LOCUS       sequence-189728-        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Cre recombinase-activated ultrasensitive protein 
            calcium sensor from CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  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 6379)
  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.CAG.Flex.GCaMP6f.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..6379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..718
                     /label=chicken beta-actin promoter
     primer_bind     997..1019
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1081..1100
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1135..1168
                     /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    1231..1264
                     /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(1275..1718,1719..1724,1725..2159,2160..2177,
                     2178..2447,2448..2453,2454..2510,2511..2537,2538..2561,
                     2562..2564,2565..2597,2598..2600,2601..2618,2619..2627))
                     /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     1834..1853
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2093..2114
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2220..2241)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2522..2531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2624..2633
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2644..2677)
                     /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(2748..2781)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2823..3411
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2876..2896)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3294..3305)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3454..3575
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3465..3484)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3519..3538
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3737..3866
                     /label=AAV2 ITR
     rep_origin      complement(4103..4691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4183..4202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4862..5653,5654..5722))
                     /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     5485..5504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5723..5827)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5853..6308)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5990..6011)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6202..6221
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
      481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
      541 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
      601 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
      781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
      901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
      961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccccataact
     1141 tcgtataaag tatcctatac gaagttatat caaaatagga agaccaatgc ttcaccatcg
     1201 acccgaattg ccaagcatca ccatcgaccc ataacttcgt ataatgtatg ctatacgaag
     1261 ttatactaga gctttcactt cgctgtcatc atttgtacaa actcttcgta gtttacctga
     1321 ccatccccat cgatgtctgc ttccctgatc atttcatcaa cctcttcatc tgttaacttc
     1381 tctccaaggt ttgtcatcac gtggcgaagc tctgctgcac tgatgtagcc attgccatcc
     1441 ttatcaaaca caccgaacgc ttctctaatt tcttcttccg tgtccctgta tttcattttt
     1501 cttgccatca ttgtcaggaa ctcagggaag tcgattgtgc cgtcaccgtc ggcatctact
     1561 tcattgatca tgtcctgcag ctctgcttct gtggggttct gccccagaga ccgcatcacc
     1621 gtccccagct ccttggttgt tattgtccca tccccgtcct tgtcaaatag ggagaattcc
     1681 tctttaaatt ctgcgatctg ctcttcagtc agttggtccg gcaggttgta ctccagcttg
     1741 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc
     1801 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
     1861 aagatggtgc gctcctggat gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
     1921 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg
     1981 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg
     2041 taggtggcat cgccctcacc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
     2101 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg
     2161 ctccctccgg taccgccctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
     2221 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttcga aagtttggac
     2281 tgcacgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     2341 tgctggtagt ggtaggcgag ctgcacgccg ccgtcctcga tgttgtggcg gatcttgaag
     2401 ttcgccttga tgccgttctt ctgcttgtcg gccttgatat agacgttctc gagtgagctc
     2461 agccgaccta tagctctgac tgcgtgacct gtcttattcc acttacgacg tgatgagtcg
     2521 accatggtgg cgagatcctt atcgtcatcg tcgtacagat cccgacccat ttgctgtcca
     2581 ccagtcatgc tagccatacc atgatgatga tgatgatgag aacccatggt ggcggatcct
     2641 agtataactt cgtataggat actttatacg aagttatcat tgggattctt cctattttga
     2701 tccaagcatc accatcgacc ctctagtcca gatctcacca tcgacccata acttcgtata
     2761 gcatacatta tacgaagtta tgtccctcga agaggttcga attcgatatc aagcttatcg
     2821 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     2881 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     2941 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3001 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3061 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3121 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3181 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3241 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3301 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3361 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3421 gtcgacccgg gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     3481 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3541 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     3601 tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat
     3661 gtggtaaaat cgataaggat cttcctagag catggctacg tagataagta gcatggcggg
     3721 ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3781 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3841 gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag
     3901 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3961 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4021 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4621 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4681 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4741 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4801 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4861 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4921 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4981 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5041 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5101 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5161 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5221 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5281 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5341 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5401 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5461 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5521 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5581 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5641 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5701 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5761 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5821 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     5881 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5941 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     6001 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     6061 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     6121 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     6181 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     6241 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     6301 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     6361 ctcttcgcta ttacgccag
//
