LOCUS       Exported                5308 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express fluorescent calcium indicator GCaMP6fu in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-GCaMP6fu
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  AUTHORS   Helassa N, Podor B, Fine A, Torok K
  TITLE     Design and mechanistic insight into ultrafast calcium indicators for
            monitoring intracellular calcium dynamics.
  JOURNAL   Sci Rep. 2016 Dec 6;6:38276. doi: 10.1038/srep38276.
  PUBMED    27922063
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"
     primer_bind     238..258
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(283..387)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      414..869
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(501..520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     711..732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    876..997
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(887..906)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     941..960
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1119..2471)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKYNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFAKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     1678..1697
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1937..1958
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2064..2085)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2366..2375
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      2468..2477
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(2525..2728)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2554..2574)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2729..3032
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3207..3795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3287..3306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4124..4171
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4196..4215
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4403..5197)
                     /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(4514..4533)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5124..5143
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5232..281)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5246..73
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5300..11)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag
       61 ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca
      121 tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact
      181 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
      241 ctaactgaca cacattccac agctggttct ttccgcctca ggactcttcc tttttcaata
      301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
      361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
      421 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      481 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      541 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      601 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      661 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      721 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      781 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      841 ttttaacaaa atattaacgc ttacaattta cgccttaaga tacattgatg agtttggaca
      901 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
      961 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     1021 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     1081 atgtggtatg gctgattatg atctagagtc gcggccgctc acttcgctgt catcatttgt
     1141 acaaactctt cgtagtttac ctgaccatcc ccatcgatgt ctgcttccct gatcatttca
     1201 tcaacctctt catctgttaa cttctctcca aggtttgtca tcacgtggcg aagctctgct
     1261 gcactgatgt agccattgcc atccttagca aacacaccga acgcttctct aatttcttct
     1321 tccgtgtccc tgtatttcat ttttcttgcc atcattgtca ggaactcagg gaagtcgatt
     1381 gtgccgtcac cgtcggcatc tacttcattg atcatgtcct gcagctctgc ttctgtgggg
     1441 ttctgcccca gagaccgcat caccgtcccc agctccttgg ttgttattgt cccatccccg
     1501 tccttgtcaa atagggagaa ttcctcttta aattctgcga tctgctcttc agtcagttgg
     1561 tccggcaggt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
     1621 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
     1681 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggatgtagcc ttcgggcatg
     1741 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
     1801 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
     1861 atgaacttca gggtcagctt gccgtaggtg gcatcgccct caccctcgcc ggacacgctg
     1921 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
     1981 agctcctcgc ccttgctcac catgctccct ccggtaccgc ccttgtacag ctcgtccatg
     2041 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     2101 ttggggtctt tcgaaagttt ggactgcacg ctcaggtagt ggttgtcggg cagcagcacg
     2161 gggccgtcgc cgatgggggt gttctgctgg tagtggtagg cgagctgcac gccgccgtcc
     2221 tcgatgttgt ggcggatctt gaagttcgcc ttgatgccgt tcttctgctt gtcggccttg
     2281 atatagacgt tctcgagtga gctcagccga cctatagctc tgactgcgtg acctgtctta
     2341 ttgtacttac gacgtgatga gtcgaccatg gtggcgagat ccttatcgtc atcgtcgtac
     2401 agatcccgac ccatttgctg tccaccagtc atgctagcca taccatgatg atgatgatga
     2461 tgagaaccca tggtggcgag atctgagtcc ggtagcgcta gcggatctga cggttcacta
     2521 aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
     2581 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
     2641 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
     2701 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
     2761 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
     2821 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     2881 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     2941 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     3001 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
     3061 atgaccccgt aattgattac tattaataac taatgcatgg cggtaatacg gttatccaca
     3121 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3181 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3241 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3301 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3361 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3421 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3481 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3541 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3601 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3661 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3721 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3781 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3841 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3901 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta acctgaggct
     3961 atggcagggc ctgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg
     4021 gggtggggtg gggaaaagga agaaacgcgg gcgtattggc cccaatgggg tctcggtggg
     4081 gtatcgacag agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca
     4141 ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc gggttccttc cggtattgtc
     4201 tccttccgtg tttcagttag cctcccccta gggtgggcga agaactccag catgagatcc
     4261 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     4321 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc
     4381 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
     4441 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     4501 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     4561 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     4621 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc
     4681 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     4741 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     4801 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     4861 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     4921 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     4981 tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca
     5041 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     5101 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     5161 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
     5221 cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     5281 agctcagagg ccgaggcggc ctcggcct
//
