AIO-mCherry

LOCUS AIO-mCherry 9728 bp ds-DNA circular SYN 10-JAN-2018 DEFINITION All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and mCherry-coupled Cas9-D10A nickase to enhance efficient and accurate genome editing. ACCESSION . VERSION . KEYWORDS AIO-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9728) AUTHORS Chiang TW, le Sage C, Larrieu D, Demir M, Jackson SP TITLE CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing. JOURNAL Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356. PUBMED 27079678 COMMENT This file is created by Vector NTI http://www.biofeng.com/ COMMENT ORIGDB|GenBank COMMENT VNTAUTHORNAME|biofeng.com| COMMENT VNTNAME|AIO-mCherry| FEATURES Location/Qualifiers source 1..9728 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 277..352 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 442..459 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 477..717 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 752..827 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(836..854) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(837..854) /label=SP6 /note="SP6 promoter, forward primer" enhancer 891..1176 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 1178..1455 /label=chicken beta-actin promoter intron 1456..1684 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1696..1705 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1705..1770 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1777..1797 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1822..5922 /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 5923..5970 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5980..6036 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 6037..6741 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(6179..6197) /label=mCherry-R /note="mCherry, reverse primer" primer_bind complement(6769..6786) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6775..6982 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6991..7131 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6991..7120 /label=AAV2 ITR rep_origin 7206..7661 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7293..7312) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7503..7524 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7678..7697) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7797..7819 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7857..7875) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7943..8047 /gene="bla" /label=AmpR promoter CDS 8048..8908 /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(8266..8285) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9079..9667 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9568..9587 /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 gggtcttcgg atccatgcga agacctgttt tagagctaga aatagcaagt 301 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt 361 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat 421 tctgcagaca aatggctcta gattaaccct cactaaaggg aaaggtcggg caggaagagg 481 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa 541 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt 601 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc 661 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa 721 acaccgggag accatcgatg agagggtctc agttttagag ctagaaatag caagttaaaa 781 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttttctat 841 agtgtcacct aaattgcagc tctggcccgt gtctcaaaat ctctggtacc cgttacataa 901 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 1021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 1081 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg 1141 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc 1201 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa 1261 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg 1321 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc 1381 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga 1441 agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc 1501 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg 1561 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg 1621 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt 1681 tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat 1741 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt 1801 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggccat cggcaccaac 1861 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg 1921 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac 1981 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga 2041 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac 2101 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag 2161 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc 2221 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc 2281 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg 2341 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag 2401 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc 2461 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag 2521 aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc 2581 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac 2641 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag 2701 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag 2761 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg 2821 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag 2881 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag 2941 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac 3001 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc 3061 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag 3121 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct 3181 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc 3241 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg 3301 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg 3361 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg 3421 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag 3481 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc 3541 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac 3601 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac 3661 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa 3721 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg 3781 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag 3841 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg 3901 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc 3961 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag 4021 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac 4081 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag 4141 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag 4201 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac 4261 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc 4321 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac 4381 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag 4441 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag 4501 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc 4561 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc 4621 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa 4681 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa 4741 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga 4801 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag 4861 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc 4921 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac 4981 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg 5041 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc 5101 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg 5161 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc 5221 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc 5281 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc 5341 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg 5401 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg 5461 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac 5521 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag 5581 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc 5641 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac 5701 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc 5761 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag 5821 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc 5881 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg 5941 aaaaaggccg gccaggcaaa aaagaaaaag ggctccggag ccacgaactt ctctctgtta 6001 aagcaagcag gagacgtgga agaaaacccc ggtcctgtga gcaagggcga ggaggataac 6061 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc 6121 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc 6181 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag 6241 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag 6301 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg 6361 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg 6421 cgcggcacca acttcccctc cgacggcccc gtaatgcaga aaaagaccat gggctgggag 6481 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg 6541 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag 6601 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac 6661 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc 6721 ggcatggacg agctgtacaa ggaattctaa ctagagctcg ctgatcagcc tcgactgtgc 6781 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 6841 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 6901 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 6961 agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag ttggccactc 7021 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 7081 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga 7141 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc 7201 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 7261 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 7321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 7381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag 7441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 7501 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga 7561 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 7621 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac 7681 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 7741 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 7801 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 7861 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 7921 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 7981 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 8041 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 8101 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 8161 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 8221 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 8281 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 8341 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 8401 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 8461 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 8521 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 8581 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 8641 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 8701 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 8761 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 8821 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 8881 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 8941 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 9001 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 9061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 9121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 9181 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 9241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 9301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 9361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 9421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 9481 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 9541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 9601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 9661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 9721 tcacatgt //