LOCUS Exported 8480 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor ZKSCAN4. ACCESSION . VERSION . KEYWORDS pZKSCAN4.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8480) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8480) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8480 /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 706..933 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 945..954 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 954..1019 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1026..1046 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1071..5171 /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 5172..5219 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5247..5264) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5253..5460 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5469..5609 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5469..5598 /label=AAV2 ITR rep_origin 5684..6139 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5771..5790) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5981..6002 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6156..6175) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6275..6297 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6335..6353) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6421..6525 /gene="bla" /label=AmpR promoter CDS 6526..7386 /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(6744..6763) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7557..8145 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8046..8065 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8207..8447 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8207..8227 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8378..8397 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8477..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 gggggcgagg ggcggggcgg ggcgaggcgg 601 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 661 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc 721 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 781 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 841 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 901 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 961 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1021 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1081 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1141 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1201 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1261 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1321 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1381 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1441 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1501 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1561 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1621 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1681 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 1741 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 1801 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 1861 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 1921 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 1981 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2041 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2101 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2161 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2221 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2281 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2341 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2401 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2461 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2521 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2581 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2641 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 2701 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 2761 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 2821 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 2881 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 2941 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3001 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3061 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3121 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3181 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3241 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3301 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3361 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3421 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3481 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3541 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3601 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3661 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 3721 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 3781 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 3841 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 3901 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 3961 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4021 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4081 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4141 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4201 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4261 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4321 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4381 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4441 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4501 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4561 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4621 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4681 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 4741 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 4801 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 4861 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 4921 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 4981 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5041 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5101 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5161 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5221 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5281 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5341 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5401 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5461 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5521 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5581 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 5641 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 5701 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 5761 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 5821 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 5881 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 5941 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6001 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6061 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6121 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6181 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6241 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6301 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6361 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6421 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6481 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6541 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6601 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 6661 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 6721 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 6781 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 6841 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 6901 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 6961 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7021 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7081 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7141 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7201 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7261 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7321 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7381 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7441 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7501 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7621 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 7681 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 7741 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 7801 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 7861 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 7921 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 7981 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8041 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8101 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 8161 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc 8221 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa 8281 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct 8341 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac 8401 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgaact 8461 ctttcacagt gacccagttt //