LOCUS Exported 8466 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 8466) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8466) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8466 /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" intron 692..919 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 931..940 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 940..1005 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1012..1032 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1057..5157 /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 5158..5205 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5233..5250) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5239..5446 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5455..5595 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5455..5584 /label=AAV2 ITR rep_origin 5670..6125 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5757..5776) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5967..5988 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6142..6161) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6261..6283 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6321..6339) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6407..6511 /gene="bla" /label=AmpR promoter CDS 6512..7372 /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(6730..6749) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7543..8131 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8032..8051 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8193..8433 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8193..8213 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8364..8383 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8463..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 cggcggggcg aggcggagag gtgcggcggc 601 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg 661 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt 721 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc 781 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta 841 agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc 901 tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga 961 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag 1021 aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg 1081 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc 1141 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga 1201 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga 1261 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag 1321 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag 1381 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac 1441 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc 1501 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg 1561 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg 1621 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag 1681 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg 1741 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc 1801 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac 1861 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg 1921 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac 1981 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac 2041 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag 2101 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag 2161 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg 2221 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc 2281 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt 2341 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc 2401 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc 2461 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag 2521 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc 2581 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac 2641 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg 2701 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc 2761 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc 2821 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat 2881 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga 2941 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag 3001 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc 3061 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc 3121 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag 3181 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc 3241 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa 3301 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc 3361 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa 3421 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag 3481 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac 3541 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac 3601 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac 3661 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc 3721 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc 3781 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag 3841 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg 3901 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat 3961 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg 4021 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg 4081 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc 4141 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag 4201 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc 4261 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg 4321 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct 4381 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag 4441 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg 4501 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg 4561 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa 4621 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc 4681 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc 4741 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc 4801 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc 4861 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa 4921 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc 4981 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc 5041 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac 5101 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa 5161 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagtaaga attcctagag 5221 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 5281 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 5341 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 5401 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac 5461 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 5521 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 5581 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 5641 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc 5701 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc 5761 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 5821 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 5881 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 5941 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 6001 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt 6061 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 6121 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 6181 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 6241 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 6301 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 6361 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 6421 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 6481 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 6541 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 6601 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 6661 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 6721 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 6781 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 6841 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6901 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 6961 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 7021 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 7081 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 7141 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 7201 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga 7261 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 7321 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 7381 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 7441 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 7501 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 7561 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 7621 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 7681 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 7741 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 7801 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 7861 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 7921 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 7981 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 8041 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 8101 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 8161 cctggccttt tgctggcctt ttgctcacat gtgagggcct atttcccatg attccttcat 8221 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca 8281 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag 8341 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg 8401 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgacggagat gagcctggac 8461 ccgttt //