LOCUS Exported 8567 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Dual expression construct expressing both dCas9VP48 and sgTetO from separate promoters. ACCESSION . VERSION . KEYWORDS pAC5-dual-dCas9VP48-sgTetO SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8567) AUTHORS Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R TITLE Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. JOURNAL Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122. PUBMED 23979020 REFERENCE 2 (bases 1 to 8567) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8567 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 270..345 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 442..727 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 729..1006 /label=chicken beta-actin promoter intron 1007..1235 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1256..1282 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1286..1306 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1316..5416 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5420..5440 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5459..5569 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" primer_bind complement(5608..5625) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5614..5821 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5830..5970 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5830..5959 /label=AAV2 ITR rep_origin 6045..6500 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6132..6151) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6342..6363 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6517..6536) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6636..6658 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6696..6714) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6782..6886 /gene="bla" /label=AmpR promoter CDS 6887..7747 /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(7105..7124) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7918..8506 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8407..8426 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg cttttctcta tcactgatag ttttagagct agaaatagca agttaaaata 301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 841 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatgtaccc 1261 atacgatgtt ccagattacg cttcgccgaa gaaaaagcgc aaggtcgaag cgtccgacaa 1321 gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga 1381 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 1441 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 1501 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 1561 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 1621 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 1681 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 1741 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 1801 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 1861 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 1921 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 1981 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc 2041 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 2101 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 2161 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 2221 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 2281 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 2341 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 2401 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 2461 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 2521 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 2581 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 2641 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 2701 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 2761 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 2821 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 2881 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 2941 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 3001 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 3061 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 3121 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 3181 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 3241 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 3301 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 3361 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 3421 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 3481 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 3541 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 3601 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 3661 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 3721 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 3781 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacg ccatcgtgcc 3841 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 3901 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 3961 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 4021 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 4081 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 4141 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 4201 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 4261 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 4321 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 4381 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 4441 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 4501 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 4561 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 4621 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 4681 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 4741 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 4801 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 4861 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 4921 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 4981 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 5041 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 5101 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 5161 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 5221 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 5281 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 5341 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 5401 tcagctggga ggcgacagcc ccaagaagaa gagaaaggtg gaggccagcg ggccggccga 5461 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct 5521 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggtaaag 5581 ggcgaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 5641 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 5701 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 5761 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg 5821 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 5881 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 5941 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc 6001 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 6061 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 6121 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 6181 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 6241 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 6301 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 6361 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 6421 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 6481 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 6541 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 6601 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 6661 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 6721 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 6781 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 6841 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 6901 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 6961 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 7021 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 7081 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 7141 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 7201 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 7261 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 7321 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 7381 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 7441 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 7501 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 7561 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 7621 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 7681 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 7741 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 7801 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 7861 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 7921 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 7981 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 8041 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 8101 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 8161 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 8221 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8281 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 8341 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 8401 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 8461 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 8521 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt //