LOCUS       sequence-318113-        6890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector A encodes pAAV-pMecp2-SaCas9-spA-pU6-sgRNA (BsaI) transgenes 
            for AAV packaging and expression of SaCas9-sgRNA in neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6890)
  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 6890)
  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-SaCas9-pU6-sgRNA
FEATURES             Location/Qualifiers
     source          1..6890
                     /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             441..3596
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3597..3644
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3651..3731
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_signal    3741..3789
                     /note="synthetic polyadenylation signal"
     promoter        3795..4043
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3795..3815
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3966..3985
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4064..4139
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4153..4293
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4153..4282
                     /label=AAV2 ITR
     rep_origin      4368..4823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4455..4474)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4665..4686
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4840..4859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4959..4981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5019..5037)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5105..5209
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5210..5278,5279..6070)
                     /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(5428..5447)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6241..6829
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6730..6749
                     /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 cctggacatc 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 agaaaacagc 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 tacccatacg
     3661 atgttccaga ttacgcttac ccatacgatg ttccagatta cgcttaccca tacgatgttc
     3721 cagattacgc ttaagaattc aataaaagat ctttattttc attagatctg tgtgttggtt
     3781 ttttgtgtgg taccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     3841 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3901 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3961 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     4021 tcttgtggaa aggacgaaac accggagacc acggcaggtc tcagttttag tactctggaa
     4081 acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat
     4141 ttttgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4201 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4261 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4321 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4381 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4441 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4501 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4561 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4621 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4681 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     4741 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4801 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     4861 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4921 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4981 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5041 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5101 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5161 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5221 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5281 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5341 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5401 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5461 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5521 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5581 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5641 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5701 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5761 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5821 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5881 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     5941 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6001 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6061 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6121 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6181 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6241 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6301 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6361 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6421 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6481 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6541 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6601 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6661 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6721 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6781 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6841 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
