LOCUS       sequence-354870-        6826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-ON /
            FLP-ON) of GCaMP6f in neurons under the control of the hSyn1 
            promoter (Kugler et al 2003).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6826)
  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 6826)
  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-VTKS4-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(6..17)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    131..355
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   357..497
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   357..486
                     /label=AAV2 ITR
     rep_origin      572..1027
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(659..678)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     869..890
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1044..1063)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1163..1185
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1223..1241)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1309..1413
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1414..1482,1483..2274)
                     /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(1632..1651)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2445..3033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2934..2953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3095..3224
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3252..3699
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    3723..3756
                     /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    3802..3850
                     /note="synthetic polyadenylation signal"
     polyA_signal    3859..4335
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4058..4077)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4370..4389)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4414..4462
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4622..4655
                     /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(4662..4695)
                     /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(4746..4779)
                     /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(4833..5276,5277..5282,5283..5717,5718..5735,
                     5736..6005,6006..6011,6012..6068,6069..6095,6096..6119,
                     6120..6122,6123..6155,6156..6158,6159..6176,6177..6185))
                     /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     5392..5411
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5651..5672
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5778..5799)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      6080..6089
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      6182..6191
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6235..6268
                     /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    6319..6352
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(6361..6826,1..123)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6414..6434)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
       61 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
      121 cgcctggatc ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
      181 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
      241 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
      301 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcagga
      361 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
      421 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
      481 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      541 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
      601 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
      661 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      721 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      781 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      841 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
      901 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
      961 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     1021 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1081 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1141 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1201 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1261 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1321 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1381 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1441 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1501 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1561 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1621 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1681 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1741 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     1801 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     1861 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     1921 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     1981 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2041 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2101 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2161 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2221 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2281 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2341 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2401 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2461 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2521 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2581 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2641 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2701 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2761 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2821 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2881 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2941 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3001 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3061 ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg
     3121 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     3181 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgctaga ctgcagaggg
     3241 ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg gggtgcctac
     3301 ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca aattgcgcat
     3361 cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc actgccagct
     3421 tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg ccgcctcagc
     3481 actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc cggccacctt
     3541 ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg cgagataggg
     3601 gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc tcagtctgcg
     3661 gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt cagatatcgt ttaaactccg
     3721 gagaagttcc tattctctag aaagtatagg aacttccctc agcaccatgg ctagcggcag
     3781 cctcggagtt tgaatagata gaataaaata tctttatttt cattccatct gtgtgttggt
     3841 tttttgtgtg agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     3901 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     3961 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     4021 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     4081 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     4141 cctcaggctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     4201 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     4261 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     4321 cccttccctg tccttctgcc tcagctaatt gagtaggggg gaggctaact gaaacacgga
     4381 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     4441 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     4501 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     4561 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccgt
     4621 ggaagttcct attctctaga aagtatagga acttcaccgg aataacttcg tataggatac
     4681 tttatacgaa gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc
     4741 tcttaataac ttcgtatagc atacattata cgaagttata acgcgccact agtgaattcg
     4801 agctcggtac ccggggatcc tctagagtcg actcacttcg ctgtcatcat ttgtacaaac
     4861 tcttcgtagt ttacctgacc atccccatcg atgtctgctt ccctgatcat ttcatcaacc
     4921 tcttcatctg ttaacttctc tccaaggttt gtcatcacgt ggcgaagctc tgctgcactg
     4981 atgtagccat tgccatcctt atcaaacaca ccgaacgctt ctctaatttc ttcttccgtg
     5041 tccctgtatt tcatttttct tgccatcatt gtcaggaact cagggaagtc gattgtgccg
     5101 tcaccgtcgg catctacttc attgatcatg tcctgcagct ctgcttctgt ggggttctgc
     5161 cccagagacc gcatcaccgt ccccagctcc ttggttgtta ttgtcccatc cccgtccttg
     5221 tcaaataggg agaattcctc tttaaattct gcgatctgct cttcagtcag ttggtccggc
     5281 aggttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc
     5341 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag
     5401 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt agccttcggg catggcggac
     5461 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag
     5521 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac
     5581 ttcagggtca gcttgccgta ggtggcatcg ccctcaccct cgccggacac gctgaacttg
     5641 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc
     5701 tcgcccttgc tcaccatgct ccctccggta ccgcccttgt acagctcgtc catgccgaga
     5761 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     5821 tctttcgaaa gtttggactg cacgctcagg tagtggttgt cgggcagcag cacggggccg
     5881 tcgccgatgg gggtgttctg ctggtagtgg taggcgagct gcacgccgcc gtcctcgatg
     5941 ttgtggcgga tcttgaagtt cgccttgatg ccgttcttct gcttgtcggc cttgatatag
     6001 acgttctcga gtgagctcag ccgacctata gctctgactg cgtgacctgt cttattccac
     6061 ttacgacgtg atgagtcgac catggtggcg agatccttat cgtcatcgtc gtacagatcc
     6121 cgacccattt gctgtccacc agtcatgcta gccataccat gatgatgatg atgatgagaa
     6181 cccatggtgg cgtcgacctg caggcatgca agctttacta gtggtggcgc tagcataact
     6241 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc
     6301 tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atggcgcgcc
     6361 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     6421 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     6481 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     6541 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     6601 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     6661 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     6721 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     6781 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacg
//
