LOCUS Exported 8484 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ZNF445. ACCESSION . VERSION . KEYWORDS pZNF445.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8484) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8484) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8484 /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 710..937 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 949..958 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 958..1023 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1030..1050 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1075..5175 /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 5176..5223 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5251..5268) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5257..5464 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5473..5613 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5473..5602 /label=AAV2 ITR rep_origin 5688..6143 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5775..5794) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5985..6006 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6160..6179) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6279..6301 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6339..6357) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6425..6529 /gene="bla" /label=AmpR promoter CDS 6530..7390 /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(6748..6767) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7561..8149 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8050..8069 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8211..8451 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8211..8231 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8382..8401 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8481..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 ggggggcggc gaggggcggg gcggggcgag 601 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc 661 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc 721 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg 781 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa 841 ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa 901 ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac 961 tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat 1021 aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag 1081 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 1141 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 1201 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 1261 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 1321 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 1381 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 1441 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 1501 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 1561 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 1621 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 1681 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 1741 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 1801 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 1861 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 1921 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 1981 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 2041 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 2101 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 2161 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 2221 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 2281 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 2341 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 2401 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 2461 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 2521 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 2581 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 2641 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 2701 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 2761 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 2821 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 2881 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 2941 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 3001 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 3061 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 3121 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 3181 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 3241 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 3301 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 3361 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 3421 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 3481 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 3541 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 3601 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 3661 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 3721 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 3781 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 3841 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 3901 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 3961 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 4021 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 4081 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 4141 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 4201 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 4261 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 4321 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 4381 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 4441 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 4501 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 4561 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 4621 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 4681 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 4741 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 4801 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 4861 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 4921 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 4981 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 5041 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 5101 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 5161 cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 5221 aagtaagaat tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat 5281 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 5341 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 5401 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg 5461 gggagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 5521 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 5581 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta 5641 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag 5701 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 5761 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 5821 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 5881 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 5941 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 6001 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc 6061 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 6121 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 6181 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 6241 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 6301 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 6361 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 6421 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 6481 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 6541 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 6601 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 6661 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 6721 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 6781 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 6841 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 6901 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 6961 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 7021 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 7081 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 7141 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 7201 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat 7261 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 7321 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 7381 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 7441 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 7501 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 7561 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 7621 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 7681 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 7741 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 7801 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 7861 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 7921 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 7981 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 8041 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 8101 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 8161 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat 8221 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa 8281 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat 8341 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg 8401 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg 8461 accatattag agattagcct gttt //