LOCUS       sequence-309833-        7251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector G encodes pAAV-pCMV-dSaCas9-VP64-spA-pU6-sgRNA (BsaI) 
            transgenes for AAV packaging and expression of CRISPR activator in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7251)
  AUTHORS   Lau CH, Ho JW, Lo PK, Tin C
  TITLE     Targeted Transgene Activation in the Brain Tissue by Systemic 
            Delivery of Engineered AAV1 Expressing CRISPRa.
  JOURNAL   Mol Ther Nucleic Acids. 2019 Jun 7;16:637-649. doi: 
            10.1016/j.omtn.2019.04.015. Epub 2019 Apr 23.
  PUBMED    31108320
REFERENCE   2  (bases 1 to 7251)
  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: "Mol Ther 
            Nucleic Acids."; date: "2019-06-7"; volume: "16:637-649. doi"; 
            pages: " 10.1016/j.omtn.2019.04.015. Epub 2019 Apr 23"
COMMENT     Sequence Label: pAAV-pCMV-dSaCas9-VP64-pU6-sgRNA
FEATURES             Location/Qualifiers
     source          1..7251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        147..450
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        451..654
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     605..625
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             673..693
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3874..3921
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3937..4086
                     /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"
     polyA_signal    4102..4150
                     /note="synthetic polyadenylation signal"
     promoter        4156..4404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4156..4176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4327..4346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4425..4500
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4514..4654
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4514..4643
                     /label=AAV2 ITR
     rep_origin      4729..5184
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4816..4835)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5026..5047
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5201..5220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5320..5342
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5380..5398)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5466..5570
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5571..5639,5640..6431)
                     /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(5789..5808)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6602..7190
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7091..7110
                     /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 gcggcctcta gatccgcgtt acataactta cggtaaatgg cccgcctggc
      181 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      241 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      301 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      361 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      421 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      481 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      541 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      601 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctaccggt
      661 gccaccatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccaag
      721 cggaactaca tcctgggcct ggccatcggc atcaccagcg tgggctacgg catcatcgac
      781 tacgagacac gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa
      841 aacaacgagg gcaggcggag caagagaggc gccagaaggc tgaagcggcg gaggcggcat
      901 agaatccaga gagtgaagaa gctgctgttc gactacaacc tgctgaccga ccacagcgag
      961 ctgagcggca tcaaccccta cgaggccaga gtgaagggcc tgagccagaa gctgagcgag
     1021 gaagagttct ctgccgccct gctgcacctg gccaagagaa gaggcgtgca caacgtgaac
     1081 gaggtggaag aggacaccgg caacgagctg tccaccaaag agcagatcag ccggaacagc
     1141 aaggccctgg aagagaaata cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc
     1201 gaagtgcggg gcagcatcaa cagattcaag accagcgact acgtgaaaga agccaaacag
     1261 ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga gcttcatcga cacctacatc
     1321 gacctgctgg aaacccggcg gacctactat gagggacctg gcgagggcag ccccttcggc
     1381 tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc actgcaccta cttccccgag
     1441 gaactgcgga gcgtgaagta cgcctacaac gccgacctgt acaacgccct gaacgacctg
     1501 aacaatctcg tgatcaccag ggacgagaac gagaagctgg aatattacga gaagttccag
     1561 atcatcgaga acgtgttcaa gcagaagaag aagcccaccc tgaagcagat cgccaaagaa
     1621 atcctcgtga acgaagagga tattaagggc tacagagtga ccagcaccgg caagcccgag
     1681 ttcaccaacc tgaaggtgta ccacgacatc aaggacatta ccgcccggaa agagattatt
     1741 gagaacgccg agctgctgga tcagattgcc aagatcctga ccatctacca gagcagcgag
     1801 gacatccagg aagaactgac caatctgaac tccgagctga cccaggaaga gatcgagcag
     1861 atctctaatc tgaagggcta taccggcacc cacaacctga gcctgaaggc catcaacctg
     1921 atcctggacg agctgtggca caccaacgac aaccagatcg ctatcttcaa ccggctgaag
     1981 ctggtgccca agaaggtgga cctgtcccag cagaaagaga tccccaccac cctggtggac
     2041 gacttcatcc tgagccccgt cgtgaagaga agcttcatcc agagcatcaa agtgatcaac
     2101 gccatcatca agaagtacgg cctgcccaac gacatcatta tcgagctggc ccgcgagaag
     2161 aactccaagg acgcccagaa aatgatcaac gagatgcaga agcggaaccg gcagaccaac
     2221 gagcggatcg aggaaatcat ccggaccacc ggcaaagaga acgccaagta cctgatcgag
     2281 aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt acagcctgga agccatccct
     2341 ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg accacatcat ccccagaagc
     2401 gtgtccttcg acaacagctt caacaacaag gtgctcgtga agcaggaaga agccagcaag
     2461 aagggcaacc ggaccccatt ccagtacctg agcagcagcg acagcaagat cagctacgaa
     2521 accttcaaga agcacatcct gaatctggcc aagggcaagg gcagaatcag caagaccaag
     2581 aaagagtatc tgctggaaga acgggacatc aacaggttct ccgtgcagaa agacttcatc
     2641 aaccggaacc tggtggatac cagatacgcc accagaggcc tgatgaacct gctgcggagc
     2701 tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc
     2761 tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc
     2821 gaggacgccc tgatcattgc caacgccgat ttcatcttca aagagtggaa gaaactggac
     2881 aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa agcaggccga gagcatgccc
     2941 gagatcgaaa ccgagcagga gtacaaagag atcttcatca ccccccacca gatcaagcac
     3001 attaaggact tcaaggacta caagtacagc caccgggtgg acaagaagcc taatagagag
     3061 ctgattaacg acaccctgta ctccacccgg aaggacgaca agggcaacac cctgatcgtg
     3121 aacaatctga acggcctgta cgacaaggac aatgacaagc tgaaaaagct gatcaacaag
     3181 agccccgaaa agctgctgat gtaccaccac gacccccaga cctaccagaa actgaagctg
     3241 attatggaac agtacggcga cgagaagaat cccctgtaca agtactacga ggaaaccggg
     3301 aactacctga ccaagtactc caaaaaggac aacggccccg tgatcaagaa gattaagtat
     3361 tacggcaaca aactgaacgc ccatctggac atcaccgacg actaccccaa cagcagaaac
     3421 aaggtcgtga agctgtccct gaagccctac agattcgacg tgtacctgga caatggcgtg
     3481 tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg
     3541 aatagcaagt gctatgagga agctaagaag ctgaagaaga tcagcaacca ggccgagttt
     3601 atcgcctcct tctacaacaa cgatctgatc aagatcaacg gcgagctgta tagagtgatc
     3661 ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca tgatcgacat cacctaccgc
     3721 gagtacctgg aaaacatgaa cgacaagagg ccccccagga tcattaagac aatcgcctcc
     3781 aagacccaga gcattaagaa gtacagcaca gacattctgg gcaacctgta tgaagtgaaa
     3841 tctaagaagc accctcagat catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc
     3901 ggccaggcaa aaaagaaaaa gggatccgga cgggctgacg cattggacga ttttgatctg
     3961 gatatgctgg gaagtgacgc cctcgatgat tttgaccttg acatgcttgg ttcggatgcc
     4021 cttgatgact ttgacctcga catgctcggc agtgacgccc ttgatgattt cgacctggac
     4081 atgctgatta actaagaatt caataaaaga tctttatttt cattagatct gtgtgttggt
     4141 tttttgtgtg gtaccgaggg cctatttccc atgattcctt catatttgca tatacgatac
     4201 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     4261 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     4321 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     4381 atcttgtgga aaggacgaaa caccggagac cacggcaggt ctcagtttta gtactctgga
     4441 aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga
     4501 tttttgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4561 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4621 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4681 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4741 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4801 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4861 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4921 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4981 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5041 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     5101 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5161 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5221 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5281 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5341 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5401 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5461 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5521 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5581 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5641 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5701 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5761 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5821 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5881 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5941 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6001 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6061 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6121 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6181 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6241 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     6301 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6361 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6421 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6481 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6541 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6601 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6661 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6721 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6781 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6841 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6901 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6961 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7021 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7081 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7141 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7201 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
