LOCUS       sequence-356199-        6612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-ON /
            FLP-ON) of GFP in cortical interneurons under the control of the Dlx
            enhancer (Dimidschstein et al 2016).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  AUTHORS   Pouchelon G, Vergara J, McMahon J, Gorissen BL, Lin JD, 
            Vormstein-Schneider D, Niehaus JL, Burbridge TJ, Wester JC, Sherer 
            M, Fernandez-Otero M, Allaway KC, Pelkey K, Chittajallu R, McBain 
            CJ, Fan M, Nasse JS, Wildenberg GA, Fishell G, Dimidschstein J
  TITLE     A versatile viral toolkit for functional discovery in the nervous 
            system.
  JOURNAL   Cell Rep Methods. 2022 May 26;2(6):100225. doi: 
            10.1016/j.crmeth.2022.100225. eCollection 2022 Jun 20.
  PUBMED    35784651
REFERENCE   2  (bases 1 to 6612)
  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: "Cell Rep 
            Methods."; date: "2022-05-26"; volume: "2(6):100225. doi"; pages: " 
            10.1016/j.crmeth.2022.100225. eCollection 2022 Jun 20"
COMMENT     Sequence Label: pAAV-VTKD4-GFP
FEATURES             Location/Qualifiers
     source          1..6612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     intron          873..1005
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     988..1007
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     protein_bind    1048..1081
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    1127..1175
                     /note="synthetic polyadenylation signal"
     polyA_signal    1184..1660
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1383..1402)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1695..1714)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1739..1787
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1947..1980
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1987..2020)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(2071..2104)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(2158..2871,2872..2874,2875..2877))
                     /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             complement(join(2161..2871,2872..2874,2875..2877))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2203..2224)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2552..2571
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2811..2832
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2874..2883
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2927..2960
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3011..3044
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3053..3641
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3106..3126)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3524..3535)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3649..3873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3875..4015
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3875..4004
                     /label=AAV2 ITR
     rep_origin      4090..4545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4177..4196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4387..4408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4562..4581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4681..4703
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4741..4759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4827..4931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4932..5000,5001..5792)
                     /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(5150..5169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5963..6551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6452..6471
                     /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 gctagactgc agagggcccg cggccgcacg cgtttattca gaatgttatg
      181 cactcacagt ggtttggcat gcatctggtg aatttttttt aacgaaaaat tagtgttggt
      241 ttcgatgtat ggtagcattc tccctaacgt aatttgaata attcagcaaa gccccactac
      301 cagctgtact tctgcagcct cttccattct tttcagcatt ataattttgg ttaattttca
      361 attttaggtc ctacgtctct gcaatttgtg tatgaataac agaataattt ccctcttttg
      421 tttcgccttt cctgttcctg aatctaaata aagatggctt tttagtatta aaagtggaag
      481 aaaattacag gtaattatct ttgacggtaa aaacgctgta atcagcgggc tacatgaaaa
      541 attactctaa ttatggctgc atttaagaga atggaaaaaa accttcttgt ggataaaaac
      601 cttaaattgt ccccaatgtc tgcttcaaat tggatggcac tgcagctgga ggctttgttc
      661 agaattgatc ctggggagct acgaacccaa agtttcacag taggaagcta gatatcgttt
      721 aaacttcctg cagcccgggc tgggcataaa agtcagggca gagccatcta ttgcttacat
      781 ttgcttctag cctgcaagtc gaggagcgca gccttccaga agcagagcgc ggcgccttaa
      841 gctgcagaag ttggtcgtga ggcactgggc acgtaagtat caaggttaca agacaggttt
      901 aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg
      961 cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt
     1021 tcaattacag ctcttaagaa agctagcgaa gttcctattc tctagaaagt ataggaactt
     1081 ccctcagcac catggctagc ggcagcctcg gagtttgaat agatagaata aaatatcttt
     1141 attttcattc catctgtgtg ttggtttttt gtgtgagatc tacgggtggc atccctgtga
     1201 cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt
     1261 cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg
     1321 gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg
     1381 ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc
     1441 ctcctgggtt caagcgattc tcctgcctca ggctcccgag ttgttgggat tccaggcatg
     1501 catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc
     1561 caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct
     1621 gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgcctcagc taattgagta
     1681 ggggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     1741 gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt
     1801 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     1861 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     1921 agccaacgtc ggggcggcag gccgtggaag ttcctattct ctagaaagta taggaacttc
     1981 tccggaataa cttcgtatag gatactttat acgaagttat gcagaatggt agctggattg
     2041 tagctgctat tagcaatatg aaacctctta ataacttcgt atagcataca ttatacgaag
     2101 ttataacgcg ccactagtga attcgagctc ggtacccggg gatcctctag agtcgactta
     2161 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     2221 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg
     2281 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     2341 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     2401 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
     2461 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
     2521 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
     2581 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     2641 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt
     2701 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
     2761 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
     2821 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt
     2881 ggcgtcgacc tgcaggcatg caagctttac tagtggtggc gctagcataa cttcgtataa
     2941 agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat
     3001 ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatggcgcg ccaatcaacc
     3061 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3121 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3181 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3241 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3301 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3361 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3421 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     3481 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3541 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3601 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggatcct gtgccttcta
     3661 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3721 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3781 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3841 gcaggcatgc tggggatgcg gtgggctcta tggcaggaac ccctagtgat ggagttggcc
     3901 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     3961 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4021 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4081 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4141 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     4201 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4261 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4321 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4381 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     4441 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     4501 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4561 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4621 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4681 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     4741 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4801 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4861 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4921 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4981 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5041 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5101 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5161 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5221 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5281 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5341 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5401 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5461 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5521 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5581 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5641 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5701 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5761 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5821 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5881 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5941 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6001 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6061 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6121 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6181 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6241 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6301 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6361 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6421 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6481 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6541 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6601 ttgctcacat gt
//
