LOCUS       sequence-461658-        6150 bp    DNA     circular SYN 14-APR-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 6150)
  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 6150)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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.GCaMP6s.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..6150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..717
                     /label=chicken beta-actin promoter
     primer_bind     996..1018
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1080..1099
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1132..1141
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1138..1146,1147..1164,1165..1167,1168..1200,1201..1203,
                     1204..1227,1228..1254,1255..1311,1312..1317,1318..1587,
                     1588..1605,1606..2040,2041..2046,2047..2490)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1234..1243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1524..1545
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1651..1672)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1912..1931)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(2501..2534)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2594..3182
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2647..2667)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3065..3076)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3225..3346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3236..3255)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3290..3309
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3508..3637
                     /label=AAV2 ITR
     rep_origin      complement(3874..4462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3954..3973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4633..5424,5425..5493))
                     /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     5256..5275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5494..5598)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5624..6079)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5761..5782)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5973..5992
                     /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 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      601 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      661 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      721 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      781 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      841 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      901 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcggggct gtccgcgggg
      961 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1021 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     1081 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc cgccaccatg
     1141 ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
     1201 cgggatctgt acgacgatga cgataaggat ctcgccacca tggtcgactc atcacgtcgt
     1261 aagtggaata agacaggtca cgcagtcaga gctataggtc ggctgagctc actcgagaac
     1321 gtctatatca aggccgacaa gcagaagaac ggcatcaagg cgaacttcca catccgccac
     1381 aacatcgagg acggcggcgt gcagctcgcc taccactacc agcagaacac ccccatcggc
     1441 gacggccccg tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa actttcgaaa
     1501 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1561 actctcggca tggacgagct gtacaagggc ggtaccggag ggagcatggt gagcaagggc
     1621 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1681 cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca cctacggcaa gctgaccctg
     1741 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1801 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1861 aagtccgcca tgcccgaagg ctacatccag gagcgcacca tcttcttcaa ggacgacggc
     1921 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1981 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2041 ctgccggacc aactgactga agagcagatc gcagaattta aagaggcttt ctccctattt
     2101 gacaaggacg gggatgggac aataacaacc aaggagctgg ggacggtgat gcggtctctg
     2161 gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt
     2221 gacggcacaa tcgacttccc tgagttcctg acaatgatgg caagaaaaat gaaatacagg
     2281 gacacggaag aagaaattag agaagcgttc ggtgtgtttg ataaggatgg caatggctac
     2341 atcagtgcag cagagcttcg ccacgtgatg acaaaccttg gagagaagtt aacagatgaa
     2401 gaggttgatg aaatgatcag ggaagcagac atcgatgggg atggtcaggt aaactacgaa
     2461 gagtttgtac aaatgatgac agcgaagtga aagctctagt ataacttcgt atagcataca
     2521 ttatacgaag ttatgggtcg atggtgatgc ttggcaattc gggtcgatgg tgaagcatat
     2581 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     2641 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     2701 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     2761 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     2821 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     2881 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     2941 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3001 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     3061 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     3121 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     3181 gcatcgatac cgtcgacccg ggcggccgct tcgagcagac atgataagat acattgatga
     3241 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     3301 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     3361 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa
     3421 cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt
     3481 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct
     3541 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     3601 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa tcggccaacg
     3661 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3721 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3781 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3841 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3901 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3961 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4021 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4081 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4141 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4201 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4261 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4321 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4381 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4441 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4501 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4561 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4621 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4681 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4741 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4801 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4861 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4921 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4981 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5041 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5101 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5161 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5221 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5281 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5341 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5401 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5461 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5521 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5581 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     5641 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5701 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5761 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5821 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5881 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5941 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     6001 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     6061 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     6121 tcggtgcggg cctcttcgct attacgccag
//
