LOCUS       sequence-310534-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector F encodes pAAV-pMecp2-dSaCas9-SID4X-spA-pU6-sgRNA (BsaI) 
            transgenes for AAV packaging and expression of CRISPR interference 
            in neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  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 7226)
  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-SID4X-pU6-sgRNA
FEATURES             Location/Qualifiers
     source          1..7226
                     /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             3654..3674
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4077..4125
                     /note="synthetic polyadenylation signal"
     promoter        4131..4379
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4131..4151
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4302..4321
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4400..4475
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4489..4629
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4489..4618
                     /label=AAV2 ITR
     rep_origin      4704..5159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4791..4810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5001..5022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5176..5195)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5295..5317
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5355..5373)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5441..5545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5546..5614,5615..6406)
                     /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(5764..5783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6577..7165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7066..7085
                     /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 agccccaaga
     3661 aaaagaggaa ggtggaggcc agcggctccg ggatgaacat ccagatgctg ctggaggccg
     3721 ctgactacct ggaacggaga gagcgcgaag ccgagcacgg atatgcttca atgctgcccg
     3781 gaagcggcat gaatattcag atgctgctgg aggctgctga ttacctggaa aggcgcgaac
     3841 gggaggccga acatggctat gcttccatgc tgcctgggtc tggaatgaat atccaaatgc
     3901 tgctggaggc agccgattac ctggaacgga gagaaagaga agccgagcac ggatacgcca
     3961 gcatgctgcc aggcagcggg atgaacatac aaatgctgct ggaggctgcc gattacctgg
     4021 agaggcgcga gagagaagct gaacatggct atgcctctat gctgccctaa gaattcaata
     4081 aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtggtacc gagggcctat
     4141 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     4201 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     4261 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     4321 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     4381 gagaccacgg caggtctcag ttttagtact ctggaaacag aatctactaa aacaaggcaa
     4441 aatgccgtgt ttatctcgtc aacttgttgg cgagattttt gcggccgcag gaacccctag
     4501 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4561 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4621 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4681 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4741 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4801 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4861 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4921 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4981 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5041 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5101 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5161 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5221 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5281 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5341 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5401 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5461 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5521 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5581 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5641 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5701 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5761 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5821 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5881 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5941 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6001 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6061 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6121 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6181 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6241 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     6301 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6361 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6421 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6481 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6541 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6601 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6661 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6721 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6781 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6841 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6901 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6961 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7021 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7081 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7141 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7201 cttttgctgg ccttttgctc acatgt
//
