LOCUS Exported 8389 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor TCFL5. ACCESSION . VERSION . KEYWORDS pTCFL5.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8389) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8389) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8389 /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 615..842 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 854..863 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 863..928 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 935..955 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 980..5080 /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 5081..5128 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5156..5173) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5162..5369 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5378..5518 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5378..5507 /label=AAV2 ITR rep_origin 5593..6048 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5680..5699) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5890..5911 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6065..6084) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6184..6206 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6244..6262) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6330..6434 /gene="bla" /label=AmpR promoter CDS 6435..7295 /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(6653..6672) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7466..8054 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7955..7974 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8116..8356 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8116..8136 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8287..8306 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8386..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 ggcggcggcg gcggccctat aaaaagcgaa 601 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg 661 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg 721 acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt 781 tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc 841 aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga 901 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat 961 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc 1021 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct 1081 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag 1141 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg 1201 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga 1261 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg 1321 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 1381 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta 1441 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa 1501 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 1561 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag 1621 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa 1681 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa 1741 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct 1801 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa 1861 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 1921 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct 1981 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag 2041 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt 2101 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag 2161 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca 2221 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga 2281 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct 2341 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc 2401 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat 2461 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 2521 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag 2581 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac 2641 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt 2701 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca 2761 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat 2821 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg 2881 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag 2941 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc 3001 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca 3061 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg 3121 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa 3181 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa 3241 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa 3301 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat 3361 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta 3421 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga 3481 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa 3541 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt 3601 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag 3661 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg 3721 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct 3781 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt 3841 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt 3901 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac 3961 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt 4021 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa 4081 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg 4141 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga 4201 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt 4261 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa 4321 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga 4381 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa 4441 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga 4501 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat 4561 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc 4621 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt 4681 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa 4741 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga 4801 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa 4861 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct 4921 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag 4981 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct 5041 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa 5101 aaaggccggc caggcaaaaa agaaaaagta agaattccta gagctcgctg atcagcctcg 5161 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 5221 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 5281 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 5341 tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg 5401 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga 5461 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg 5521 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa 5581 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 5641 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt 5701 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 5761 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt 5821 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 5881 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt 5941 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt 6001 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc 6061 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 6121 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 6181 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 6241 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 6301 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 6361 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 6421 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 6481 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 6541 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 6601 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 6661 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 6721 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 6781 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 6841 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 6901 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 6961 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 7021 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 7081 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 7141 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 7201 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 7261 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 7321 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 7381 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 7441 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 7501 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 7561 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 7621 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 7681 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 7741 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 7801 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 7861 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 7921 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 7981 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 8041 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 8101 cttttgctca catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac 8161 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa 8221 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 8281 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 8341 atcttgtgga aaggacgaaa caccgctcga tggagatcaa gtgatgttt //