LOCUS       sequence-483392-        7863 bp    DNA     circular SYN 14-AUG-2025
DEFINITION  To produce the AAV vector that expresses GFP under the control of 
            the mGAD65 promoter. The mGAD65 promoter has highly specificity to 
            GABAergic interneurons..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7863)
  AUTHORS   Hoshino C, Konno A, Hosoi N, Kaneko R, Mukai R, Nakai J, Hirai H
  TITLE     GABAergic neuron-specific whole-brain transduction by AAV-PHP.B 
            incorporated with a new GAD65 promoter.
  JOURNAL   Mol Brain. 2021 Feb 15;14(1):33. doi: 10.1186/s13041-021-00746-1.
  PUBMED    33588899
REFERENCE   2  (bases 1 to 7863)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol 
            Brain."; date: "2021-02-15"; volume: "14(1):33. doi"; pages: " 
            10.1186/s13041-021-00746-1"
COMMENT     Sequence Label: pAAV mGAD65(delE1) GFP WPRE SV40pA
FEATURES             Location/Qualifiers
     source          1..7863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(108..127)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     162..181
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   277..406
                     /label=AAV2 ITR
     rep_origin      777..1290
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(864..883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1074..1095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1772..1791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1891..1913
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1951..1969)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2037..2141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2142..2210,2211..3002)
                     /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(2360..2379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3173..3761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3662..3681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      6584..6593
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6590..6592,6593..6595,6596..7309)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6590..6592,6593..6595,6596..7306)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6635..6656)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6896..6915)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7243..7264
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(7325..7863,1..50)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7378..7398)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7796..7807)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgatgccg
       61 tatacggtac cacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
      121 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
      181 caatgtatct tatcatgtct gtcgaccccc aacgcgttgg agctctagag catggctacg
      241 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
      301 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
      361 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgg cgtaatagcg
      421 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgat
      481 tccgttgcaa tggctggcgg taatattgtt ctggatatta ccagcaaggc cgatagtttg
      541 agttcttcta ctcaggcaag tgatgttatt actaatcaaa gaagtattgc gacaacggtt
      601 aatttgcgtg atggacagac tcttttactc ggtggcctca ctgattataa aaacacttct
      661 caggattctg gcgtaccgtt cctgtctaaa atccctttaa tcggcctcct gtttagctcc
      721 cgctctgatt ctaacgagga aagcacgtta tacgtgctcg tcaaagcaac catagtacgc
      781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      961 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1081 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1141 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1201 gaattttaac aaaatattaa cgcttacaat ttaaatattt gcttatacaa tcttcctgtt
     1261 tttggggctt ttctgattat caaccggggt acatatgatt gacatgctag ttttacgatt
     1321 accgttcatc gattctcttg tttgctccag actctcaggc aatgacctga tagcctttgt
     1381 agagacctct caaaaatagc taccctctcc ggcatgaatt tatcagctag aacggttgaa
     1441 tatcatattg atggtgattt gactgtctcc ggcctttctc acccgtttga atctttacct
     1501 acacattact caggcattgc atttaaaata tatgagggtt ctaaaaattt ttatccttgc
     1561 gttgaaataa aggcttctcc cgcaaaagta ttacagggtc ataatgtttt tggtacaacc
     1621 gatttagctt tatgctctga ggctttattg cttaattttg ctaattcttt gccttgcctg
     1681 tatgatttat tggatgttgg aatcgcctga tgcggtattt tctccttacg catctgtgcg
     1741 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     1801 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     1861 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     1921 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1981 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     2041 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2101 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2161 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2221 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2281 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2341 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     2401 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2461 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2521 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2581 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2641 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     2701 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2761 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2821 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2881 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2941 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3001 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3061 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3181 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3241 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3301 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3361 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3421 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3481 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3541 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3601 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3661 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3721 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3781 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3841 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3901 gaacgaccga gcgcagcgag tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     3961 ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc
     4021 catgctctag actcgaggct ttgcttcctg ggcactacag ccccacctga agcaggtgcg
     4081 ttagggagga gttgggctct gcagcttgcc agattttaat tctggcttcc agcggaggac
     4141 cctgcacccc acggtcctcg caagagtgtc tgggaaggtt tattcggttg ctgagtagag
     4201 acgcaacaga cagatctacc tcagcccctg caccttggct tgataaggct ggaaaaggaa
     4261 gattgcaacg tcgtggttgt ttccaaaccc tttttctatc tagccttttt cagtctctca
     4321 acatttaagc agcaaaagaa ctcgagagag aaaacttgta ctaaatatcc ttctttgacc
     4381 ggccctctcc tctccccact acggggattt ctgggtggct cacagtggac aaggaatagg
     4441 taatcccacc aaaccttgag gtgggctaaa ctggagattt tggtatgggt ctcctactgt
     4501 aaagagaaag ccttccgggc taacggtatt cctttcgcag tctactgcct cctttgtgaa
     4561 ctaatgactg taattattac ctcccagagc tcttttgtta tcaacaaccc ggagccagag
     4621 gggggtgggg gatgggccct ttgggaaaag ggaaaaaaat ttacaaagca aattgctgtc
     4681 tacgaagggt tggtttttta aaaagacaaa cccaattcca gcctgcattc gaagtgggga
     4741 gtggcaaaat tttttttcaa agtatgataa gattttaaaa atactcagag ctgtgggatt
     4801 caattgcatt tccacagtat cgctttaagt tactattttt gacgaagtaa aattcagatg
     4861 ttgaaaatct gtatctcttg ttttcatcca tttcttcctt ggaaggtgtg tgtgtgtgtg
     4921 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtttaagaa cctttttttt tatttccgac
     4981 cactcatcca tggtaaaaag cttcaacccc agccagctgg cttgaagaga attattttgc
     5041 aggatttagt atggatttaa acaaagactc aggctctcca agtcttgttg gcaaggctgg
     5101 gcggaagcgc cctttccagg agctgttacc tctgccagct gaagccacca ggcctggagg
     5161 accctaaaga cttactaagt tgaggcctag ggtcaaggtg actgccttcc cttagagaca
     5221 cgccttgggt agcacctcag cagtgccaat ctcaccgccc tccccctccc cacctcgggc
     5281 cactcaccgg acctcagtgg gcatcctcta gctagatcct ccctggcccc cctggattct
     5341 ggaaaccttg ggaagaagat aaagctttgt ggagagaatg cacttggtca tctgcagtta
     5401 ttcgaggtct ccggatccct aacaaaataa gtgatcctgg tcaccaagac taaaggtccc
     5461 agggtttctc ctccggcaac agctttggtg actcatttga aaaactcaaa gaaggaataa
     5521 aggactatgg agagcaggga agctgcagtc cctcgctcgg agagaaggaa aggagatcgg
     5581 gaacggttta aagcagcact ttcctgcaag tctttcttga gctctctgta agcctggaac
     5641 tatgggttta gtataggcaa gaggaaacgg aagcggagcc agagggcccc aaatcttcct
     5701 caaggcgccg tccctgcatt taagagcctt tgggaaaagg gaaacagaaa ggattttttc
     5761 attcctttat tagaaaacac aaacgaaact acttctttct gggctgggaa attgccactg
     5821 ttgtgttcct aggcacctgc tgatggcaag aactcctctt gggaagggac gaggtcagtg
     5881 ggttgagtat ccctgaaaag atgggagtgc cttttcctgg gatttggaaa aactgattcg
     5941 cttactttga gactccctga atcctgtgta tgtgtcagct ctcacccact ccaagaagtt
     6001 tctgttcgtt ttatttattt aatttttctc agcctgaggt cctcagtgat agattccagc
     6061 gtggatttta attgcttcaa tcagcagcct ttctcttcag ccgtcagtca aaacctggac
     6121 gatgggtctg gggacccagg catgactgca cagaaaccgt acgaccccca cccccacccc
     6181 ccaggctggc tcagctcagc aggcagacat taggctctgc taaggagatg aaatgagttc
     6241 gttggtgtgg aagcggtcca ccttcctccc tccttggttc cttccccaag ctttggagct
     6301 cttcctgatt gaggccaggt ggcctgggct cttgaggtca cagcgacctc agcactggtt
     6361 tggaggaagg cgggcgcgag aaccactttt gctcggcagc agccagagac agccctcaaa
     6421 caatgagcca atcgatgcac acgtgaaatt gccctcggcc accattccaa tcttgccaag
     6481 gtctccctgt ccctggggtc acctaggcca gcgtgctgag ggctgtggta gcaccctgga
     6541 cagcgcgttc cggcagaagc agacctggag gaattcaccg gtcgccacca tggtgagcaa
     6601 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     6661 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     6721 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     6781 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     6841 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     6901 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     6961 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     7021 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     7081 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     7141 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     7201 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     7261 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcat
     7321 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     7381 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     7441 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     7501 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     7561 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     7621 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     7681 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     7741 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     7801 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     7861 tct
//
