LOCUS Exported 8463 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ZNF324. ACCESSION . VERSION . KEYWORDS pZNF324.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8463) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8463) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8463 /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 689..916 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 928..937 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 937..1002 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1009..1029 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1054..5154 /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 5155..5202 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5230..5247) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5236..5443 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5452..5592 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5452..5581 /label=AAV2 ITR rep_origin 5667..6122 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5754..5773) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5964..5985 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6139..6158) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6258..6280 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6318..6336) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6404..6508 /gene="bla" /label=AmpR promoter CDS 6509..7369 /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(6727..6746) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7540..8128 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8029..8048 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8190..8430 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8190..8210 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8361..8380 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8460..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 cggggcgagg cggagaggtg cggcggcagc 601 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc 661 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc 721 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca 781 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg 841 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga 901 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac 961 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag 1021 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac 1081 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1141 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1201 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1261 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1321 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1381 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1441 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1501 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1561 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1621 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1681 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1741 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1801 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1861 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1921 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1981 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2041 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2101 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2161 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2221 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2281 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2341 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2401 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2461 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2521 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2581 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2641 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2701 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2761 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2821 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2881 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2941 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3001 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3061 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3121 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3181 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3241 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3301 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3361 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3421 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3481 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3541 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3601 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3661 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3721 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3781 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3841 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3901 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3961 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4021 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4081 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4141 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4201 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4261 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4321 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4381 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4441 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4501 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4561 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4621 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4681 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4741 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4801 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4861 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4921 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4981 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 5041 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 5101 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg 5161 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt cctagagctc 5221 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 5281 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 5341 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 5401 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc 5461 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 5521 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 5581 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5641 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5701 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt 5761 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5821 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5881 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5941 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc 6001 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa 6061 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 6121 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 6181 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 6241 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 6301 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 6361 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 6421 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 6481 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 6541 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 6601 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 6661 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 6721 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 6781 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 6841 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 6901 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 6961 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 7021 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 7081 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 7141 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 7201 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg 7261 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 7321 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 7381 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 7441 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 7501 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 7561 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 7621 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 7681 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 7741 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 7801 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 7861 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 7921 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 7981 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 8041 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 8101 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 8161 ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt 8221 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa 8281 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt 8341 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt 8401 tcttggcttt atatatcttg tggaaaggac gaaacaccgc tgtgacctca gacctccgcg 8461 ttt //