LOCUS Exported 8508 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor TCF15. ACCESSION . VERSION . KEYWORDS pTCF15.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8508) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8508) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8508 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 169..454 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 456..733 /label=chicken beta-actin promoter intron 734..962 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 974..983 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 983..1048 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1055..1075 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1100..5200 /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 5201..5248 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5276..5293) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5282..5489 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5498..5638 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5498..5627 /label=AAV2 ITR rep_origin 5713..6168 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5800..5819) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6010..6031 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6185..6204) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6304..6326 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6364..6382) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6450..6554 /gene="bla" /label=AmpR promoter CDS 6555..7415 /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(6773..6792) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7586..8174 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8075..8094 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8236..8476 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8236..8256 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8407..8426 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8505..72 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 61 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg 121 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact 181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt 241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 361 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca 421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt 481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 541 attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg 601 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc 661 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag 721 cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg 781 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg 841 acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt 901 tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc 961 aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga 1021 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat 1081 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc 1141 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct 1201 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag 1261 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg 1321 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga 1381 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg 1441 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 1501 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta 1561 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa 1621 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 1681 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag 1741 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa 1801 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa 1861 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct 1921 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa 1981 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 2041 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct 2101 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag 2161 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt 2221 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag 2281 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca 2341 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga 2401 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct 2461 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc 2521 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat 2581 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 2641 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag 2701 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac 2761 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt 2821 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca 2881 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat 2941 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg 3001 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag 3061 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc 3121 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca 3181 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg 3241 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa 3301 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa 3361 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa 3421 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat 3481 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta 3541 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga 3601 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa 3661 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt 3721 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag 3781 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg 3841 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct 3901 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt 3961 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt 4021 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac 4081 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt 4141 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa 4201 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg 4261 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga 4321 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt 4381 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa 4441 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga 4501 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa 4561 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga 4621 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat 4681 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc 4741 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt 4801 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa 4861 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga 4921 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa 4981 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct 5041 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag 5101 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct 5161 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa 5221 aaaggccggc caggcaaaaa agaaaaagta agaattccta gagctcgctg atcagcctcg 5281 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 5341 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 5401 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 5461 tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg 5521 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga 5581 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg 5641 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa 5701 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 5761 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 5821 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 5881 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt 5941 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 6001 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt 6061 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 6121 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc 6181 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 6241 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 6301 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 6361 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 6421 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 6481 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 6541 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 6601 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 6661 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 6721 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 6781 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 6841 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 6901 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 6961 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 7021 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 7081 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 7141 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 7201 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 7261 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 7321 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 7381 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 7441 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 7501 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 7561 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 7621 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 7681 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 7741 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 7801 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 7861 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 7921 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 7981 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 8041 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 8101 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 8161 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 8221 cttttgctca catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac 8281 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa 8341 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 8401 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 8461 atcttgtgga aaggacgaaa caccacggag atgagcctgg acccgttt //