LOCUS       sequence-355075-        7245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-ON /
            FLP-ON) of GCaMP6f 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 7245)
  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 7245)
  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-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..7245
                     /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..2601,2602..2607,2608..3042,3043..3060,
                     3061..3330,3331..3336,3337..3393,3394..3420,3421..3444,
                     3445..3447,3448..3480,3481..3483,3484..3501,3502..3510))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2717..2736
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2976..2997
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3103..3124)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3405..3414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3507..3516
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3560..3593
                     /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    3644..3677
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3686..4274
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3739..3759)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4157..4168)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4282..4506
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4508..4648
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4508..4637
                     /label=AAV2 ITR
     rep_origin      4723..5178
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4810..4829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5020..5041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5195..5214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5314..5336
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5374..5392)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5460..5564
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5565..5633,5634..6425)
                     /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(5783..5802)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6596..7184
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7085..7104
                     /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 agtcgactca
     2161 cttcgctgtc atcatttgta caaactcttc gtagtttacc tgaccatccc catcgatgtc
     2221 tgcttccctg atcatttcat caacctcttc atctgttaac ttctctccaa ggtttgtcat
     2281 cacgtggcga agctctgctg cactgatgta gccattgcca tccttatcaa acacaccgaa
     2341 cgcttctcta atttcttctt ccgtgtccct gtatttcatt tttcttgcca tcattgtcag
     2401 gaactcaggg aagtcgattg tgccgtcacc gtcggcatct acttcattga tcatgtcctg
     2461 cagctctgct tctgtggggt tctgccccag agaccgcatc accgtcccca gctccttggt
     2521 tgttattgtc ccatccccgt ccttgtcaaa tagggagaat tcctctttaa attctgcgat
     2581 ctgctcttca gtcagttggt ccggcaggtt gtactccagc ttgtgcccca ggatgttgcc
     2641 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     2701 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     2761 gatgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     2821 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg
     2881 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     2941 accctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     3001 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atgctccctc cggtaccgcc
     3061 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     3121 catgtgatcg cgcttctcgt tggggtcttt cgaaagtttg gactgcacgc tcaggtagtg
     3181 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtaggc
     3241 gagctgcacg ccgccgtcct cgatgttgtg gcggatcttg aagttcgcct tgatgccgtt
     3301 cttctgcttg tcggccttga tatagacgtt ctcgagtgag ctcagccgac ctatagctct
     3361 gactgcgtga cctgtcttat tccacttacg acgtgatgag tcgaccatgg tggcgagatc
     3421 cttatcgtca tcgtcgtaca gatcccgacc catttgctgt ccaccagtca tgctagccat
     3481 accatgatga tgatgatgat gagaacccat ggtggcgtcg acctgcaggc atgcaagctt
     3541 tactagtggt ggcgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt
     3601 aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt
     3661 atgctatacg aagttatggc gcgccaatca acctctggat tacaaaattt gtgaaagatt
     3721 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3781 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3841 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3901 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3961 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4021 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4081 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     4141 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4201 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4261 ggccgcctcc ccgcctggat cctgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4321 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4381 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4441 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     4501 ctatggcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4561 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4621 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4681 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     4741 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     4801 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4861 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4921 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4981 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5041 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     5101 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5161 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5221 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5281 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5341 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5401 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5461 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5521 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5581 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5641 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5701 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5761 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5821 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5881 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5941 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6001 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6061 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6121 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6181 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6241 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6301 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6361 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6421 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6481 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6541 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6601 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6661 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6721 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6781 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6841 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6901 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6961 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7021 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7081 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7141 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7201 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
