LOCUS       sequence-189734-        6060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of ultrasensitive protein calcium sensor from CAG 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6060)
  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 6060)
  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.GCaMP6m.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..6060
                     /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"
     regulatory      1133..1142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1139..1147,1148..1165,1166..1168,1169..1201,1202..1204,
                     1205..1228,1229..1255,1256..1312,1313..1318,1319..1588,
                     1589..1606,1607..2041,2042..2047,2048..2491)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6m
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKGSYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1235..1244
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1525..1546
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1652..1673)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1913..1932)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2504..3092
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2557..2577)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2975..2986)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3135..3256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3146..3165)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3200..3219
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3418..3547
                     /label=AAV2 ITR
     rep_origin      complement(3784..4372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3864..3883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4543..5334,5335..5403))
                     /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     5166..5185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5404..5508)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5534..5989)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5671..5692)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5883..5902
                     /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 ccgccaccat
     1141 gggttctcat catcatcatc atcatggtat ggctagcatg actggtggac agcaaatggg
     1201 tcgggatctg tacgacgatg acgataagga tctcgccacc atggtcgact catcacgtcg
     1261 taagtggaat aagacaggtc acgcagtcag agctataggt cggctgagct cactcgagaa
     1321 cgtctatatc aaggccgaca agcagaagaa cggcatcaag gcgaacttca agatccgcca
     1381 caacatcgag gacggcggcg tgcagctcgc ctaccactac cagcagaaca cccccatcgg
     1441 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca aactttcgaa
     1501 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1561 cactctcggc atggacgagc tgtacaaggg cggtaccgga gggagcatgg tgagcaaggg
     1621 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1681 ccacaagttc agcgtgtccg gcgagggtga gggcgatgcc acctacggca agctgaccct
     1741 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1801 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1861 caagtccgcc atgcccgaag gctacatcca ggagcgcacc atcttcttca aggacgacgg
     1921 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1981 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2041 cctgccggac caactgactg aagagcagat cgcagaattt aaagaggctt tctccctatt
     2101 tgacaaggac ggggatggga caataacaac caaggagctg gggacggtga tgcggtctct
     2161 ggggcagaac cccacagaag cagagctgca ggacatgatc aatgaagtag atgccgacgg
     2221 tgacggcaca atcgacttcc ctgagttcct gacaatgatg gcaagaaaag ggagctacag
     2281 ggacacggaa gaagaaatta gagaagcgtt cggtgtgttt gataaggatg gcaatggcta
     2341 catcagtgca gcagagcttc gccacgtgat gacaaacctt ggagagaagt taacagatga
     2401 agaggttgat gaaatgatca gggaagcaga catcgatggg gatggtcagg taaactacga
     2461 agagtttgta caaatgatga cagcgaagtg aaagcttatc gataatcaac ctctggatta
     2521 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2581 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2641 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2701 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2761 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     2821 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2881 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     2941 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3001 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3061 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacccg ggcggccgct
     3121 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3181 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3241 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3301 gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga
     3361 tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg
     3421 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     3481 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     3541 cgcgcagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3601 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3661 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3721 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3781 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3841 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3901 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3961 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4021 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4081 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4141 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4201 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4261 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4321 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4381 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4441 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4501 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4561 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4621 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4681 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4741 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4801 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4861 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4921 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4981 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5041 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5101 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5161 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5221 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5281 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5341 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5401 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5461 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5521 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5581 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5641 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5701 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     5761 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5821 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5881 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5941 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     6001 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
//
