LOCUS Exported 10694 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION ""All-in-one"" CRISPR/Cas9 (wt) plasmid for cloning of custom gRNA with blue/white screening. ACCESSION . VERSION . KEYWORDS All_in_one_CRISPR/Cas9_LacZ SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10694) AUTHORS Findlay SD, Vincent KM, Berman JR, Postovit LM TITLE A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells. JOURNAL PLoS One. 2016 Apr 18;11(4):e0153901. doi: 10.1371/journal.pone.0153901. eCollection 2016. PUBMED 27089539 REFERENCE 2 (bases 1 to 10694) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10694 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 144..161 /label=L4440 /note="L4440 vector, forward primer" protein_bind 278..299 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 314..344 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 352..368 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 376..392 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 412..430 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 412..429 /label=SP6 /note="SP6 promoter, forward primer" promoter 442..460 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 591..611 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 762..781 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 825..846 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 861..891 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 899..915 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 923..939 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" CDS 935..1213 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEW" primer_bind 958..978 /label=T3 /note="T3 promoter, forward primer" promoter 960..978 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter complement(1026..1044) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1054..1071) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1054..1070) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1063..1085) /label=M13/pUC Forward /note="In lacZ gene" enhancer 1276..1655 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1656..1859 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1810..1830 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" promoter 1904..1922 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 1936..1945 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1942..6045 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6061..6081 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6082..6108 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(6125..6145) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 6130..6459 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6310..6445 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6372..6391 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 6517..6526 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6523..7233 /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(6668..6686) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 6936..6955 /label=mCherry-F /note="mCherry, forward primer" misc_feature 7249..7822 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(7416..7433) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" CDS 7858..8661 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(7921..7940) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 8531..8550 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 8793..8914 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8830..8849) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8884..8903 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(8963..8979) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 8987..9003 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." CDS 9065..9925 /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(9283..9302) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 10096..10684 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10585..10604 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 61 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 121 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 181 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 241 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt 301 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg 361 gataacaatt tcacacagga aacagctatg acatgattac gaattgcaac gatttaggtg 421 acactataga agagaaggaa ttaatacgac tcactatagg gagagagaga gaattaccct 481 cactaaaggg aggagaagca tgaattcccc agtggaaaga cgcgcaggca aaacgcacca 541 cgtgacggag cgtgaccgcg cgccgagcgc gcgccaaggt cgggcaggaa gagggcctat 601 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa 661 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat 721 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg 781 taactcgaga cgcggtggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 841 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 901 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca 961 attaaccctc actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa 1021 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga 1081 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 1141 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 1201 tggcgaatgg taggacgcgc cctgtagcgg cgcgtctcac gctgcttcgc gatgtacggg 1261 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt 1321 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 1381 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 1441 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 1501 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 1561 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 1621 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 1681 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 1741 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 1801 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 1861 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata 1921 gggagaccca agcttgccac catggacaag aagtacagca tcggcctgga catcggtacc 1981 aacagcgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaagttcaag 2041 gtgctgggca acaccgaccg ccacagcatc aagaagaacc tgatcggcgc cctgctgttc 2101 gacagcggcg agaccgccga ggccacccgc ctgaagcgca ccgcccgccg ccgctacacc 2161 cgccgcaaga accgcatctg ctacctgcag gagatcttca gcaacgagat ggccaaggtg 2221 gacgacagct tcttccaccg cctggaggag agcttcctgg tggaggagga caagaagcac 2281 gagcgccacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 2341 accatctacc acctgcgcaa gaagctggtg gacagcaccg acaaggccga cctgcgcctg 2401 atctacctgg ccctggccca catgatcaag ttccgcggcc acttcctgat cgagggcgac 2461 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 2521 cagctgttcg aggagaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgagc 2581 gcccgcctga gcaagagccg ccgcctggag aacctgatcg cccagctgcc cggcgagaag 2641 aagaacggcc tgttcggcaa cctgatcgcc ctgagcctgg gcctgacccc caacttcaag 2701 agcaacttcg acctggccga ggacgccaag ctgcagctga gcaaggacac ctacgacgac 2761 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt cctggccgcc 2821 aagaacctga gcgacgccat cctgctgagc gacatcctgc gcgtgaacac cgagatcacc 2881 aaggcccccc tgagcgccag catgatcaag cgctacgacg agcaccacca ggacctgacc 2941 ctgctgaagg ccctggtgcg ccagcagctg cccgagaagt acaaggagat cttcttcgac 3001 cagagcaaga acggctacgc cggctacatc gacggcggcg ccagccagga ggagttctac 3061 aagttcatca agcccatcct ggagaagatg gacggcaccg aggagctgct ggtgaagctg 3121 aaccgcgagg acctgctgcg caagcagcgc accttcgaca acggcagcat cccccaccag 3181 atccacctgg gcgagctgca cgccatcctg cgccgccagg aggacttcta ccccttcctg 3241 aaggacaacc gcgagaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc 3301 cccctggccc gcggcaacag ccgcttcgcc tggatgaccc gcaagagcga ggagaccatc 3361 accccctgga acttcgagga ggtggtggac aagggcgcca gcgcccagag cttcatcgag 3421 cgcatgacca acttcgacaa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 3481 ctgtacgagt acttcaccgt gtacaacgag ctgaccaagg tgaagtacgt gaccgagggc 3541 atgcgcaagc ccgccttcct gagcggcgag cagaagaagg ccatcgtgga cctgctgttc 3601 aagaccaacc gcaaggtgac cgtgaagcag ctgaaggagg actacttcaa gaagatcgag 3661 tgcttcgaca gcgtggagat cagcggcgtg gaggaccgct tcaacgccag cctgggcacc 3721 taccacgacc tgctgaagat catcaaggac aaggacttcc tggacaacga ggagaacgag 3781 gacatcctgg aggacatcgt gctgaccctg accctgttcg aggaccgcga gatgatcgag 3841 gagcgcctga agacctacgc ccacctgttc gacgacaagg tgatgaagca gctgaagcgc 3901 cgccgctaca ccggctgggg ccgcctgagc cgcaagctta tcaacggcat ccgcgacaag 3961 cagagcggca agaccatcct ggacttcctg aagagcgacg gcttcgccaa ccgcaacttc 4021 atgcagctga tccacgacga cagcctgacc ttcaaggagg acatccagaa ggcccaggtg 4081 agcggccagg gcgacagcct gcacgagcac atcgccaacc tggccggcag ccccgccatc 4141 aagaagggca tcctgcagac cgtgaaggtg gtggacgagc tggtgaaggt gatgggccgc 4201 cacaagcccg agaacatcgt gatcgagatg gcccgcgaga accagaccac ccagaagggc 4261 cagaagaaca gccgcgagcg catgaagcgc atcgaggagg gcatcaagga gctgggcagc 4321 cagatcctga aggagcaccc cgtggagaac acccagctgc agaacgagaa gctgtacctg 4381 tactacctgc agaacggccg cgacatgtac gtggaccagg agctggacat caaccgcctg 4441 agcgactacg acgtggacca catcgtgccc cagagcttcc tgaaggacga cagcatcgac 4501 aacaaggtgc tgacccgcag cgacaagaac cgcggcaaga gcgacaacgt gcccagcgag 4561 gaggtggtga agaagatgaa gaactactgg cgccagctgc tgaacgccaa gctgatcacc 4621 cagcgcaagt tcgacaacct gaccaaggcc gagcgcggcg gcctgagcga gctggacaag 4681 gccggcttca tcaagcgcca gctggtggag acccgccaga tcaccaagca cgtggcccag 4741 atcctggaca gccgcatgaa caccaagtac gacgagaacg acaagctgat ccgcgaggtg 4801 aaggtgatca ccctgaagag caagctggtg agcgacttcc gcaaggactt ccagttctac 4861 aaggtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtggtg 4921 ggcaccgccc tgatcaagaa gtaccccaag ctggagagcg agttcgtgta cggcgactac 4981 aaggtgtacg acgtgcgcaa gatgatcgcc aagagcgagc aggagatcgg caaggccacc 5041 gccaagtact tcttctacag caacatcatg aacttcttca agaccgagat caccctggcc 5101 aacggcgaga tccgcaagcg ccccctgatc gagaccaacg gcgagaccgg cgagatcgtg 5161 tgggacaagg gccgcgactt cgccaccgtg cgcaaggtgc tgagcatgcc ccaggtgaac 5221 atcgtgaaga agaccgaggt gcagaccggc ggcttcagca aggagagcat cctgcccaag 5281 cgcaacagcg acaagctgat cgcccgcaag aaggactggg accccaagaa gtacggcggc 5341 ttcgacagcc ccaccgtggc ctacagcgtg ctggtggtgg ccaaggtgga gaagggcaag 5401 agcaagaagc tgaagagcgt gaaggagctg ctgggcatca ccatcatgga gcgcagcagc 5461 ttcgagaaga accccatcga cttcctggag gccaagggct acaaggaggt gaagaaggac 5521 ctgatcatca agctgcccaa gtacagcctg ttcgagctgg agaacggccg caagcgcatg 5581 ctggccagcg ccggcgagct gcagaagggc aacgagctgg ccctgcccag caagtacgtg 5641 aacttcctgt acctggccag ccactacgag aagctgaagg gcagccccga ggacaacgag 5701 cagaagcagc tgttcgtgga gcagcacaag cactacctgg acgagatcat cgagcagatc 5761 agcgagttca gcaagcgcgt gatcctggcc gacgccaacc tggacaaggt gctgagcgcc 5821 tacaacaagc accgcgacaa gcccatccgc gagcaggccg agaacatcat ccacctgttc 5881 accctgacca acctgggcgc ccccgccgcc ttcaagtact tcgacaccac catcgaccgc 5941 aagcgctaca ccagcaccaa ggaggtgctg gacgccaccc tgatccacca gagcatcacc 6001 ggtctgtacg agacccgcat cgacctgagc cagctgggcg gcgacggcgg ctccggacct 6061 ccaaagaaaa agagaaaagt atacccctac gacgtgcccg actacgccta ataactcgag 6121 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 6181 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 6241 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 6301 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 6361 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 6421 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata 6481 tccattttcg gatctgatcg gcgcgccata ccggtcgcca ccatggtgag caagggcgag 6541 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc 6601 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc 6661 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg 6721 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac 6781 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 6841 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag 6901 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg 6961 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc 7021 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac 7081 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc 7141 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac 7201 tccaccggcg gcatggacga gctgtacaag taggcggccg cgatcccgcc cctctccctc 7261 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta 7321 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc 7381 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct 7441 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt 7501 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa 7561 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg 7621 gatagttgtg gaaagagtca aatggctcac ctcaagcgta ttcaacaagg ggctgaagga 7681 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac 7741 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt 7801 cctttgaaaa acacgatgat aagcttgcca caacccgcga taattcctgc agccaatatg 7861 ggatcggcca ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 7921 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 7981 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 8041 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 8101 gcagtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 8161 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 8221 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 8281 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 8341 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgtatg 8401 cccgacggcg atgatctcgt cgtgactcat ggcgatgcct gcttgccgaa tatcatggtg 8461 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 8521 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 8581 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 8641 cttcttgacg agttcttctg aacgcggtgc tacgagattt cgattccacc gccgccttct 8701 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 8761 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 8821 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 8881 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 8941 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 9001 caattccaca caacatacga gccggaagca taaaggtaag cctgaatatt gaaaaaggaa 9061 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 9121 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 9181 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 9241 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 9301 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 9361 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 9421 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 9481 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 9541 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 9601 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 9661 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 9721 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 9781 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 9841 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 9901 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 9961 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 10021 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 10081 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 10141 aaaaccaccg ctaccagcgg tggttttttt gccggatcaa gagctaccaa ctctttttcc 10201 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 10261 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 10321 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 10381 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 10441 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 10501 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 10561 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 10621 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 10681 gaaaaacgcc agca //