LOCUS       sequence-309836-        7211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector D encodes pAAV-pMecp2-dSaCas9-VP160-spA-pU6-sgRNA (BsaI) 
            transgenes for AAV packaging and expression of CRISPR activator in 
            neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7211)
  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 7211)
  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-pMecp2-dSaCas9-VP160-pU6-sgRNA
FEATURES             Location/Qualifiers
     source          1..7211
                     /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        149..377
                     /label=Mecp2 promoter
                     /note="promoter for selective expression in neurons (Adachi
                     et al., 2005; Gray et al., 2011)"
     regulatory      384..393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             396..416
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3597..3644
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3660..4043
                     /codon_start=1
                     /product="10 tandem repeats of the minimal activation 
                     domain of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP160
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS
                     DALDDFDLDMLGSDALDDFDLDML"
     polyA_signal    4062..4110
                     /note="synthetic polyadenylation signal"
     promoter        4116..4364
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4116..4136
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4287..4306
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4385..4460
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4474..4614
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4474..4603
                     /label=AAV2 ITR
     rep_origin      4689..5144
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4776..4795)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4986..5007
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5161..5180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5280..5302
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5340..5358)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5426..5530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5531..5599,5600..6391)
                     /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(5749..5768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6562..7150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7051..7070
                     /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 gaaagcttag ctgaatgggg tccgcctctt ttccctgcct
      181 aaacagacag gaactcctgc caattgaggg cgtcaccgct aaggctccgc cccagcctgg
      241 gctccacaac caatgaaggg taatctcgac aaagagcaag gggtggggcg cgggcgcgca
      301 ggtgcagcag cacacaggct ggtcgggagg gcggggcgcg acgtctgccg tgcggggtcc
      361 cggcatcggt tgcgcgcacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta
      421 tccacggagt cccagcagcc aagcggaact acatcctggg cctggccatc ggcatcacca
      481 gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc
      541 tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa
      601 ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca
      661 acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg
      721 gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga
      781 gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca
      841 aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc
      901 tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg
      961 actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc
     1021 agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac
     1081 ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg
     1141 gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc
     1201 tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc
     1261 tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca
     1321 ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag
     1381 tgaccagcac cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca
     1441 ttaccgcccg gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc
     1501 tgaccatcta ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc
     1561 tgacccagga agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc
     1621 tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga
     1681 tcgctatctt caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag
     1741 agatccccac caccctggtg gacgacttca tcctgagccc cgtcgtgaag agaagcttca
     1801 tccagagcat caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca
     1861 ttatcgagct ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc
     1921 agaagcggaa ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag
     1981 agaacgccaa gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc
     2041 tgtacagcct ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg
     2101 tggaccacat catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg
     2161 tgaagcagga agaagccagc aagaagggca accggacccc attccagtac ctgagcagca
     2221 gcgacagcaa gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca
     2281 agggcagaat cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt
     2341 tctccgtgca gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag
     2401 gcctgatgaa cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt
     2461 ccatcaatgg cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga
     2521 acaaggggta caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct
     2581 tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg
     2641 aaaagcaggc cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca
     2701 tcacccccca ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg
     2761 tggacaagaa gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg
     2821 acaagggcaa caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca
     2881 agctgaaaaa gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc
     2941 agacctacca gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt
     3001 acaagtacta cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc
     3061 ccgtgatcaa gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg
     3121 acgactaccc caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg
     3181 acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca
     3241 aaaaagaaaa ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga
     3301 agatcagcaa ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca
     3361 acggcgagct gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga
     3421 acatgatcga catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca
     3481 ggatcattaa gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc
     3541 tgggcaacct gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa
     3601 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc gggcgcgccg
     3661 acgcgctgga cgatttcgat ctcgacatgc tgggttctga tgccctcgat gactttgacc
     3721 tggatatgtt gggaagcgac gcattggatg actttgatct ggacatgctc ggctccgatg
     3781 ctctggacga tttcgatctc gatatgttag ggtcagacgc actggatgat ttcgaccttg
     3841 atatgttggg aagcgatgcc cttgatgatt tcgacctgga catgctcggc agcgacgccc
     3901 tggacgattt cgatctggac atgctggggt ccgatgcctt ggatgatttt gacttggata
     3961 tgctggggag tgatgccctg gacgactttg acctggacat gctgggctcc gatgcgctcg
     4021 atgacttcga tttggatatg ttgtatatcg attaagaatt caataaaaga tctttatttt
     4081 cattagatct gtgtgttggt tttttgtgtg gtaccgaggg cctatttccc atgattcctt
     4141 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     4201 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     4261 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     4321 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac cacggcaggt
     4381 ctcagtttta gtactctgga aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc
     4441 tcgtcaactt gttggcgaga tttttgcggc cgcaggaacc cctagtgatg gagttggcca
     4501 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4561 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     4621 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4681 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4741 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     4801 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4861 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4921 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4981 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     5041 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     5101 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     5161 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     5221 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5281 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     5341 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6241 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6541 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6601 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6661 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6721 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6781 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6841 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6901 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6961 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7021 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7081 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7141 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7201 tgctcacatg t
//
