LOCUS       sequence-355044-        6362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-OFF 
            / 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 6362)
  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 6362)
  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-VTKD6-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6362
                     /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    complement(1048..1081)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1082..1115
                     /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)"
     CDS             complement(join(1250..1693,1694..1699,1700..2134,2135..2152,
                     2153..2422,2423..2428,2429..2485,2486..2512,2513..2536,
                     2537..2539,2540..2572,2573..2575,2576..2593,2594..2602))
                     /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     1809..1828
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2068..2089
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2195..2216)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2497..2506
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2599..2608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2643..2676)
                     /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(2761..2794)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2803..3391
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2856..2876)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3274..3285)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3399..3623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3625..3765
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3625..3754
                     /label=AAV2 ITR
     rep_origin      3840..4295
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3927..3946)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4137..4158
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4312..4331)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4431..4453
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4491..4509)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4577..4681
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4682..4750,4751..5542)
                     /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(4900..4919)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5713..6301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6202..6221
                     /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 agctagcata acttcgtata gcatacatta tacgaagtta
     1081 tgaagttcct attctctaga aagtatagga acttcgcaga atggtagctg gattgtagct
     1141 gctattagca atatgaaacc tcttagaagt tcctattctt caaaaggtat aggaacttca
     1201 acgcgcccta gaattcgagc tcggtacccg gggatcctct agagtcgact cacttcgctg
     1261 tcatcatttg tacaaactct tcgtagttta cctgaccatc cccatcgatg tctgcttccc
     1321 tgatcatttc atcaacctct tcatctgtta acttctctcc aaggtttgtc atcacgtggc
     1381 gaagctctgc tgcactgatg tagccattgc catccttatc aaacacaccg aacgcttctc
     1441 taatttcttc ttccgtgtcc ctgtatttca tttttcttgc catcattgtc aggaactcag
     1501 ggaagtcgat tgtgccgtca ccgtcggcat ctacttcatt gatcatgtcc tgcagctctg
     1561 cttctgtggg gttctgcccc agagaccgca tcaccgtccc cagctccttg gttgttattg
     1621 tcccatcccc gtccttgtca aatagggaga attcctcttt aaattctgcg atctgctctt
     1681 cagtcagttg gtccggcagg ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
     1741 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
     1801 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggatgtagc
     1861 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga
     1921 agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc
     1981 cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcaccctcgc
     2041 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
     2101 ccccggtgaa cagctcctcg cccttgctca ccatgctccc tccggtaccg cccttgtaca
     2161 gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat
     2221 cgcgcttctc gttggggtct ttcgaaagtt tggactgcac gctcaggtag tggttgtcgg
     2281 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtag gcgagctgca
     2341 cgccgccgtc ctcgatgttg tggcggatct tgaagttcgc cttgatgccg ttcttctgct
     2401 tgtcggcctt gatatagacg ttctcgagtg agctcagccg acctatagct ctgactgcgt
     2461 gacctgtctt attccactta cgacgtgatg agtcgaccat ggtggcgaga tccttatcgt
     2521 catcgtcgta cagatcccga cccatttgct gtccaccagt catgctagcc ataccatgat
     2581 gatgatgatg atgagaaccc atggtggcgt cgacctgcag gcatgcaagc ttggtggcta
     2641 gcgaagttcc tatactttct agagaatagg aacttcttgc cttaacccag aaattatcac
     2701 tgttattctt tagaatggtg caaagagaag ttcctatacc ttttgaagaa taggaacttc
     2761 ataacttcgt atagcataca ttatacgaag ttatggcgcg ccaatcaacc tctggattac
     2821 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     2881 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     2941 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3001 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3061 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3121 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3181 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     3241 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3301 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3361 agtcggatct ccctttgggc cgcctccccg cctggatcct gtgccttcta gttgccagcc
     3421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3601 tggggatgcg gtgggctcta tggcaggaac ccctagtgat ggagttggcc actccctctc
     3661 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3721 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     3781 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     3841 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3901 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3961 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4021 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4081 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4141 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4201 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4261 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4321 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4381 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4441 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4501 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4561 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4621 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4681 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4741 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4801 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4861 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4921 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4981 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5041 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5101 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5161 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5221 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5281 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5341 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5401 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5461 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5521 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5581 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5641 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5701 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5761 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5821 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5881 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5941 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6001 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6061 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6121 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6181 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6241 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6301 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6361 gt
//
