LOCUS       Exported                5308 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express fluorescent calcium indicator GCaMP3bright in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-GCaMP3bright
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  AUTHORS   Helassa N, Zhang XH, Conte I, Scaringi J, Esposito E, Bradley J, 
            Carter T, Ogden D, Morad M, Torok K
  TITLE     Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid 
            Intracellular Calcium Dynamics.
  JOURNAL   Sci Rep. 2015 Nov 3;5:15978. doi: 10.1038/srep15978.
  PUBMED    26527405
REFERENCE   2  (bases 1 to 5308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5308
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      616..625
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             631..648
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             652..684
                     /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             688..711
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      718..727
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             739..795
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     1008..1029
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1135..1156)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1396..1415)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1531..1974
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREAAIDGDGQVNYEEFVQMMTAK"
     polyA_signal    2096..2217
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2133..2152)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2187..2206
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2224..2679)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2361..2382)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2573..2592
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2706..2810
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2812..3169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2835..2855)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3020..3155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3082..3101
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3204..3998
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3258..3277)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3868..3887
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4186..4205)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4230..4277
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4606..5194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5095..5114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgccac catgggttct catcatcatc atcatcatgg tatggctagc
      661 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatctcgcc
      721 accatggtcg actcatcacg tcgtaagtgg aataagacag gtcacgcagt cagagctata
      781 ggtcggctga gctcactcga gaacgtctat atcaaggccg acaagcagaa gaacggcatc
      841 aaggcgaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgcctaccac
      901 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      961 agcgtccagt ccaaactttc gaaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1021 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcggtacc
     1081 ggagggagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1141 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg tgagggcgat
     1201 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1261 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1321 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacat ccaggagcgc
     1381 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1441 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1501 ctggggcaca agctggagta caacacgcgt gaccaactga ctgaagagca gatcgcagaa
     1561 tttaaagagg ctttctccct atttgacaag gacggggatg ggacaataac aaccaaggag
     1621 ctggggacgg tgatgcggtc tctggggcag aaccccacag aagcagagct gcaggacatg
     1681 atcaatgaag tagatgccga cggtgatggc acaatcgact tccctgagtt cctgacaatg
     1741 atggcaagaa aaatgaaaga cacagacagt gaagaagaaa ttagagaagc gttccgtgtg
     1801 tttgataagg atggcaatgg ctacatcagt gcagcagagc ttcgccacgt gatgacaaac
     1861 cttggagaga agttaacaga tgaagaggtt gatgaaatga tcagggaagc agccatcgat
     1921 ggggatggtc aggtaaacta cgaagagttt gtacaaatga tgacagcgaa gtgagcggcc
     1981 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     2041 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     2101 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2161 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     2221 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2281 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2341 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2401 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2461 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2521 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2581 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2641 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     2701 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2761 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     2821 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2941 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3001 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3061 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3121 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     3181 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3241 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3301 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3361 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     3421 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3481 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3541 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3601 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3661 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3721 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3781 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3841 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3901 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3961 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4021 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4081 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4141 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     4201 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     4261 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     4321 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     4381 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     4441 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4501 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4561 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4621 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4681 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4741 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4801 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4861 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4921 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4981 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5041 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5101 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5161 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5221 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5281 tgtggataac cgtattaccg ccatgcat
//
