LOCUS Exported 8508 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ZNF696. ACCESSION . VERSION . KEYWORDS pZNF696.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8508) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8508) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8508 /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 734..961 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 973..982 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 982..1047 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1054..1074 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1099..5199 /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 5200..5247 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5275..5292) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5281..5488 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5497..5637 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5497..5626 /label=AAV2 ITR rep_origin 5712..6167 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5799..5818) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6009..6030 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6184..6203) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6303..6325 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6363..6381) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6449..6553 /gene="bla" /label=AmpR promoter CDS 6554..7414 /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(6772..6791) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7585..8173 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8074..8093 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8235..8475 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8235..8255 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8406..8425 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8505..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 gggggggcgc gcgccggggg gggggggggg 601 gggggggggg gggggggggg sggggcggrg rggkgcggcg gcagccaatc agagcggcgc 661 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag 721 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc 781 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga 841 cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt 901 ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca 961 ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat 1021 attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc 1081 cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct 1141 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg 1201 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc 1261 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg 1321 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac 1381 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg 1441 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 1501 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat 1561 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac 1621 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 1681 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga 1741 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat 1801 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac 1861 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg 1921 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac 1981 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc 2041 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg 2101 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc 2161 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc 2221 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga 2281 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac 2341 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac 2401 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg 2461 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc 2521 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg 2581 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 2641 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga 2701 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc 2761 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc 2821 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac 2881 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt 2941 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg 3001 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga 3061 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc 3121 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag 3181 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc 3241 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag 3301 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag 3361 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag 3421 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc 3481 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac 3541 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac 3601 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag 3661 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc 3721 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga 3781 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc 3841 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg 3901 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg 3961 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg 4021 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc 4081 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg 4141 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag 4201 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc 4261 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat 4321 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg 4381 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac 4441 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac 4501 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag 4561 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag 4621 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc 4681 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc 4741 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc 4801 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa 4861 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag 4921 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac 4981 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg 5041 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg 5101 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg 5161 tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa 5221 aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga tcagcctcga 5281 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 5341 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 5401 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 5461 gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg 5521 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 5581 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc 5641 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa 5701 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 5761 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc 5821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 5881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc 5941 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 6001 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc 6061 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta 6121 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct 6181 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 6241 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 6301 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 6361 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 6421 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 6481 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 6541 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 6601 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 6661 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 6721 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 6781 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 6841 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 6901 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 6961 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 7021 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 7081 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 7141 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 7201 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 7261 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 7321 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 7381 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 7441 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 7501 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 7561 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 7621 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 7681 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 7741 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 7801 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 7861 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 7921 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 7981 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 8041 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 8101 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 8161 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 8221 ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca 8281 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa 8341 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta 8401 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata 8461 tcttgtggaa aggacgaaac accgcgagtg agccggggct gcgggttt //