LOCUS       sequence-198271-        5910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Cre recombinase-activated ultrasensitive protein 
            calcium sensor from Synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  AUTHORS   Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, 
            Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, 
            Kim DS
  TITLE     Ultrasensitive fluorescent proteins for imaging neuronal activity.
  JOURNAL   Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038/nature12354.
  PUBMED    23868258
REFERENCE   2  (bases 1 to 5910)
  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"; doi: "10"; journalName: 
            "Nature"; date: "2013-07-18- 18"; volume: "499"; issue: "7458"; 
            pages: "295-300"
COMMENT     Sequence Label: pAAV.Syn.Flex.GCaMP6f.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..5910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   50..179
                     /label=AAV2 ITR
     promoter        261..708
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    738..771
                     /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    834..867
                     /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(878..1321,1322..1327,1328..1762,1763..1780,
                     1781..2050,2051..2056,2057..2113,2114..2140,2141..2164,
                     2165..2167,2168..2200,2201..2203,2204..2221,2222..2230))
                     /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     1437..1456
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1696..1717
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1823..1844)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2125..2134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2227..2236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2247..2280)
                     /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(2351..2384)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2426..3014
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2479..2499)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2897..2908)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3017..3033)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3018..3034)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3063..3184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3074..3093)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3128..3147
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3683..4271)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3763..3782)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4442..5233,5234..5302))
                     /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     5065..5084
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5303..5407)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5433..5888)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5570..5591)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5782..5801
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tgcgcgctcg
       61 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc
      121 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt
      181 gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta ggaagatctc
      241 tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag gcggggtggg
      301 ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa
      361 attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca
      421 ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc
      481 cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac tccccttccc
      541 ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc
      601 gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct
      661 cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc gaattcaagc
      721 tgctagcaag gatccccata acttcgtata aagtatccta tacgaagtta tatcaaaata
      781 ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cccataactt
      841 cgtataatgt atgctatacg aagttatact agagctttca cttcgctgtc atcatttgta
      901 caaactcttc gtagtttacc tgaccatccc catcgatgtc tgcttccctg atcatttcat
      961 caacctcttc atctgttaac ttctctccaa ggtttgtcat cacgtggcga agctctgctg
     1021 cactgatgta gccattgcca tccttatcaa acacaccgaa cgcttctcta atttcttctt
     1081 ccgtgtccct gtatttcatt tttcttgcca tcattgtcag gaactcaggg aagtcgattg
     1141 tgccgtcacc gtcggcatct acttcattga tcatgtcctg cagctctgct tctgtggggt
     1201 tctgccccag agaccgcatc accgtcccca gctccttggt tgttattgtc ccatccccgt
     1261 ccttgtcaaa tagggagaat tcctctttaa attctgcgat ctgctcttca gtcagttggt
     1321 ccggcaggtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
     1381 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
     1441 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gatgtagcct tcgggcatgg
     1501 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc
     1561 cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
     1621 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc accctcgccg gacacgctga
     1681 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
     1741 gctcctcgcc cttgctcacc atgctccctc cggtaccgcc cttgtacagc tcgtccatgc
     1801 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
     1861 tggggtcttt cgaaagtttg gactgcacgc tcaggtagtg gttgtcgggc agcagcacgg
     1921 ggccgtcgcc gatgggggtg ttctgctggt agtggtaggc gagctgcacg ccgccgtcct
     1981 cgatgttgtg gcggatcttg aagttcgcct tgatgccgtt cttctgcttg tcggccttga
     2041 tatagacgtt ctcgagtgag ctcagccgac ctatagctct gactgcgtga cctgtcttat
     2101 tccacttacg acgtgatgag tcgaccatgg tggcgagatc cttatcgtca tcgtcgtaca
     2161 gatcccgacc catttgctgt ccaccagtca tgctagccat accatgatga tgatgatgat
     2221 gagaacccat ggtggcggat cctagtataa cttcgtatag gatactttat acgaagttat
     2281 cattgggatt cttcctattt tgatccaagc atcaccatcg accctctagt ccagatctca
     2341 ccatcgaccc ataacttcgt atagcataca ttatacgaag ttatgtccct cgaagaggtt
     2401 cgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     2461 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2521 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2581 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2641 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2701 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2761 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     2821 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     2881 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     2941 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3001 ggccgcctcc ccgcatcgat accgtcgacc tcgacccggg cggccgcttc gagcagacat
     3061 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3121 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     3181 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     3241 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc
     3301 atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga
     3361 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgacgggctt tgcccgggcg
     3421 gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag
     3481 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3541 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3601 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3661 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3721 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3781 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3841 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3901 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3961 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4021 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4081 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4141 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4201 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4261 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4321 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4381 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4441 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4501 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4561 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4621 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4681 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4741 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4801 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4861 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4921 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4981 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5041 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5101 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5161 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5221 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5281 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5341 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5401 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     5461 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5521 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5581 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5641 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5701 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5761 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5821 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5881 gggcgcgtcc cattcgccat tcaggctgcg
//
