LOCUS       sequence-355032-        6380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-ON /
            FLP-OFF) 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 6380)
  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 6380)
  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-VTKD5-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6380
                     /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)"
     protein_bind    complement(1088..1121)
                     /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(1172..1205)
                     /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(1259..1702,1703..1708,1709..2143,2144..2161,
                     2162..2431,2432..2437,2438..2494,2495..2521,2522..2545,
                     2546..2548,2549..2581,2582..2584,2585..2602,2603..2611))
                     /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     1818..1837
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2077..2098
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2204..2225)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2506..2515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2608..2617
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2661..2694
                     /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    2745..2778
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2779..2812
                     /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)"
     misc_feature    2821..3409
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2874..2894)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3292..3303)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3417..3641
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3643..3783
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3643..3772
                     /label=AAV2 ITR
     rep_origin      3858..4313
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3945..3964)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4155..4176
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4330..4349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4449..4471
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4509..4527)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4595..4699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4700..4768,4769..5560)
                     /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(4918..4937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5731..6319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6220..6239
                     /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 ctccggaata acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt
     1141 gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa
     1201 gttataacgc gccactagtg aattcgagct cggtacccgg ggatcctcta gagtcgactc
     1261 acttcgctgt catcatttgt acaaactctt cgtagtttac ctgaccatcc ccatcgatgt
     1321 ctgcttccct gatcatttca tcaacctctt catctgttaa cttctctcca aggtttgtca
     1381 tcacgtggcg aagctctgct gcactgatgt agccattgcc atccttatca aacacaccga
     1441 acgcttctct aatttcttct tccgtgtccc tgtatttcat ttttcttgcc atcattgtca
     1501 ggaactcagg gaagtcgatt gtgccgtcac cgtcggcatc tacttcattg atcatgtcct
     1561 gcagctctgc ttctgtgggg ttctgcccca gagaccgcat caccgtcccc agctccttgg
     1621 ttgttattgt cccatccccg tccttgtcaa atagggagaa ttcctcttta aattctgcga
     1681 tctgctcttc agtcagttgg tccggcaggt tgtactccag cttgtgcccc aggatgttgc
     1741 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
     1801 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
     1861 ggatgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
     1921 agcggctgaa gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg
     1981 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     2041 caccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
     2101 tgggcaccac cccggtgaac agctcctcgc ccttgctcac catgctccct ccggtaccgc
     2161 ccttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     2221 ccatgtgatc gcgcttctcg ttggggtctt tcgaaagttt ggactgcacg ctcaggtagt
     2281 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtagg
     2341 cgagctgcac gccgccgtcc tcgatgttgt ggcggatctt gaagttcgcc ttgatgccgt
     2401 tcttctgctt gtcggccttg atatagacgt tctcgagtga gctcagccga cctatagctc
     2461 tgactgcgtg acctgtctta ttccacttac gacgtgatga gtcgaccatg gtggcgagat
     2521 ccttatcgtc atcgtcgtac agatcccgac ccatttgctg tccaccagtc atgctagcca
     2581 taccatgatg atgatgatga tgagaaccca tggtggcgtc gacctgcagg catgcaagct
     2641 ttactagtgg tggcgctagc ataacttcgt ataaagtatc ctatacgaag ttatttgcct
     2701 taacccagaa attatcactg ttattcttta gaatggtgca aagaataact tcgtataatg
     2761 tatgctatac gaagttatga agttcctatt ctctagaaag tataggaact tcggcgcgcc
     2821 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     2881 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     2941 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3001 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3061 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3121 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3181 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     3241 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3301 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3361 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggatcctgt
     3421 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3481 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3541 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3601 agacaatagc aggcatgctg gggatgcggt gggctctatg gcaggaaccc ctagtgatgg
     3661 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3721 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3781 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3841 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3901 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     3961 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4021 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     4081 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4141 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     4201 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     4261 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4321 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4381 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4441 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4501 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4561 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4621 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4681 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4741 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4801 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4861 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4921 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4981 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5041 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5101 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5161 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5221 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5281 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5341 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5401 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5461 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5521 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5581 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5641 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5701 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5761 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5821 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5881 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5941 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6001 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6061 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6361 ctggcctttt gctcacatgt
//
