restriction sites for insertion of guide RNA sequence.. ACCESSION . VERSION . KEYWORDS pX458M-53BP1-DN1S SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10582) AUTHORS Jayavaradhan R, Pillis DM, Goodman M, Zhang F, Zhang Y, Andreassen PR, Malik P TITLE CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites. JOURNAL Nat Commun. 2019 Jun 28;10(1):2866. doi: 10.1038/s41467-019-10735-7. PUBMED 31253785 REFERENCE 2 (bases 1 to 10582) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10582 /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" enhancer 450..735 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 737..1014 /label=chicken beta-actin promoter intron 1015..1242 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1254..1263 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1263..1328 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1335..1355 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1380..5480 /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 6765..6812 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6828..6881 /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 6882..7595 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 6882..7595 /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(6924..6945) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(7185..7204) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7532..7553 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(7623..7640) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7629..7836 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 7845..7985 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7845..7974 /label=AAV2 ITR rep_origin 8060..8515 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8147..8166) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8357..8378 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8532..8551) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8651..8673 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8711..8729) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8797..8901 /gene="bla" /label=AmpR promoter CDS 8902..9762 /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(9120..9139) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9933..10521 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10422..10441 /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 taagagctat gctggaaaca gcatagcaag 301 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg 361 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa 421 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct 481 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc 541 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 601 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 661 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 721 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc 781 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg 841 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg 901 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt 961 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc 1021 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg 1081 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc 1141 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 1201 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca 1261 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg 1321 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg 1381 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca 1441 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca 1501 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca 1561 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc 1621 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg 1681 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca 1741 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac 1801 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga 1861 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg 1921 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca 1981 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc 2041 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga 2101 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg 2161 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga 2221 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc 2281 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga 2341 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc 2401 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct 2461 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa 2521 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc 2581 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca 2641 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga 2701 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat 2761 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg 2821 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc 2881 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata 2941 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg 3001 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga 3061 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg 3121 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca 3181 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga 3241 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc 3301 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc 3361 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt 3421 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc 3481 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg 3541 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga 3601 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg 3661 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga 3721 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg 3781 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata 3841 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg 3901 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca 3961 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact 4021 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca 4081 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg 4141 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta 4201 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc 4261 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc 4321 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc 4381 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga 4441 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca 4501 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc 4561 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca 4621 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga 4681 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca 4741 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt 4801 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag 4861 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc 4921 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact 4981 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga 5041 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact 5101 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc 5161 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc 5221 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca 5281 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg 5341 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg 5401 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc 5461 tgtctcagct gggaggcgac acagggtcca caggatccac aggcagcaca gggagcatgg 5521 gaccacatgg ccatgtctta catcgtcaca tgagaacaat ccgggaagta cgcacacttg 5581 tcactcgtgt cattacagat gtgtattatg tggatggaac agaagtagaa agaaaagtaa 5641 ctgaggagac tgaagagcca attgtagagt gtcaggagtg tgaaactgaa gtttcccctt 5701 cacagactgg gggctcctca ggtgacctgg gggatatcag ctccttctcc tccaaggcat 5761 ccagcttaca ccgcacatca agtgggacaa gtctctcagc tatgcacagc agtggaagct 5821 cagggaaagg agccggacca ctcagaggga aaaccagcgg gacagaaccc gcagattttg 5881 ccttacccag ctcccgagga ggcccaggaa aactgagtcc tagaaaaggg gtcagtcaga 5941 cagggacgcc agtgtgtgag gaggatggtg atgcaggcct tggcatcaga cagggaggga 6001 aggctccagt cacgcctcgt gggcgtgggc gaaggggccg cccaccttct cggaccactg 6061 gaaccagaga aacagctgtg cctggcccct tgggcataga ggacatttca cctaacttgt 6121 caccagatga taaatccttc agccgtgtcg tgccccgagt gccagactcc accagacgaa 6181 cagatgtggg tgctggtgct ttgcgtcgta gtgactctcc agaaattcct ttccaggctg 6241 ctgctggccc ttctgatggc ttagatgcct cctctccagg aaatagcttt gtagggctcc 6301 gtgttgtagc caagtggtca tccaatggct acttttactc tgggaaaatc acacgagatg 6361 tcggagctgg gaagtataaa ttgctctttg atgatgggta cgaatgtgat gtgttgggca 6421 aagacattct gttatgtgac cccatcccgc tggacactga agtgacggcc ctctcggagg 6481 atgagtattt cagtgcagga gtggtgaaag gacataggaa ggagtctggg gaactgtact 6541 acagcattga aaaagaaggc caaagaaagt ggtataagcg aatggctgtc atcctgtcct 6601 tggagcaagg aaacagactg agagagcagt atgggcttgg cccctatgaa gcagtaacac 6661 ctcttacaaa ggcagcagat atcagcttag acaatttggt ggaagggaag cggaaacgga 6721 gatctaacgt cagctcccca gccaccccta ctgcctcctc gagcaaaagg ccggcggcca 6781 cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa 6841 gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc 6901 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 6961 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 7021 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 7081 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 7141 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 7201 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 7261 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 7321 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 7381 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 7441 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 7501 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 7561 ccgggatcac tctcggcatg gacgagctgt acaaggaatt ctaactagag ctcgctgatc 7621 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 7681 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 7741 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 7801 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat 7861 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt 7921 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct 7981 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 8041 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 8101 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc 8161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 8221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt 8281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 8341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg 8401 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag 8461 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg 8521 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 8581 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 8641 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 8701 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 8761 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 8821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 8881 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 8941 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 9001 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 9061 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 9121 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 9181 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 9241 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 9301 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 9361 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 9421 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 9481 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 9541 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 9601 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc 9661 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 9721 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 9781 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 9841 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 9901 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 9961 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 10021 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 10081 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 10141 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 10201 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 10261 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 10321 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 10381 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 10441 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 10501 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 10561 tgctggcctt ttgctcacat gt //