LOCUS Exported 11505 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION A modified version of plasmid pX330 (#42230): Added are a ccdB stuffer and a Puromycin selection cassette. ACCESSION . VERSION . KEYWORDS pX330-Puro-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11505) AUTHORS Pritchard CEJ, Kroese LJ, Huijbers IJ TITLE Direct Generation of Conditional Alleles Using CRISPR/Cas9 in Mouse Zygotes. JOURNAL Methods Mol Biol. 2017;1642:21-35. doi: 10.1007/978-1-4939-7169-5_2. PUBMED 28815491 REFERENCE 2 (bases 1 to 11505) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11505 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 1..76 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 173..458 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 460..737 /label=chicken beta-actin promoter intron 738..965 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 977..986 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 986..1051 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1058..1078 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1103..5203 /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 5204..5251 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5279..5296) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5285..5492 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 5510..6009 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(5545..5568) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 5942..5961 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 6049..6648 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(6049..6068) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6545..6565 /label=Puro-F /note="Puromycin resistance gene, forward primer" rep_origin 7264..7719 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7351..7370) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7561..7582 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7736..7755) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7855..7877 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7915..7933) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8001..8105 /gene="bla" /label=AmpR promoter CDS 8106..8966 /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(8324..8343) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9137..9725 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9626..9645 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 9787..10027 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 9787..9807 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 9958..9977 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 10067..10097 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 10151..10810 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(10217..10236) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 11152..11457 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 11336..11355 /label=ccdB-fwd /note="ccdB gene, forward primer" ORIGIN 1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 61 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag 121 tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta cccgttacat 181 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 241 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 301 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 361 acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt 421 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct 481 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt 541 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg 601 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg 661 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc 721 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg 781 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc 841 gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat 901 ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt 961 ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact acaaggatca 1021 tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg 1081 tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa 1141 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt 1201 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga 1261 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag 1321 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga 1381 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga 1441 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac 1501 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat 1561 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct 1621 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca 1681 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc 1741 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa 1801 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag 1861 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga 1921 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa 1981 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa 2041 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct 2101 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca 2161 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa 2221 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa 2281 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat 2341 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa 2401 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc 2461 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac 2521 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg 2581 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct 2641 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat 2701 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa 2761 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg 2821 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata 2881 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga 2941 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga 3001 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg 3061 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca 3121 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat 3181 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc 3241 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa 3301 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca 3361 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca 3421 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca 3481 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta 3541 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc 3601 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa 3661 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga 3721 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca 3781 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc 3841 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat 3901 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa 3961 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa 4021 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg 4081 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa 4141 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc 4201 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa 4261 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg 4321 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat 4381 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag 4441 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt 4501 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc 4561 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt 4621 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct 4681 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct 4741 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa 4801 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca 4861 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag 4921 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta 4981 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac 5041 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa 5101 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg 5161 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac 5221 gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc ctagagctcg ctgatcagcc 5281 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 5341 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 5401 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 5461 gattgggaag agaatagcag gcatgctggg gagcggccgc aattctaccg ggtaggggag 5521 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc 5581 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct 5641 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc 5701 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt 5761 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc 5821 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg 5881 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg 5941 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg 6001 ggcctttcga cctgcatcca tctagatctc gagcagctga agcttaccat gaccgagtac 6061 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc 6121 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag 6181 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg 6241 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg 6301 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc 6361 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc 6421 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg 6481 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 6541 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 6601 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac 6661 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcgatgat atcagatccc 6721 cgggatgcag aaattgatga tctattaaac aataaagatg tccactaaaa tggaagtttt 6781 tcctgtcata ctttgttaag aagggtgaga acagagtacc tacattttga atggaaggat 6841 tggagctacg ggggtggggg tggggtggga ttagataaat gcctgctctt tactgaaggc 6901 tctttactat tgctttatga taatgtttca tagttggata tcataattta aacaagcaaa 6961 accaaattaa gggccagctc attcctccca ctcatgatct atagatctat agatctctcg 7021 tgggatcatt gtttttctct tgattcccac tttgtggttc taagtactgt ggtttccaaa 7081 tgtgtcagtt tcatagcctg aagaacgaga tcagcagcct ctgttccaca tacacttcat 7141 tctcagtatt gttttgccaa gttctaattc catcagaagc tcctgcaggg gcgcctgatg 7201 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat 7261 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 7321 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg 7381 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 7441 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg 7501 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 7561 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt 7621 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat 7681 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa 7741 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 7801 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 7861 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 7921 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 7981 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 8041 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 8101 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 8161 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 8221 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 8281 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 8341 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 8401 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 8461 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 8521 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 8581 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 8641 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 8701 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 8761 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 8821 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 8881 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 8941 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 9001 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 9061 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 9121 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 9181 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 9241 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 9301 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 9361 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 9421 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 9481 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 9541 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 9601 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 9661 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 9721 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 9781 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 9841 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 9901 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 9961 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 10021 aaaggacgaa acaccgggtc ttccggccgc attaggcacc ccaggcttta cactttatgc 10081 ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa 10141 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca 10201 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt 10261 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc 10321 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat 10381 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga 10441 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct 10501 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg 10561 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga 10621 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta 10681 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 10741 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg 10801 cggggcgtaa agatctggat ccggcttact aaaagccaga taacagtatg cgtatttgcg 10861 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag 10921 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc 10981 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc 11041 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc 11101 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt 11161 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat 11221 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca 11281 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg 11341 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc 11401 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg 11461 tcaggctccc ttatacacag ccagtctgca ggtcgaggaa gacct //