LOCUS       Exported                6404 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Green fluorescent indicator for calcium imaging in the mitochondria 
            (low affinity variant).
ACCESSION   .
VERSION     .
KEYWORDS    pCMV CEPIA4mt
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2014-06-13"; volume: "5:4153. doi"; pages: " 
            10.1038/ncomms5153"
FEATURES             Location/Qualifiers
     source          1..6404
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(37..625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(117..136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(796..1656)
                     /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     1419..1438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1657..1761)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1826..2202
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2204..2407
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2358..2378
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     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             2660..2677
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2681..2713
                     /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             2717..2740
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      2747..2756
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2768..2824
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     3037..3058
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3164..3185)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             3560..4000
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKDLGTVLRSLGQNPTEA
                     ELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVWDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGEGQVNYEEFVQMMTAK"
     CDS             4010..4039
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(4075..4092)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4081..4305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4351..4779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4438..4457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4648..4669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4788..4808)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4793..5122
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4973..5108
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5035..5054
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5148..5942
                     /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="MVEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5202..5221)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5812..5831
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6116..6237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6153..6172)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6207..6226
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6270..6287)
                     /label=L4440
                     /note="L4440 vector, forward primer"