LOCUS       sequence-375110-        7427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual AAV vector AAV-dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7427)
  AUTHORS   Wang G, Chow RD, Bai Z, Zhu L, Errami Y, Dai X, Dong MB, Ye L, Zhang
            X, Renauer PA, Park JJ, Shen L, Ye H, Fuchs CS, Chen S
  TITLE     Multiplexed activation of endogenous genes by CRISPRa elicits potent
            antitumor immunity.
  JOURNAL   Nat Immunol. 2019 Nov;20(11):1494-1505. doi: 
            10.1038/s41590-019-0500-4. Epub 2019 Oct 14.
  PUBMED    31611701
REFERENCE   2  (bases 1 to 7427)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41590-019-0500-4"; journalName: "Nat Immunol"; date: 
            "2019-11"; volume: "20"; issue: "11"; pages: "1494-1505"
COMMENT     Sequence Label: pGW011b Dual AAV vector pAAV-EFS-dCAS9-spA
FEATURES             Location/Qualifiers
     source          1..7427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        194..405
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      424..433
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             436..483
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(484..507,508..510,511..3066,3067..3069,3070..4584)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4588..4608
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4625..4673
                     /note="synthetic polyadenylation signal"
     repeat_region   4690..4830
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4690..4819
                     /label=AAV2 ITR
     rep_origin      4905..5360
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4992..5011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5202..5223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5377..5396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5496..5518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5556..5574)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5642..5746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5747..5815,5816..6607)
                     /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(5965..5984)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6778..7366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7267..7286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttctagc taggtcttga aaggagtggg aattggctcc
      181 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      241 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      301 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
      361 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcgcg
      421 tacgccacca tggccaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
      481 aaggacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
      541 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
      601 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
      661 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
      721 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
      781 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
      841 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
      901 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
      961 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     1021 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     1081 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     1141 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac
     1201 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     1261 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     1321 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     1381 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     1441 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     1501 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     1561 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     1621 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     1681 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     1741 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     1801 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     1861 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     1921 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     1981 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     2041 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     2101 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     2161 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     2221 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     2281 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     2341 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     2401 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     2461 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     2521 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     2581 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     2641 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     2701 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     2761 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     2821 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     2881 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     2941 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccac
     3001 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     3061 gacaaggccc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     3121 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     3181 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     3241 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     3301 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     3361 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     3421 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     3481 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     3541 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     3601 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     3661 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     3721 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     3781 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     3841 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     3901 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     3961 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     4021 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     4081 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     4141 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     4201 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     4261 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     4321 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     4381 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     4441 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     4501 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     4561 gacctgtctc agctgggagg cgacagccct aagaaaaaga ggaaggtgag ctgagaattc
     4621 aataaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt gtgcggaccg
     4681 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4741 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4801 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4861 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4921 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4981 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5041 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5101 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5161 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5221 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5281 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5341 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5401 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5461 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5521 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5581 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5641 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5701 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5761 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5821 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5881 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5941 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6001 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6061 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6121 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6181 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6241 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6301 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6361 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6421 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6481 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6541 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6601 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6661 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6721 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6781 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6841 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6901 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6961 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7021 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7081 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7141 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7201 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7261 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7321 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7381 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
