LOCUS Exported 8698 bp ds-DNA circular SYN 03-7-2014 DEFINITION . ACCESSION . VERSION . KEYWORDS pX330S-5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8698) AUTHORS . TITLE Direct Submission JOURNAL Exported 2014”N73“ú from SnapGene Viewer 2.4.3 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8698 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 295..572 /note="chicken beta-actin promoter" CDS 822..887 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 894..914 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS 939..5039 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="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 5040..5087 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" polyA_signal 5121..5328 /note="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" repeat_region 5337..5477 /note="AAV2 ITR" /note="inverted terminal repeat of adeno-associated virus serotype 2" rep_origin 5552..6007 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" CDS 6628..7419 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /note="SmR" /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 7602..8190 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" promoter 8265..8505 /label="U6 Promoter" /note="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 8532..8607 /note="gRNA scaffold" /note="guide RNA scaffold for the CRISPR/Cas9 system" ORIGIN 1 tgagacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat 241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc 601 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 661 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 721 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 781 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg 841 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga 901 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc 961 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca 1021 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg 1081 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca 1141 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg 1201 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag 1261 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc 1321 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg 1381 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc 1441 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg 1501 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca 1561 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc 1621 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga 1681 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg 1741 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc 1801 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga 1861 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc 1921 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag 1981 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc 2041 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac 2101 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca 2161 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt 2221 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc 2281 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga 2341 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc 2401 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc 2461 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat 2521 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg 2581 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact 2641 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg 2701 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca 2761 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca 2821 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga 2881 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg 2941 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg 3001 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc 3061 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg 3121 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga 3181 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga 3241 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca 3301 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg 3361 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg 3421 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg 3481 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca 3541 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg 3601 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga 3661 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa 3721 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc 3781 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg 3841 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc 3901 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg 3961 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa 4021 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg 4081 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa 4141 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca 4201 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt 4261 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta 4321 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag 4381 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca 4441 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag 4501 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg 4561 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc 4621 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc 4681 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga 4741 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca 4801 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata 4861 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca 4921 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc 4981 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca 5041 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag 5101 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 5161 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 5221 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 5281 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga 5341 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 5401 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 5461 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 5521 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 5581 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5641 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5701 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5761 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5821 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 5881 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 5941 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 6001 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 6061 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 6121 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 6181 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 6241 ataataatgg tttcttagac gtcaggtggc acttccagcc aggacagaaa tgcctcgact 6301 tcgctgctac ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc 6361 cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc 6421 aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg 6481 gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg 6541 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc 6601 agtcgcccta aaacaaagtt aaacattatg agggaagcgg tgatcgccga agtatcgact 6661 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta 6721 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg 6781 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg 6841 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt 6901 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa 6961 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg 7021 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag 7081 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg 7141 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc 7201 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg 7261 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat 7321 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac 7381 tacgtgaaag gcgagatcac caaggtagtc ggcaaataaa agcattggta actgtcagac 7441 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 7501 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 7561 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 7621 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 7681 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 7741 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 7801 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 7861 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 7921 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 7981 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 8041 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 8101 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 8161 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 8221 ctggcctttt gctggccttt tgctcaggtc tcatagaggc atgtgagggc ctatttccca 8281 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt 8341 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg 8401 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt 8461 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt 8521 cgagaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa 8581 cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa 8641 aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagag //