LOCUS       sequence-194578-        7231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSa Cas9 fused to gold standard VP64 activator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7231)
  AUTHORS   Vora S, Cheng J, Xiao R, VanDusen NJ, Quintino L, Pu WT, 
            Vandenberghe LH, Chavez A, Church G
  TITLE     Rational design of a compact CRISPR-Cas9 activator for AAV-mediated 
            delivery
  JOURNAL   bioRxiv 2018. 298620
REFERENCE   2  (bases 1 to 7231)
  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"; journalName: "bioRxiv 
            2018. 298620"
COMMENT     Sequence Label: pAAV-dSa-VP64
FEATURES             Location/Qualifiers
     source          1..7231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     127..146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(163..618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     512..531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   693..833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   704..833
                     /label=AAV2 ITR
     enhancer        853..1232
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1233..1436
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1387..1407
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1455..1464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1467..1487
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4668..4715
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4746..4895
                     /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"
     CDS             4920..4940
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4971..4988)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4977..5184
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5197..5326
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5388..5976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5468..5487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6147..6938,6939..7007))
                     /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     6770..6789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7008..7112)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7180..7198
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
       61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
      181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
      241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
      301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
      421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag
      661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct
      721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
      781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
      841 cctcgaggcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      901 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      961 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     1021 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     1081 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     1141 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     1201 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     1261 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     1321 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     1381 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
     1441 ggctaactac cggtgccacc atggccccaa agaagaagcg gaaggtcggt atccacggag
     1501 tcccagcagc caagcggaac tacatcctgg gcctggccat cggcatcacc agcgtgggct
     1561 acggcatcat cgactacgag acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag
     1621 aggccaacgt ggaaaacaac gagggcaggc ggagcaagag aggcgccaga aggctgaagc
     1681 ggcggaggcg gcatagaatc cagagagtga agaagctgct gttcgactac aacctgctga
     1741 ccgaccacag cgagctgagc ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc
     1801 agaagctgag cgaggaagag ttctctgccg ccctgctgca cctggccaag agaagaggcg
     1861 tgcacaacgt gaacgaggtg gaagaggaca ccggcaacga gctgtccacc aaagagcaga
     1921 tcagccggaa cagcaaggcc ctggaagaga aatacgtggc cgaactgcag ctggaacggc
     1981 tgaagaaaga cggcgaagtg cggggcagca tcaacagatt caagaccagc gactacgtga
     2041 aagaagccaa acagctgctg aaggtgcaga aggcctacca ccagctggac cagagcttca
     2101 tcgacaccta catcgacctg ctggaaaccc ggcggaccta ctatgaggga cctggcgagg
     2161 gcagcccctt cggctggaag gacatcaaag aatggtacga gatgctgatg ggccactgca
     2221 cctacttccc cgaggaactg cggagcgtga agtacgccta caacgccgac ctgtacaacg
     2281 ccctgaacga cctgaacaat ctcgtgatca ccagggacga gaacgagaag ctggaatatt
     2341 acgagaagtt ccagatcatc gagaacgtgt tcaagcagaa gaagaagccc accctgaagc
     2401 agatcgccaa agaaatcctc gtgaacgaag aggatattaa gggctacaga gtgaccagca
     2461 ccggcaagcc cgagttcacc aacctgaagg tgtaccacga catcaaggac attaccgccc
     2521 ggaaagagat tattgagaac gccgagctgc tggatcagat tgccaagatc ctgaccatct
     2581 accagagcag cgaggacatc caggaagaac tgaccaatct gaactccgag ctgacccagg
     2641 aagagatcga gcagatctct aatctgaagg gctataccgg cacccacaac ctgagcctga
     2701 aggccatcaa cctgatcctg gacgagctgt ggcacaccaa cgacaaccag atcgctatct
     2761 tcaaccggct gaagctggtg cccaagaagg tggacctgtc ccagcagaaa gagatcccca
     2821 ccaccctggt ggacgacttc atcctgagcc ccgtcgtgaa gagaagcttc atccagagca
     2881 tcaaagtgat caacgccatc atcaagaagt acggcctgcc caacgacatc attatcgagc
     2941 tggcccgcga gaagaactcc aaggacgccc agaaaatgat caacgagatg cagaagcgga
     3001 accggcagac caacgagcgg atcgaggaaa tcatccggac caccggcaaa gagaacgcca
     3061 agtacctgat cgagaagatc aagctgcacg acatgcagga aggcaagtgc ctgtacagcc
     3121 tggaagccat ccctctggaa gatctgctga acaacccctt caactatgag gtggaccaca
     3181 tcatccccag aagcgtgtcc ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg
     3241 aagaagccag caagaagggc aaccggaccc cattccagta cctgagcagc agcgacagca
     3301 agatcagcta cgaaaccttc aagaagcaca tcctgaatct ggccaagggc aagggcagaa
     3361 tcagcaagac caagaaagag tatctgctgg aagaacggga catcaacagg ttctccgtgc
     3421 agaaagactt catcaaccgg aacctggtgg ataccagata cgccaccaga ggcctgatga
     3481 acctgctgcg gagctacttc agagtgaaca acctggacgt gaaagtgaag tccatcaatg
     3541 gcggcttcac cagctttctg cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt
     3601 acaagcacca cgccgaggac gccctgatca ttgccaacgc cgatttcatc ttcaaagagt
     3661 ggaagaaact ggacaaggcc aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg
     3721 ccgagagcat gcccgagatc gaaaccgagc aggagtacaa agagatcttc atcacccccc
     3781 accagatcaa gcacattaag gacttcaagg actacaagta cagccaccgg gtggacaaga
     3841 agcctaatag agagctgatt aacgacaccc tgtactccac ccggaaggac gacaagggca
     3901 acaccctgat cgtgaacaat ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa
     3961 agctgatcaa caagagcccc gaaaagctgc tgatgtacca ccacgacccc cagacctacc
     4021 agaaactgaa gctgattatg gaacagtacg gcgacgagaa gaatcccctg tacaagtact
     4081 acgaggaaac cgggaactac ctgaccaagt actccaaaaa ggacaacggc cccgtgatca
     4141 agaagattaa gtattacggc aacaaactga acgcccatct ggacatcacc gacgactacc
     4201 ccaacagcag aaacaaggtc gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc
     4261 tggacaatgg cgtgtacaag ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa
     4321 actactacga agtgaatagc aagtgctatg aggaagctaa gaagctgaag aagatcagca
     4381 accaggccga gtttatcgcc tccttctaca acaacgatct gatcaagatc aacggcgagc
     4441 tgtatagagt gatcggcgtg aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg
     4501 acatcaccta ccgcgagtac ctggaaaaca tgaacgacaa gaggcccccc aggatcatta
     4561 agacaatcgc ctccaagacc cagagcatta agaagtacag cacagacatt ctgggcaacc
     4621 tgtatgaagt gaaatctaag aagcaccctc agatcatcaa aaagggcaaa aggccggcgg
     4681 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ggaggccagc ggttccggac
     4741 gggctgacgc attggacgat tttgatctgg atatgctggg aagtgacgcc ctcgatgatt
     4801 ttgaccttga catgcttggt tcggatgccc ttgatgactt tgacctcgac atgctcggca
     4861 gtgacgccct tgatgatttc gacctggaca tgctgattaa ctctagaagt tccggatctc
     4921 cgaaaaagaa acgcaaagtt ggttaagaat tcctagagct cgctgatcag cctcgactgt
     4981 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     5041 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     5101 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     5161 agagaatagc aggcatgctg gggagctaga ggccgcagga acccctagtg atggagttgg
     5221 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     5281 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggacat gtgagcaaaa
     5341 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5401 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5461 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5521 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5581 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5641 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5701 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5761 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5821 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5881 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5941 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6001 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggct tccacgctca
     6301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7021 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7081 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7141 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7201 tttcgtctcg cgcgtttcgg tgatgacggt g
//
