LOCUS Exported 9288 bp ds-DNA circular SYN 08-JUN-2021 DEFINITION Cas9n (D10A nickase mutant) from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA. ACCESSION . VERSION . KEYWORDS pSpCas9n(BB)-2A-GFP (PX461) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9288) AUTHORS Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F TITLE Genome engineering using the CRISPR-Cas9 system. JOURNAL Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. PUBMED 24157548 REFERENCE 2 (bases 1 to 9288) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 8, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9288 /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..1232 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1244..1253 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1253..1318 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1325..1345 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1370..5470 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5471..5518 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5534..5587 /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 5588..6301 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 5588..6301 /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(5630..5651) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5891..5910) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6238..6259 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(6329..6346) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6335..6542 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6551..6691 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6551..6680 /label=AAV2 ITR rep_origin 6766..7221 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6853..6872) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7063..7084 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7238..7257) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7357..7379 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7417..7435) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7503..7607 /gene="bla" /label=AmpR promoter CDS 7608..8468 /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(7826..7845) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8639..9227 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9128..9147 /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 cgcgccrggg gggggggggg gggggggggg ggggggsggg gsgrggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct 1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga 1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc 1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc 1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa 1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat 1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta 1381 cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta 1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa 1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa 1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat 1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt 1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga 1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag 1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg 1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt 1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg 1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct 2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag 2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca 2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca 2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat 2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata 2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga 2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg 2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg 2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt 2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg 2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac 2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat 2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg 2821 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga 2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac 2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa 3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa 3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga 3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga 3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact 3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga 3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa 3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc 3421 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa 3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc 3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga 3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag 3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga 3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca 3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga 3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag 3901 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg 3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca 4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag 4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg 4141 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga 4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga 4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca 4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga 4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag 4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt 4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac 4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa 4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt 4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga 4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt 4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg 4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa 4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga 4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga 5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct 5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta 5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc 5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca 5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa 5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc 5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct 5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga 5521 attcggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc 5581 tggcccagtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 5641 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 5701 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 5761 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 5821 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 5881 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 5941 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 6001 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 6061 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 6121 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 6181 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 6241 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 6301 ggaattctaa ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 6361 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 6421 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 6481 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 6541 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 6601 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 6661 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 6721 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6781 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6841 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6901 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6961 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 7021 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 7081 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga 7141 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 7201 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 7261 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 7321 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 7381 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 7441 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 7501 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 7561 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 7621 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 7681 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 7741 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7801 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7861 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7921 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7981 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 8041 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 8101 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 8161 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 8221 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 8281 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 8341 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 8401 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 8461 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 8521 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 8581 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 8641 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 8701 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 8761 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 8821 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8881 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8941 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 9001 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 9061 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 9121 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 9181 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 9241 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt //