LOCUS       sequence-109678-        5970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosol-targeted version of the Ca2+ indicator GCaMP3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5970)
  AUTHORS   Mehta S, Aye-Han NN, Ganesan A, Oldach L, Gorshkov K, Zhang J
  TITLE     Calmodulin-controlled spatial decoding of oscillatory Ca2+ signals 
            by calcineurin.
  JOURNAL   eLife. 2014 Jul 23;3:e03765. doi: 10.7554/eLife.03765.
  PUBMED    25056880
REFERENCE   2  (bases 1 to 5970)
  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"; journalName: "eLife."; 
            date: "2014-07-23"; volume: "3:e03765. doi"; pages: " 
            10.7554/eLife.03765"
COMMENT     Sequence Label: pcDNA3-Cyto-GCaMP3
FEATURES             Location/Qualifiers
     source          1..5970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      13..22
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             34..51
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             55..87
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(91..99,100..114)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(121..129,130..147,148..150,151..183,184..186,187..210,
                     211..237,238..294,295..300,301..570,571..588,589..1023,
                     1024..1026,1027..1029,1030..1470)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             join(121..129,130..147,148..150,151..183,184..186,187..210,
                     211..237,238..294,295..300,301..570,571..588,589..1023,
                     1024..1026,1027..1029,1030..1470)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      217..226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     507..528
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(634..655)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(895..914)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1477..1503
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1522..1540)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1523..1540)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1560..1577)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1566..1790
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1836..2264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1923..1942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2133..2154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2273..2293)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2278..2608
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2459..2594
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2521..2540
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2675..3469
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2729..2748)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3339..3358
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3643..3764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3680..3699)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3734..3753
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3813..3829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3813..3829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3826..3848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3837..3853
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3861..3867,3868..3885,3886..3891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3906..3927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4044..4061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4215..4803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4295..4314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4974..5765,5766..5834))
                     /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     5597..5616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5835..5939)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aagcttgcgg ccgccaccat ggtgcggggt tctcatcatc atcatcatca tggtatggct
       61 agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatccc
      121 atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg
      181 ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt
      241 cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag
      301 aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc
      361 cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc
      421 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg
      481 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      541 atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag
      601 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      661 ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc
      721 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      781 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
      841 ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac
      901 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
      961 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1021 aacacgcgtg accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta
     1081 tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct
     1141 ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac
     1201 ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaagac
     1261 acagacagtg aagaagaaat tagagaagcg ttccgtgtgt ttgataagga tggcaatggc
     1321 tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat
     1381 gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac
     1441 gaagagtttg tacaaatgat gacagcgaag gaattcctgc cccccctgga gcgcctgacc
     1501 ctgtaatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     1561 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1621 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1681 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     1741 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     1801 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     1861 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1921 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1981 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2041 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2101 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2161 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta
     2221 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2281 tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta tgcaaagcat
     2341 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2401 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2461 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2521 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     2581 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     2641 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     2701 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     2761 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     2821 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     2881 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     2941 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3001 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3061 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3121 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3181 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3241 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3301 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3361 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3421 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     3481 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3541 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3601 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     3661 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3721 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     3781 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3841 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3901 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3961 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4021 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4081 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4141 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4201 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4261 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4321 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4381 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     4441 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4501 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4561 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4621 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4681 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4741 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4801 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4861 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4921 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4981 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5041 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5101 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5161 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5221 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5281 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5341 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5401 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5461 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5521 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5581 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5641 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5701 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5761 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5821 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5881 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5941 acatttcccc gaaaagtgcc acctgacgtc
//
