LOCUS       sequence-360815-        6263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector to express nuclear localized eGFP from a Gad65 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6263)
  AUTHORS   de Solis CA, Hosek MP, Holehonnur R, Ho A, Banerjee A, Luong JA, 
            Jones LE, Chaturvedi D, Ploski JE
  TITLE     Adeno-associated viral serotypes differentially transduce inhibitory
            neurons within the rat amygdala.
  JOURNAL   Brain Res. 2017 Oct 1;1672:148-162. doi: 
            10.1016/j.brainres.2017.07.023. Epub 2017 Jul 29.
  PUBMED    28764932
REFERENCE   2  (bases 1 to 6263)
  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: "Brain 
            Res."; date: "2017-10-1"; volume: "1672:148-162. doi"; pages: " 
            10.1016/j.brainres.2017.07.023. Epub 2017 Jul 29"
COMMENT     Sequence Label: pAAV-Gad65P-eGFP-3XNLS-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1214..1223
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1220..1222,1223..1225,1226..1936)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1220..1222,1223..1225,1226..1936)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1265..1286)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1526..1545)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1873..1894
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1946..1966
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1970..1990
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1994..2014
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2066..2654
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2119..2139)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2537..2548)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2681..2698)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2687..2911
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3121..3450)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3135..3270
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3435..3455
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3696..4284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3776..3795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4455..5246,5247..5315))
                     /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     5078..5097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5316..5420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5488..5506
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5544..5566)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5666..5685
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5899..5918
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6208..6226
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6208..6225
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   join(6247..6263,1..113)
                     /label=AAV2 ITR
ORIGIN
        1 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
       61 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt
      121 aatgattaac ccgccatgct acttatctac gtagccatgc tctaggtcac caagactaaa
      181 ggtcccaggg tttctcctcc ggcaacagct ttggtgactc atttgaaaaa ctcaaagaag
      241 gaataaagga ctatggagag cagggaagct gcagtccctc gctcggagag aaggaaagga
      301 gatcgggaac ggtttaaagc agcactttcc tgcaagtctt tcttgagctc tctgtaagcc
      361 tggaactatg ggtttagtat aggcaagagg aaacggaagc ggagccagag ggccccaaat
      421 cttcctcaag gcgccgtccc tgcatttaag agcctttggg aaaagggaaa cagaaaggat
      481 tttttcattc ctttattaga aaacacaaac gaaactactt ctttctgggc tgggaaattg
      541 ccactgttgt gttcctaggc acctgctgat ggcaagaact cctcttggga agggacgagg
      601 tcagtgggtt gagtatccct gaaaagatgg gagtgccttt tcctgggatt tggaaaaact
      661 gattcgctta ctttgagact ccctgaatcc tgtgtatgtg tcagctctca cccactccaa
      721 gaagtttctg ttcgttttat ttatttaatt tttctcagcc tgaggtcctc agtgatagat
      781 tccagcgtgg attttaattg cttcaatcag cagcctttct cttcagccgt cagtcaaaac
      841 ctggacgatg ggtctgggga cccaggcatg actgcacaga aaccgtacga cccccacccc
      901 caccccccag gctggctcag ctcagcaggc agacattagg ctctgctaag gagatgaaat
      961 gagttcgttg gtgtggaagc ggtccacctt cctccctcct tggttccttc cccaagcttt
     1021 ggagctcttc ctgattgagg ccaggtggcc tgggctcttg aggtcacagc gacctcagca
     1081 ctggtttgga ggaaggcggg cgcgagaacc acttttgctc ggcagcagcc agagacagcc
     1141 ctcaaacaat gagccaatcg atgcacacgt gaaattgccc tcggccacca ttccaatctt
     1201 gccaagaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1261 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1321 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1381 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1441 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1501 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1561 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1621 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1681 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1741 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1801 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1861 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1921 ggacgagctg tacaagggat ccgatccaaa aaagaagaga aaggtagatc caaaaaagaa
     1981 gagaaaggta gatccaaaaa agaagagaaa ggtatagtcg agtggagctc gcgactagtc
     2041 gcaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     2101 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2161 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2221 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2281 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2341 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2401 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     2461 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     2521 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     2581 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     2641 ggccgcctcc ccgcatcgat accgtcgact cgctgatcag cctcgactgt gccttctagt
     2701 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2761 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2821 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2881 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2941 tcgactagag catggctacg tagataagta gcatggcggg ttaatcatta actacaagga
     3001 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3061 gcgaccaaag gtcgcccgac gcccgggcgg cctcagtgag cgagcgagcg cgcagagctt
     3121 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     3181 cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     3241 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     3301 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     3361 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     3421 tggggacttt ccacacccta actgacacac attccacagc tgcattaatg aatcggccaa
     3481 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3541 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3601 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3661 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3721 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3781 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3841 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3901 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3961 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4021 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4081 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4141 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4201 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4261 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4321 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4381 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4441 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4501 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4561 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4621 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4681 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4741 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4801 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4861 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4921 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4981 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5041 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5101 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5161 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5221 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5281 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5341 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5401 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     5461 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     5521 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     5581 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     5641 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5701 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5761 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5821 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5881 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5941 gccggtgatg ccggccacga tgcgtccggc gtagaggatc tggctagcga tgaccctgct
     6001 gattggttcg ctgaccattt ccgggtgcgg gacggcgtta ccagaaactc agaaggttcg
     6061 tccaaccaaa ccgactctga cggcagttta cgagagagat gatagggtct gcttcagtaa
     6121 gccagatgct acacaattag gcttgtacat attgtcgtta gaacgcggct acaattaata
     6181 cataacctta tgtatcatac acatacgatt taggtgacac tatagaatac acggaattaa
     6241 ttctagctgc gcgctcgctc gct
//
