LOCUS       40924_12425        6404 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Green fluorescent indicator for calcium imaging in the mitochondria 
            (low affinity variant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6404)
  AUTHORS   Suzuki J, Kanemaru K, Ishii K, Ohkura M, Okubo Y, Iino M
  TITLE     Imaging intraorganellar Ca(2+) at subcellular resolution using 
            CEPIA.
  JOURNAL   Nat Commun. 2014 Jun 13;5:4153. doi: 10.1038/ncomms5153.
  PUBMED    24923787
REFERENCE   2  (bases 1 to 6404)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6404)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat
            Commun."; date: "2014-06-13"; pages: "
            10.1038/ncomms5153"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(37..625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(799..1656)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1657..1761)
                     /label=AmpR promoter
     enhancer        1825..2202
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2204..2407
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             2450..2524
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2546..2620
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2654..4000
                     /codon_start=1
                     /label=GCaMP6f
                     /note="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGYYKTRAEVKFEGDTLVNRIVL
                     KGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKDLGTVLRSL
                     GQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVWDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGEGQVNYEEFVQMMTAK"
     CDS             4010..4039
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    4081..4305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4351..4779
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4793..5122
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5148..5939
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MVEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    6116..6249
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6270..6287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
       61 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
      121 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
      181 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
      241 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgta tgcacgaacc
      301 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
      361 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
      421 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
      481 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
      541 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
      601 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
      661 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
      721 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
      781 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
      841 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
      901 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
      961 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1021 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1081 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     1141 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     1201 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     1261 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     1321 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     1381 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     1441 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     1501 accgctgttg agatccagtt cgatgtaacc cactcgggca cccaactgat cttcagcatc
     1561 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     1621 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     1681 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     1741 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcagatcgac
     1801 ggatcgggag atcggtaccg aattcacatt gattattgag tagttattaa tagtaatcaa
     1861 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1921 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1981 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2041 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2101 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2161 ctacttggca gtacatctac ggttagtcat cgctattacc atagtgatgc ggttttggca
     2221 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2281 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2341 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2401 cagagctttc tggctaacta gagaacccac tgcttactgg cacgtgacca tgtccgtcct
     2461 gacgccgctg ctgctgcggg gcttgacagg ctcggcccgg cggctcccag tgccgcgcgc
     2521 caaaattcat tcactggggg accccatgag cgtgctcacc ccactcctgc tgcgggggct
     2581 gaccggcagc gctaggcggc tgccagtccc gcgggccaag atccacagtc tcggcgatcc
     2641 cggatccatg cggggttctc atcatcatca tcatcatggt atggctagca tgactggtgg
     2701 acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga
     2761 ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag
     2821 ctcactcgag aacgtctata tcatggccga caagcagaag aacggcatca aggcgaactt
     2881 caagatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa
     2941 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3001 caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3061 cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat
     3121 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     3181 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg
     3241 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     3301 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     3361 gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt
     3421 caaggacgac ggctactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     3481 gaaccgcatc gtgctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     3541 gctggagtac aacacgcgtg accaactgac tgaagagcag atcgcagaat ttaaagaggc
     3601 tttctcccta tttgacaagg acggggatgg gacaataaca accaaggatc tggggacggt
     3661 gctgcggtct ctggggcaga accccacaga agcagagctc caggacatga tcaatgaagt
     3721 agatgccgac ggtaatggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa
     3781 aatgaaagac acagacagtg aagaagaaat tcgcgaagcg ttccgtgtgt gggataagga
     3841 tggcaatggc tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa
     3901 gttaacagat gaagaggttg atgaaatgat cagggaagca gatatcgatg gagaaggtca
     3961 ggtaaactac gaagagtttg tacaaatgat gacagcgaag gcggccgcag aacaaaaact
     4021 catctcagaa gaggatctga atggggccgc atagtctaga agctcgctga tcagcctcga
     4081 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     4141 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     4201 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     4261 gggaagacaa tagcaggcat gctggggatg gcccgggctc tatggcttct gaggcggaaa
     4321 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     4381 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4441 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4501 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctctcc cccaaaaaac
     4561 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4621 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4681 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4741 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     4801 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4861 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4921 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4981 cctaactccg cccagttccg cccattctcc gcccctaggc tgactaattt tttttattta
     5041 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     5101 tggaggccta ggcttttgca aaaagctccc cccccgggag gtccacaatg gttgaacaag
     5161 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5221 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5281 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactccag gacgaggcag
     5341 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5401 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5461 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5521 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5581 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5641 tcgcgccagc cgaactgttc gccaggctca aggcgcgtat gcccgacggc gaggatctcg
     5701 tcgtgactca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5761 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5821 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     5881 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     5941 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     6001 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6061 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     6121 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6181 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     6241 tgtctgtata ccgtcgatct ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6301 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6361 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaa
//
