LOCUS Exported 8478 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ZFP1. ACCESSION . VERSION . KEYWORDS pZFP1.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8478) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8478) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8478 /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 704..931 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 943..952 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 952..1017 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1024..1044 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1069..5169 /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 5170..5217 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5245..5262) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5251..5458 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5467..5607 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5467..5596 /label=AAV2 ITR rep_origin 5682..6137 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5769..5788) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5979..6000 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6154..6173) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6273..6295 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6333..6351) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6419..6523 /gene="bla" /label=AmpR promoter CDS 6524..7384 /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(6742..6761) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7555..8143 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8044..8063 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8205..8445 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8205..8225 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8376..8395 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8475..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 gggcgagggg cggggcgggg cgaggcggag 601 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 661 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 721 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 781 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct 841 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct 901 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag 961 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg 1021 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac 1081 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac 1141 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag 1201 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag 1261 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc 1321 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc 1381 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag 1441 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc 1501 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg 1561 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc 1621 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc 1681 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg 1741 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc 1801 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag 1861 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag 1921 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc 1981 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac 2041 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag 2101 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc 2161 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc 2221 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc 2281 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg 2341 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc 2401 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg 2461 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc 2521 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag 2581 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc 2641 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa 2701 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa 2761 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat 2821 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac 2881 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg 2941 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac 3001 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag 3061 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc 3121 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa 3181 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc 3241 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac 3301 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga 3361 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa 3421 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag 3481 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac 3541 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc 3601 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc 3661 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag 3721 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga 3781 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg 3841 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag 3901 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat 3961 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac 4021 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa 4081 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc 4141 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt 4201 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca 4261 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa 4321 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc 4381 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac 4441 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg 4501 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg 4561 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag 4621 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag 4681 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa 4741 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg 4801 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac 4861 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct 4921 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag 4981 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag 5041 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc 5101 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg 5161 ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa 5221 gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 5281 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 5341 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 5401 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc 5461 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 5521 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 5581 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc 5641 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 5701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt 5761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 5821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 5881 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 5941 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 6001 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc 6061 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 6121 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 6181 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 6241 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 6301 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 6361 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 6421 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 6481 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 6541 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 6601 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 6661 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 6721 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 6781 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 6841 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 6901 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 6961 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 7021 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 7081 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 7141 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 7201 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc 7261 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 7321 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 7381 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7441 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 7501 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 7561 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 7621 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 7681 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 7741 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 7801 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 7861 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 7921 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 7981 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 8041 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 8101 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 8161 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca 8221 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt 8281 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg 8341 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt 8401 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accggaaacc 8461 ctgaaactcc agccgttt //