LOCUS Exported 10249 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression vector for sgRNA and for Expression of Cas9 linked via T2A to mCherry linked to the Ad4 E4orf6 gene via P2A. ACCESSION . VERSION . KEYWORDS pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10249) 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 10249) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10249 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 7..247 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7..27 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 178..197 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 274..349 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 446..731 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 733..1010 /label=chicken beta-actin promoter intron 1011..1239 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1251..1260 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1260..1325 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1332..1352 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1377..5477 /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 5478..5525 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5529..5582 /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 5589..6296 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(5734..5752) /label=mCherry-R /note="mCherry, reverse primer" CDS 6303..6359 /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 6366..7265 /codon_start=1 /gene="E4" /product="adenovirus 4 protein that mediates the ubiquitination and proteasomal degradation of DNA ligase IV (Baker et al., 2007)" /label=E4orf6 /translation="MSGNSSIMTRSRTRLALSRHHPYQPPATLPRCEETESRASLVEDH PVLPDCDTLSMHNVSSVRGLPCSAGFTVLQEFPVPWDMVLTPEELRVLKTCMSVCLCCA NIDLFSSQLIHGRERWVIHCHCQNPGSLRCMAGGTVLAIWFRRIIQGCMFNQRVMWYRE VVNLHMPKEIMYMGSVFWRGRHLIYIRIWYDGHVGSIVPQMSFGWSTLNYGLLNNLVVL CCTYCSDLSEIRIRCCARRTRRLMLRAIGIMRRESLDPDPLSSSLTERRRQRLLRGLMR HNRPIPFADYDSHRSYSR" primer_bind complement(7290..7307) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7296..7503 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 7512..7652 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7512..7641 /label=AAV2 ITR rep_origin 7727..8182 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7814..7833) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8024..8045 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8199..8218) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8318..8340 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8378..8396) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8464..8568 /gene="bla" /label=AmpR promoter CDS 8569..9429 /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(8787..8806) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9600..10188 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10089..10108 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ccccacgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 241 aaaggacgaa acaccgggtc ttcgagaaga cctgttttag agctagaaat agcaagttaa 301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt 361 agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct 421 gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct 481 gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt 541 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 601 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg 661 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 721 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 781 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 841 gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga 901 ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg 961 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg 1021 cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 1081 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1141 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt 1201 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg 1261 actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg 1321 ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca 1381 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg 1441 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca 1501 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc 1561 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc 1621 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag 1681 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg 1741 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg 1801 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca 1861 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca 1921 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg 1981 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg 2041 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg 2101 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca 2161 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg 2221 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga 2281 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca 2341 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc 2401 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca 2461 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga 2521 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc 2581 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc 2641 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga 2701 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg 2761 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg 2821 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc 2881 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg 2941 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg 3001 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc 3061 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg 3121 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg 3181 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc 3241 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt 3301 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga 3361 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc 3421 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga 3481 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc 3541 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg 3601 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa 3661 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc 3721 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa 3781 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt 3841 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc 3901 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga 3961 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact 4021 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg 4081 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg 4141 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt 4201 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg 4261 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc 4321 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta 4381 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg 4441 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca 4501 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga 4561 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg 4621 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag 4681 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa 4741 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg 4801 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc 4861 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg 4921 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc 4981 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg 5041 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg 5101 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca 5161 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg 5221 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca 5281 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg 5341 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc 5401 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt 5461 ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga 5521 aaaagcttga gggcagagga agtctgctaa catgcggtga cgtggaggag aatcccggcc 5581 ctgctagcat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc 5641 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg 5701 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc 5761 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg 5821 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt 5881 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc 5941 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg 6001 gccccgtaat gcagaagaaa accatgggct gggaggcctc ctccgagcgg atgtaccccg 6061 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact 6121 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct 6181 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac 6241 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagctcg 6301 aggcaacaaa cttctcacta ctcaaacaag caggtgacgt ggaggagaat cccgggcctc 6361 ttaagatgtc aggaaatagt agcatcatga ctcgctcacg cacaagactg gcactgtcaa 6421 ggcaccaccc atatcagccc cccgcaactc tgcctagatg cgaggaaaca gagtctcggg 6481 ctagtctggt ggaagaccac cctgtcctgc cagactgcga tactctgagt atgcataacg 6541 tgagctccgt ccgcggactg ccctgttcag cagggttcac tgtgctgcag gagtttcccg 6601 tgccttggga catggtcctg acccctgagg aactgagggt gctgaagaca tgcatgtctg 6661 tctgtctgtg ctgtgccaac attgatctgt tctctagtca gctgatccac ggaagagaga 6721 ggtgggtcat ccactgccat tgtcagaatc ccgggagtct gagatgcatg gccggcggaa 6781 cagtgctggc tatctggttc aggagaatca ttcagggctg tatgtttaac cagcgagtga 6841 tgtggtacag ggaggtggtc aatctgcata tgccaaaaga aatcatgtat atgggcagcg 6901 tgttctggcg cggacgacac ctgatctaca ttcggatctg gtatgacggg catgtgggct 6961 ccattgtccc ccagatgtcc tttgggtggt ctactctgaa ttacggcctg ctgaacaatc 7021 tggtggtcct gtgctgtacc tattgttcag atctgagcga gattaggatc agatgctgtg 7081 cacgacgaac ccgaaggctg atgctgcgag caattggcat catgagacgg gagagcctgg 7141 acccagatcc cctgtcaagc tccctgacag aacgccgaag gcagcggctg ctgcgcggac 7201 tgatgaggca taacaggcca atccccttcg cagactacga ctcccatcgg tcctactccc 7261 gctaggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 7321 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 7381 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 7441 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 7501 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 7561 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 7621 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 7681 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 7741 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 7801 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 7861 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 7921 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 7981 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 8041 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 8101 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 8161 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 8221 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 8281 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 8341 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 8401 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 8461 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 8521 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 8581 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 8641 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 8701 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 8761 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 8821 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 8881 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 8941 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 9001 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 9061 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 9121 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 9181 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 9241 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 9301 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 9361 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 9421 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 9481 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 9541 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 9601 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 9661 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 9721 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 9781 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 9841 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 9901 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 9961 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 10021 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 10081 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 10141 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 10201 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt //