LOCUS Exported 10765 bp ds-DNA linear SYN 01-JUN-2021 DEFINITION Expression vector for a sgRNA against the mouse Rosa26 locus and Cas9 linked via T2A to BFP linked to the Ad4 E1B55K gene via P2A. ACCESSION . VERSION . KEYWORDS pU6-sgRosa26-1_CBh-Cas9-T2A-BFP-P2A-Ad4E1B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10765) AUTHORS Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R TITLE Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. JOURNAL Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198. PUBMED 25803306 REFERENCE 2 (bases 1 to 10765) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 1, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10765 /organism="synthetic DNA construct" /mol_type="other DNA" intron 103..330 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 342..351 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 351..416 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 423..443 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 468..4568 /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 4569..4616 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4620..4673 /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 4680..5396 /codon_start=1 /product="enhanced blue variant of GFP (Ai et al., 2007)" /label=EBFP2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL EFRTAAGITLGMDELYK" primer_bind complement(4725..4746) /label=EGFP-N /note="EGFP, reverse primer" CDS 5403..5459 /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 5466..6914 /codon_start=1 /product="adenovirus 4 E1B 55K protein, which mediates the ubiquitination and proteasomal degradation of DNA ligase IV (Baker et al., 2007)" /label=E1B /translation="MESRNPFQQGLPAGFLSSSFVENMEVPAPECNLRLLAGTAARHSE DPESPAAGGSRRESESRPGPSGGGVADLFPELHRVLTRSSSGRERGIKRERHDETNHRI ELTVGLMSRKRPETVWWYEVQSTGTDEVSVMHERFSLEQVKTCWLEPEDDWEVAIRNYA KLALRPDRKYKITKLINIRNACYISGNGAEVEICLQDRVAFRCCMMNMYPGVVDMDGVT FMNMRFRGDGYNGTVFMANTKLTVHGCSFFGFNNTCIEAWGQVGVKGCSFSANWMGVVG RTKSMLSVKKCLFERCHLGVMSEGEARIRHCASTETGCFVLCKGNAKIKHNMICGASDE RGYQMLTCAGGNSHMLAAVHVASHSRKPWPEFEHNVMTRCNMHLGARRGMFMPYQCNLN YVKVLLEPDVMSRVSLTGVFDMNVEVWKILRYDEYKTRCRACECGGKHARFQPVCVDVT EDLRPDHLVLSCTGTEFGSSGEESD" primer_bind complement(6942..6959) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6948..7155 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 7164..7304 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7164..7293 /label=AAV2 ITR rep_origin 7379..7834 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7466..7485) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7676..7697 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7851..7870) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7970..7992 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8030..8048) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8116..8220 /gene="bla" /label=AmpR promoter CDS 8221..9081 /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(8439..8458) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9252..9840 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9741..9760 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 9907..10147 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 9907..9927 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 10078..10097 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 10177..10252 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 10349..10634 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" ORIGIN 1 ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg 61 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc 121 cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc 181 gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg 241 agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg 301 gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg 361 accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg 421 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca 481 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca 541 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga 601 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga 661 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct 721 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc 781 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg 841 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca 901 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg 961 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca 1021 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg 1081 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga 1141 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc 1201 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc 1261 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt 1321 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc 1381 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg 1441 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga 1501 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg 1561 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca 1621 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg 1681 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc 1741 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct 1801 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga 1861 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg 1921 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga 1981 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca 2041 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa 2101 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag 2161 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc 2221 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact 2281 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt 2341 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca 2401 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc 2461 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg 2521 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag 2581 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca 2641 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg 2701 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag 2761 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag 2821 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc 2881 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc 2941 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct 3001 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca 3061 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc 3121 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag 3181 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc 3241 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga 3301 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt 3361 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg 3421 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa 3481 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg 3541 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt 3601 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa 3661 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag 3721 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca 3781 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact 3841 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg 3901 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga 3961 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg 4021 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc 4081 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac 4141 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga 4201 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc 4261 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta 4321 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg 4381 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt 4441 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca 4501 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg 4561 gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagcttg 4621 agggcagagg aagtctgcta acatgcggtg acgtggagga gaatcccggc cctgctagca 4681 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 4741 gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg cgagggcgat gccaccaacg 4801 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 4861 tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc 4921 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 4981 tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 5041 tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga cggcaacatc ctggggcaca 5101 agctggagta caacttcaac agccacaaca tctatatcat ggccgtcaag cagaagaacg 5161 gcatcaaggt gaacttcaag atccgccaca acgtggagga cggcagcgtg cagctcgccg 5221 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacagccact 5281 acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat cacatggtcc 5341 tgctggagtt ccgcaccgcc gccgggatca ctctcggcat ggacgagctg tacaagctcg 5401 aggcaacaaa cttctcacta ctcaaacaag caggtgacgt ggaggagaat cccgggcctc 5461 ttaagatgga gagccgaaat ccttttcagc agggactgcc agccgggttc ctgtcttcat 5521 cctttgtgga gaatatggag gtgccagccc ccgagtgcaa cctgcgactg ctggcaggaa 5581 ccgcagctcg ccacagtgag gatcctgaat caccagcagc cggcggaagt aggagagaga 5641 gtgaatcacg accaggacct tcaggaggag gagtcgctga cctgttccca gaactgcatc 5701 gcgtgctgac acgaagctcc tctggccgag agaggggaat caagagagag cggcacgatg 5761 aaaccaatca taggattgag ctgacagtgg ggctgatgtc tcgcaaacga cctgagactg 5821 tctggtggta cgaagtgcag agcaccggca cagacgaagt gtccgtcatg cacgagaggt 5881 tttctctgga acaggtgaag acctgttggc tggagcctga agacgattgg gaggtggcca 5941 tcagaaacta cgccaagctg gctctgagac cagaccggaa gtacaagatc actaagctga 6001 tcaacatccg gaatgcttgc tatatcagcg gcaatggagc agaggtcgaa atttgtctgc 6061 aggatagggt ggccttcaga tgctgtatga tgaacatgta ccccggggtg gtcgacatgg 6121 atggcgtgac tttcatgaat atgaggttta gaggggatgg ctataacggc accgtcttca 6181 tggctaatac aaagctgact gtgcatggat gctccttctt tgggtttaac aatacctgta 6241 tcgaggcttg gggacaagtg ggggtcaagg gctgcagctt ctccgcaaac tggatgggag 6301 tggtcgggag gacaaaatca atgctgagcg tgaagaaatg cctgtttgaa agatgtcacc 6361 tgggcgtgat gagcgaggga gaagcacgga ttcgccattg tgcctccact gagaccggct 6421 gcttcgtgct gtgcaaagga aacgcaaaga tcaaacacaa tatgatttgc ggagcctctg 6481 acgagagagg gtaccagatg ctgacatgtg ccgggggcaa cagtcatatg ctggctgcag 6541 tgcacgtcgc ttctcatagt cgaaagccat ggcccgagtt cgaacacaac gtgatgacta 6601 gatgcaatat gcatctgggg gcccggcgcg gcatgtttat gccataccag tgtaacctga 6661 attatgtgaa agtcctgctg gaacccgacg tgatgtcccg ggtgtctctg acaggcgtgt 6721 tcgatatgaa tgtggaggtc tggaagatcc tgcgctacga cgaatataaa actcgatgca 6781 gggcctgcga gtgtggaggg aagcacgctc ggtttcagcc cgtgtgcgtg gacgtgacag 6841 aggatctgcg ccctgatcat ctggtcctgt cctgcactgg cactgagttc gggtcaagcg 6901 gggaagaaag cgactaggaa ttcctagagc tcgctgatca gcctcgactg tgccttctag 6961 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 7021 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 7081 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag 7141 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct 7201 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 7261 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta 7321 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac 7381 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 7441 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 7501 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 7561 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca 7621 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 7681 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa 7741 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac 7801 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc 7861 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 7921 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 7981 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 8041 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 8101 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 8161 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 8221 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 8281 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 8341 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 8401 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 8461 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 8521 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 8581 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 8641 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 8701 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 8761 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 8821 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 8881 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc 8941 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 9001 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 9061 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 9121 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 9181 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 9241 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 9301 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 9361 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 9421 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 9481 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 9541 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 9601 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 9661 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 9721 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 9781 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 9841 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 9901 tcatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 9961 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 10021 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 10081 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 10141 aaaggacgaa acaccgactc cagtctttct agaagagttt tagagctaga aatagcaagt 10201 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt 10261 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat 10321 tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg 10381 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc 10441 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 10501 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 10561 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 10621 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc 10681 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg 10741 cggggggggg gggggggcgc gcgcc //