LOCUS       sequence-271272-        6696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for calcium imaging and photoactivation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6696)
  AUTHORS   Lee D, Kume M, Holy TE
  TITLE     Sensory coding mechanisms revealed by optical tagging of 
            physiologically defined neuronal types.
  JOURNAL   Science. 2019 Dec 13;366(6471):1384-1389. doi: 
            10.1126/science.aax8055.
  PUBMED    31831669
REFERENCE   2  (bases 1 to 6696)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2019-12-13- 13"; volume: "366"; 
            issue: "6471"; pages: "1384-1389"
COMMENT     Sequence Label: pAAV2-GCaMP5g-P2A-PAmCherry
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        223..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1008..1027
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1082..1091
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1088..1096,1097..1114,1115..1117,1118..1150,1151..1153,
                     1154..1177,1178..1204,1205..1261,1262..1267,1268..1537,
                     1538..1555,1556..1990,1991..1996,1997..2437)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             join(1088..1096,1097..1114,1115..1117,1118..1150,1151..1153,
                     1154..1177,1178..1204,1205..1261,1262..1267,1268..1537,
                     1538..1555,1556..1990,1991..1996,1997..2437)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1184..1193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1474..1495
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1601..1622)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1862..1881)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2453..2509
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      2516..2525
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2522..3232
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein"
                     /label=PAmCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2667..2685)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2935..2954
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3258..3379
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3269..3288)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3323..3342
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3465..3482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3471..3678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3708..3848
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3708..3837
                     /label=AAV2 ITR
     rep_origin      3934..4389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4021..4040)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4231..4252
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4456..4475)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4575..4597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4635..4653)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4721..4825
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4826..4894,4895..5686)
                     /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(5044..5063)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5857..6445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6346..6365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtagatct gaattcggta cctagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      541 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      601 tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggcggaga ggtgcggcgg
      661 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      721 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      781 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      841 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat
      901 gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagctagagc
      961 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     1021 tattgtgctg tctcatcatt ttggcaaaga attcctcgaa gatccgaagg ggttcaagct
     1081 tgccaccatg ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca
     1141 gcaaatgggt cgggatctgt acgacgatga cgataaggat ctcgccacca tggtcgactc
     1201 atcacgtcgt aagtggaata agacaggtca cgcagtcaga gctataggtc ggctgagctc
     1261 actcgagaac gtctatatca aggccgacaa gcagaagaac ggcatcaagg cgaacttcaa
     1321 gatccgccac aacatcgagg acggcggcgt gcagctcgcc taccactacc agcagaacac
     1381 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa
     1441 actttcgaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1501 cgccgggatc actctcggca tggacgagct gtacaagggc ggtaccggag ggagcatggt
     1561 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1621 cgtaaacggc cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca cctacggcaa
     1681 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1741 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1801 cgacttcttc aagtccgcca tgcccgaagg ctacatccag gagcgcacca tcttcttcaa
     1861 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1921 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1981 ggagtacaac ctgccggacc aactgactga agagcagatc gcagaattta aagaggcttt
     2041 ctccctattt gacaaggacg gggatgggac aataacaacc aaggagctgg ggacggtgat
     2101 gcggtctctg gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga
     2161 tgccgacggt gacggcacaa tcgacttccc tgagttcctg acaatgatgg caagaaaaat
     2221 gaaatacaca gacagtgaag aagaaattag agaagcgttc cgtgtgtttg ataaggatgg
     2281 caatggctac atcagtgcag cagagcttcg ccacgtgatg acaaaccttg gagagaagtt
     2341 aacagatgaa gaggttgatg aaatgatcag ggaagcagac atcgatgggg atggtcaggt
     2401 aaactacgaa gagtttgtac aaatgatgac agcgaaggaa ttcggaagcg gagctactaa
     2461 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggacctg gatccgccac
     2521 catggtgagc aagggcgagg aggataacat ggccatcatt aaggagttca tgcgcttcaa
     2581 ggtgcacatg gaggggtccg tgaacggcca cgtgttcgag atcgagggcg agggcgaggg
     2641 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     2701 cttcacctgg gacatcctgt cccctcaatt catgtacggc tccaatgcct acgtgaagca
     2761 ccccgccgac atccccgact actttaagct gtccttcccc gagggcttca agtgggagcg
     2821 cgtgatgaaa ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     2881 cggtgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     2941 aatgcagaag aagaccatgg gctgggaggc cctctccgag cggatgtacc ccgaggacgg
     3001 cgccctgaag ggcgaggtca agccgagagt gaagctgaag gacggcggcc actacgacgc
     3061 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     3121 caaccgcaag ttggacatca cctcacacaa cgaggactac accatcgtgg aacagtacga
     3181 acgtgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aagcggccgc
     3241 gcggatccca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3301 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3361 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3421 ggaggtgtgg gaggtttttt agtcgactag agctcgctga tcagcctcga ctgtgccttc
     3481 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3541 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3601 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3661 tagcaggcat gctggggaga gatctcacgt gcggaccgag cggccgcagg aacccctagt
     3721 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3781 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3841 cctgcagggg cgcctgatgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     3901 tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg
     3961 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4021 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4081 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4141 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4201 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4261 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     4321 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     4381 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4441 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4501 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4561 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4621 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4681 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4741 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4801 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4861 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4921 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4981 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5041 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5101 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5161 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5221 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5281 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5341 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5401 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5461 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5521 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5581 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5641 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5701 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5761 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5821 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5881 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5941 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6001 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6061 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6121 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6181 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6241 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6301 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6361 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6421 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6481 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6541 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     6601 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     6661 ttggccgatt cattaatgca gctggggctc acatgt
//
