LOCUS Exported 8483 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor REST. ACCESSION . VERSION . KEYWORDS pREST.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8483) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8483) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8483 /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 709..936 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 948..957 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 957..1022 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1029..1049 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1074..5174 /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 5175..5222 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5250..5267) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5256..5463 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5472..5612 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5472..5601 /label=AAV2 ITR rep_origin 5687..6142 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5774..5793) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5984..6005 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6159..6178) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6278..6300 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6338..6356) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6424..6528 /gene="bla" /label=AmpR promoter CDS 6529..7389 /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(6747..6766) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7560..8148 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8049..8068 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8210..8450 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8210..8230 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8381..8400 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8480..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 gggcggggcg aggggcgggg cggggcgagg 601 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 661 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg 721 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 781 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 841 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat 901 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact 961 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 1021 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 1081 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 1141 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 1201 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 1261 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 1321 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 1381 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 1441 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 1501 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 1561 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 1621 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 1681 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 1741 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 1801 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 1861 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 1921 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 1981 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 2041 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 2101 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 2161 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 2221 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 2281 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 2341 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 2401 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 2461 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 2521 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 2581 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 2641 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 2701 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 2761 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 2821 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 2881 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 2941 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 3001 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 3061 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 3121 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 3181 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 3241 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 3301 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 3361 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 3421 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 3481 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 3541 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 3601 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 3661 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 3721 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 3781 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 3841 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 3901 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 3961 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 4021 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 4081 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 4141 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 4201 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 4261 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 4321 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 4381 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 4441 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 4501 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 4561 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 4621 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 4681 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 4741 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 4801 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 4861 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 4921 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 4981 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 5041 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 5101 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 5161 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 5221 agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 5281 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 5341 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 5401 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 5461 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 5521 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 5581 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 5641 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 5701 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 5761 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 5821 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 5881 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 5941 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 6001 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg 6061 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 6121 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 6181 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 6241 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 6301 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 6361 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 6421 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 6481 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 6541 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 6601 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 6661 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 6721 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 6781 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 6841 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 6901 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6961 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 7021 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 7081 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 7141 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 7201 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 7261 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 7321 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 7381 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 7441 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 7501 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 7561 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 7621 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 7681 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc 7741 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 7801 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 7861 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7921 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7981 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 8041 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 8101 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 8161 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt 8221 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat 8281 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt 8341 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt 8401 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg 8461 agtaatgaaa ctttgaacag ttt //