LOCUS Exported 9014 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Encodes dCas9(C) with DHFR-PP7-VP64 fused to BDKRB2. ACCESSION . VERSION . KEYWORDS pBDKRB2_TCS(Q'G)_NLS-HA-dCas9(C)_T2A_DHFR-PCP-VP64 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9014) AUTHORS Baeumler TA, Ahmed AA, Fulga TA TITLE Engineering Synthetic Signaling Pathways with Programmable dCas9-Based Chimeric Receptors. JOURNAL Cell Rep. 2017 Sep 12;20(11):2639-2653. doi: 10.1016/j.celrep.2017.08.044. PUBMED 28903044 REFERENCE 2 (bases 1 to 9014) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9014 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 12..297 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 299..576 /label=chicken beta-actin promoter intron 577..804 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 2177..2197 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2198..2218 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2237..2263 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2291..4789 /codon_start=1 /product="C-terminal portion of catalytically inactive Streptococcus pyogenes dCas9 carrying the N863A mutation (Zetsche et al., 2015)" /label=dCas9(C) /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGD" CDS 4793..4813 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4835..4888 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4889..5362 /codon_start=1 /gene="folA" /product="E. coli dihydrofolate reductase" /label=ecDHFR /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER" CDS 5828..5848 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5873..6022 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" primer_bind complement(6056..6073) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6062..6269 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6278..6418 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6278..6407 /label=AAV2 ITR rep_origin 6493..6948 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6580..6599) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6790..6811 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6965..6984) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7084..7106 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7144..7162) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7230..7334 /gene="bla" /label=AmpR promoter CDS 7335..8195 /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(7553..7572) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8366..8954 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8855..8874 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg 121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc 241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc 301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt 361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg 421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg 481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg 541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc 601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta 661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag 721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac 781 ctgcctgaaa tcactttttt tcaggttgga ccggtgcgcc accatgaaga cgatcatcgc 841 cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat 901 cgatatgttc tcaccctgga agatttctat gttcctttct gtgcgcgagg acagcgtgcc 961 cacaactgcg agttttagcg ccgacatgct taacgtgacc ctgcagggcc ccactctcaa 1021 cgggacattt gctcagtcta aatgtcctca agtggaatgg ctcggctggt tgaacactat 1081 ccagccccct ttcctttggg ttctttttgt tctggccacg ctggaaaaca tctttgttct 1141 ctctgtgttt tgcctccata agagctcctg tacggtggcc gaaatttacc tgggtaattt 1201 ggccgcagcg gatctcattt tggcctgtgg cctgcctttc tgggctatta ctatcagcaa 1261 taattttgat tggttgtttg gcgagacctt gtgtcgcgtg gtgaatgcaa ttatcagtat 1321 gaatctgtac agctcaatct gcttccttat gctcgtcagc attgacaggt acctcgccct 1381 tgtaaagact atgagcatgg gccgcatgag gggcgtacgc tgggctaagc tctatagcct 1441 ggtgatttgg ggttgcaccc tcctcctcag ttcacctatg ctggtcttcc gaacgatgaa 1501 ggagtactct gacgagggcc acaatgttac cgcatgcgtt atcagttacc catctttgat 1561 ttgggaggtc ttcacaaaca tgctgctgaa cgtggtgggt tttttgctcc ccctttctgt 1621 gattaccttc tgtacaatgc agatcatgca agttttgcga aacaacgaaa tgcaaaaatt 1681 taaggagata cagaccgagc gaagggccac agtgctggtc ttggtcgttt tgctgttgtt 1741 cattatctgt tggctgcctt tccagatcag cactttcctg gacacgttgc acagactcgg 1801 aatcctgagt agttgtcagg acgaaaggat catcgacgtt atcactcaaa tcgcgtcctt 1861 catggcatac tccaatagct gcttgaaccc attggtgtat gttattgtgg gaaaacggtt 1921 taggaagaag agctgggaag tgtaccaagg ggtgtgccaa aagggaggct gccgatcaga 1981 gccaatccaa atggaaaata gcatgggtac cctgagaacc tccattagcg tggagcgaca 2041 gatccacaag ctccaggact gggctggctc tagacagatc gataccggtg gacgcacccc 2101 acccagcctg ggtccccaag atgagtcctg caccaccgcc agctcctccc tggccaagga 2161 cacttcatcg accggtgaga acctgtactt ccagggcccc aagaagaaga ggaaggtggg 2221 gggaggcagc actagttacc catacgacgt accagattac gctggaggtt ctggatccgg 2281 cggtggctcg aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 2341 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 2401 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 2461 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 2521 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 2581 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 2641 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 2701 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 2761 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 2821 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 2881 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 2941 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 3001 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 3061 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 3121 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 3181 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 3241 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc 3301 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 3361 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 3421 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 3481 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 3541 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 3601 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 3661 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 3721 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 3781 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 3841 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 3901 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 3961 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc 4021 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 4081 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 4141 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 4201 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 4261 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 4321 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata 4381 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 4441 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 4501 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 4561 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 4621 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga 4681 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat 4741 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gccccaagaa 4801 gaagagaaag gtggaggcca gcagcggcag tggagagggc agaggaagtc tgctaacatg 4861 cggtgacgtc gaggagaatc ctggcccaat gatcagtctg attgcggcgt tagcggtaga 4921 ttacgttatc ggcatggaaa acgccatgcc gtggaacctg cctgccgatc tcgcctggtt 4981 taaacgcaac accttaaata aacccgtgat tatgggccgc catacctggg aatcaatcgg 5041 tcgtccgttg ccaggacgca aaaatattat cctcagcagt caaccgagta cggacgatcg 5101 cgtaacgtgg gtgaagtcgg tggatgaagc catcgcggcg tgtggtgacg taccagaaat 5161 catggtgatt ggcggcggtc gcgttattga acagttcttg ccaaaagcgc aaaaactgta 5221 tctgacgcat atcgacgcag aagtggaagg cgacacccat ttcccggatt acgagccgga 5281 tgactgggaa tcggtattca gcgaattcca cgatgctgat gcgcagaact ctcacagcta 5341 ttgctttgag attctggagc ggcgaggtgg aggaggttca ggaggtggag gctctggagg 5401 tggaggcagc tccaaaacca tcgttctttc ggtcggcgag gctactcgca ctctgactga 5461 gatccagtcc accgcagacc gtcagatctt cgaagagaag gtcgggcctc tggtgggtcg 5521 gctgcgcctc acggcttcgc tccgtcaaaa cggagccaag accgcgtatc gcgtcaacct 5581 aaaactggat caggcggacg tcgttgattc cggacttccg aaagtgcgct acactcaggt 5641 atggtcgcac gacgtgacaa tcgttgcgaa tagcaccgag gcctcgcgca aatcgttgta 5701 cgatttgacc aagtccctcg tcgcgacctc gcaggtcgaa gatcttgtcg tcaaccttgt 5761 gccgctgggc cgtagcgctg gaggaggtgg aagcggagga ggaggaagcg gaggaggagg 5821 tagcggacct aagaaaaaga ggaaggtggc ggccgctgga tccggacggg ctgacgcatt 5881 ggacgatttt gatctggata tgctgggaag tgacgccctc gatgattttg accttgacat 5941 gcttggttcg gatgcccttg atgactttga cctcgacatg ctcggcagtg acgcccttga 6001 tgatttcgac ctggacatgc tgattaacta agaattccta gagctcgctg atcagcctcg 6061 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 6121 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 6181 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 6241 tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg 6301 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga 6361 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg 6421 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa 6481 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 6541 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt 6601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 6661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt 6721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 6781 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt 6841 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt 6901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc 6961 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 7141 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 7201 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 7261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 7321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 7381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 7441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 7501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 7561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 7621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 7681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 7741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 7801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 7861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 7921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 7981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 8041 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 8101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 8161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 8221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 8281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 8341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 8401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 8461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 8521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 8581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 8641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 8701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 8761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 8821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 8881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 8941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 9001 cttttgctca catg //