LOCUS       sequence-354869-        5943 bp    DNA     circular SYN 15-APR-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-OFF 
            / 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 5943)
  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 5943)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-VTKS6-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..5943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   193..322
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        350..797
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(821..854)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    855..888
                     /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(1023..1466,1467..1472,1473..1907,1908..1925,
                     1926..2195,2196..2201,2202..2258,2259..2285,2286..2309,
                     2310..2312,2313..2345,2346..2348,2349..2366,2367..2375))
                     /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     1582..1601
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1841..1862
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1968..1989)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2270..2279
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2372..2381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2416..2449)
                     /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(2534..2567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2576..3164
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2629..2649)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3047..3058)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3172..3396
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3398..3538
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3398..3527
                     /label=AAV2 ITR
     rep_origin      3613..4068
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3700..3719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3910..3931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4085..4104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4204..4226
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4264..4282)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4350..4454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4455..4523,4524..5315)
                     /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(4673..4692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5486..5943,1..131)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
       61 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      181 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
      241 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      301 aactccatca ctaggggttc ctgctagact gcagagggcc ctgcgtatga gtgcaagtgg
      361 gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac cccgacccac
      421 tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag agggggaggg
      481 gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt
      541 cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac
      601 tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg
      661 gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc gaccatctgc
      721 gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt
      781 cgtgcctgag agcgcagtca gatatcgttt aaactccgga ataacttcgt atagcataca
      841 ttatacgaag ttatgaagtt cctattctct agaaagtata ggaacttcgc agaatggtag
      901 ctggattgta gctgctatta gcaatatgaa acctcttaga agttcctatt cttcaaaagg
      961 tataggaact tcaacgcgcc ctagaattcg agctcggtac ccggggatcc tctagagtcg
     1021 actcacttcg ctgtcatcat ttgtacaaac tcttcgtagt ttacctgacc atccccatcg
     1081 atgtctgctt ccctgatcat ttcatcaacc tcttcatctg ttaacttctc tccaaggttt
     1141 gtcatcacgt ggcgaagctc tgctgcactg atgtagccat tgccatcctt atcaaacaca
     1201 ccgaacgctt ctctaatttc ttcttccgtg tccctgtatt tcatttttct tgccatcatt
     1261 gtcaggaact cagggaagtc gattgtgccg tcaccgtcgg catctacttc attgatcatg
     1321 tcctgcagct ctgcttctgt ggggttctgc cccagagacc gcatcaccgt ccccagctcc
     1381 ttggttgtta ttgtcccatc cccgtccttg tcaaataggg agaattcctc tttaaattct
     1441 gcgatctgct cttcagtcag ttggtccggc aggttgtact ccagcttgtg ccccaggatg
     1501 ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc
     1561 tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc
     1621 tcctggatgt agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg
     1681 gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc
     1741 acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg
     1801 ccctcaccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
     1861 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatgct ccctccggta
     1921 ccgcccttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     1981 aggaccatgt gatcgcgctt ctcgttgggg tctttcgaaa gtttggactg cacgctcagg
     2041 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     2101 taggcgagct gcacgccgcc gtcctcgatg ttgtggcgga tcttgaagtt cgccttgatg
     2161 ccgttcttct gcttgtcggc cttgatatag acgttctcga gtgagctcag ccgacctata
     2221 gctctgactg cgtgacctgt cttattccac ttacgacgtg atgagtcgac catggtggcg
     2281 agatccttat cgtcatcgtc gtacagatcc cgacccattt gctgtccacc agtcatgcta
     2341 gccataccat gatgatgatg atgatgagaa cccatggtgg cgtcgacctg caggcatgca
     2401 agcttggtgg ctagcgaagt tcctatactt tctagagaat aggaacttct tgccttaacc
     2461 cagaaattat cactgttatt ctttagaatg gtgcaaagag aagttcctat accttttgaa
     2521 gaataggaac ttcataactt cgtatagcat acattatacg aagttatggc gcgccaatca
     2581 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2641 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2701 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     2761 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     2821 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     2881 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     2941 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
     3001 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3061 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3121 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggat cctgtgcctt
     3181 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3241 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3301 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3361 atagcaggca tgctggggat gcggtgggct ctatggcagg aacccctagt gatggagttg
     3421 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3481 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3541 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3601 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3661 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     3721 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3781 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3841 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3901 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     3961 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     4021 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4081 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4141 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4201 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4261 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4321 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4381 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4441 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4501 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4561 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4621 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4681 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4741 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4801 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4861 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4921 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4981 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5041 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5101 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5161 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5221 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5281 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5341 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5401 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5461 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5521 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5581 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5641 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5701 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5761 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5821 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5881 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5941 tcg
//
