LOCUS Exported 8474 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ZBTB5. ACCESSION . VERSION . KEYWORDS pZBTB5.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8474) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8474) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8474 /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 700..927 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 939..948 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 948..1013 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1020..1040 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1065..5165 /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 5166..5213 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5241..5258) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5247..5454 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5463..5603 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5463..5592 /label=AAV2 ITR rep_origin 5678..6133 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5765..5784) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5975..5996 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6150..6169) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6269..6291 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6329..6347) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6415..6519 /gene="bla" /label=AmpR promoter CDS 6520..7380 /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(6738..6757) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7551..8139 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8040..8059 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8201..8441 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8201..8221 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8372..8391 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8471..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 ggggggcggg gcggggcgag gcggagaggt 601 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 661 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt 721 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg 781 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc 841 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag 901 cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac tataaggacc 961 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc 1021 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca 1081 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg 1141 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc 1201 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa 1261 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca 1321 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg 1381 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg 1441 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg 1501 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc 1561 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc 1621 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg 1681 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg 1741 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg 1801 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga 1861 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg 1921 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga 1981 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg 2041 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt 2101 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag 2161 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg 2221 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca 2281 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg 2341 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc 2401 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca 2461 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt 2521 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg 2581 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag 2641 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg 2701 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg 2761 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt 2821 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc 2881 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg 2941 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag 3001 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga 3061 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg 3121 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg 3181 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc 3241 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc 3301 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga 3361 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg 3421 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc 3481 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg 3541 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc 3601 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga 3661 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc 3721 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg 3781 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga 3841 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg 3901 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc 3961 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg 4021 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg 4081 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc 4141 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca 4201 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg 4261 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc 4321 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca 4381 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg 4441 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg 4501 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca 4561 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct 4621 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg 4681 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg 4741 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg 4801 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg 4861 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc 4921 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg 4981 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact 5041 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc 5101 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag 5161 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat 5221 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 5281 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 5341 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 5401 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc 5461 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga 5521 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 5581 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg 5641 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta 5701 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg 5761 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 5821 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 5881 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt 5941 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 6001 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc 6061 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 6121 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 6181 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 6241 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 6301 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat 6361 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga 6421 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 6481 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 6541 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 6601 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 6661 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 6721 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 6781 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 6841 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 6901 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 6961 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 7021 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 7081 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 7141 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 7201 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg 7261 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 7321 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 7381 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 7441 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 7501 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 7561 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 7621 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 7681 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 7741 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 7801 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 7861 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 7921 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 7981 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 8041 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 8101 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 8161 ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat ttcccatgat 8221 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 8281 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 8341 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 8401 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg aaacagctag 8461 agcaaagtgc gttt //