LOCUS       sequence-205712-        7684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid to target the mCherry-roGFP2 sensor to 
            the mitochondrial matrix..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7684)
  AUTHORS   Norcross S, Trull KJ, Snaider J, Doan S, Tat K, Huang L, Tantama M
  TITLE     Extending roGFP Emission via Forster-Type Resonance Energy Transfer 
            Relay Enables Simultaneous Dual Compartment Ratiometric Redox 
            Imaging in Live Cells.
  JOURNAL   ACS Sens. 2017 Nov 22;2(11):1721-1729. doi: 
            10.1021/acssensors.7b00689. Epub 2017 Nov 7.
  PUBMED    29072071
REFERENCE   2  (bases 1 to 7684)
  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"; doi: 
            "10.1021/acssensors.7b00689"; journalName: "ACS Sens"; date: 
            "2017-11-22- 22"; volume: "2"; issue: "11"; pages: "1721-1729"
COMMENT     Sequence Label: GW1-mito-mCherry-roGFP2
FEATURES             Location/Qualifiers
     source          1..7684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        111..490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        491..694
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     645..665
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     691..715
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          830..1656
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             1862..1936
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1958..2032
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2054..2128
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2150..2224
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2279..2986
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2424..2442)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2692..2711
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(3008..3010,3011..3724)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3050..3071)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3311..3330)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3658..3679
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3850..3984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3887..3906)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3941..3960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4007..4023)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4007..4023)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4020..4042)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4031..4047
                     /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(4055..4061,4062..4079,4080..4085))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4237..4254)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4408..4996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4488..4507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5167..5958,5959..6027))
                     /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     5790..5809
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6084..6597)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6279..6300)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6491..6510
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6769..6787
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6825..6847)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6947..6966
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7198..7220
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7212..7229
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7213..7229
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cattggttat atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatccata
       61 tcataatatg tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt
      121 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      181 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      241 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      301 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      361 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      421 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      481 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      541 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      601 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      661 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg
      721 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
      781 cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc
      841 ctatagagtc tataggccca cccccttggc ttcttatgca tgctatactg tttttggctt
      901 ggggtctata cacccccgct tcctcatgtt ataggtgatg gtatagctta gcctataggt
      961 gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc cattactaat
     1021 ccataacatg gctctttgcc acaactctct ttattggcta tatgccaata cactgtcctt
     1081 cagagactga cacggactct gtatttttac aggatggggt ctcatttatt atttacaaat
     1141 tcacatatac aacaccaccg tccccagtgc ccgcagtttt tattaaacat aacgtgggat
     1201 ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta gcggcggagc
     1261 ttctacatcc gagccctgct cccatgcctc cagcgactca tggtcgctcg gcagctcctt
     1321 gctcctaaca gtggaggcca gacttaggca cagcacgatg cccaccacca ccagtgtgcc
     1381 gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag ctcggggagc gggcttgcac
     1441 cgctgacgca tttggaagac ttaaggcagc ggcagaagaa gatgcaggca gctgagttgt
     1501 tgtgttctga taagagtcag aggtaactcc cgttgcggtg ctgttaacgg tggagggcag
     1561 tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata gctgacagac
     1621 taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtccttgac acgaagcttc
     1681 ccggccacag caatcgatcc atgacacaac cagtgacaag cagtccccca gtgaaaccgc
     1741 atccgcatcc gagtccgata ccgataaaga ttctgaatcg gagtgagtgc cgcgtccgag
     1801 agcgttccct gtccacgtcc accatcggcg gagcaggtgt gcctgaggcc tctagaccac
     1861 catgagcgtg ctgacaccat tgttgctcag aggcctcact ggttcagcca ggcggctgcc
     1921 agtcccaaga gctaagatcc atagtctggg agatccaatg tcagttctca ctcccctttt
     1981 gcttagggga ctgaccggaa gcgctcggcg gttgcctgtc ccacgcgcca agattcactc
     2041 cctgggcgac cctatgagtg tccttactcc tcttttgctg agaggcttga cagggagcgc
     2101 tcggaggctc ccagtgcctc gcgcaaagat ccattctctc ggagatccca tgtccgtcct
     2161 cactcctctg ctgctgcgcg ggcttacagg ttccgctcgc cgcctgcctg ttcctcgggc
     2221 aaagatacac agtctgggcg atcctcaccg gcgtggagga gcatctggtg gagctagtat
     2281 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     2341 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     2401 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     2461 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     2521 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     2581 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     2641 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     2701 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     2761 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     2821 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     2881 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     2941 cgccgagggt cgtcactcta ccggcggcat ggacgagctg tacaagggag gttcaggagg
     3001 tcgatccgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     3061 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     3121 ctacggcaag ctgaccctga agttcatctc caccaccggc aagctgcccg tgccctggcc
     3181 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     3241 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     3301 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     3361 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3421 gcacaagctg gagtacaact acaactgcca caacgtctat atcatggccg acaagcagaa
     3481 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     3541 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     3601 ccactacctg agcacctgct ccgccctgag caaagacccc aacgagaagc gcgatcacat
     3661 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     3721 gtaataaact cgacgaattc attgatcata atcagccata ccacatttgt agaggtttta
     3781 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3841 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3901 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3961 aatgtatctt atcatgtctg gatccgtacc gagctcgcgt aatcatgtca tagctgtttc
     4021 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4081 gtaaagcctg gggtgcctaa tgagtgagct aactacatta attgcgttgc gctcactgcc
     4141 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4201 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4261 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4321 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4381 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4441 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4501 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4561 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4621 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4681 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4741 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4801 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4861 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4921 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4981 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5041 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5101 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5161 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5221 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5281 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5341 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5401 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5461 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5521 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5581 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5641 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5701 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5761 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5821 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5881 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5941 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6001 ggcgacacgg aaatgttgaa tactcatact cttccttttt caataagcgg ccgcggccat
     6061 gccggccact agtctcgagg gccaccccgg ttgataatca gaaaagcccc aaaaacagga
     6121 agattgtata agcaaatatt taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     6181 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     6241 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     6301 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     6361 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6421 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     6481 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     6541 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg
     6601 gatccctcga gttattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6661 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6721 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6781 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     6841 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     6901 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     6961 attgtactga gagtgcacca tatgaagacg tcgcctcctc actacttctg gaatagctca
     7021 gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcaggg cgccattcgc
     7081 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     7141 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     7201 agtcacgacg ttgtaaaacg acggccagtg cccgtgtcaa ggacggtgac tgcagtgaat
     7261 aataaaatgt gtgtttgtcc gaaatacgcg ttttgagatt tctgtcgccg actaaattca
     7321 tgtcgcgcga tagtggtgtt tatcgccgat agagatggcg atattggaaa aatcgatatt
     7381 tgaaaatatg gcatattgaa aatgtcgccg atgtgagttt ctgtgtaact gatatcgcca
     7441 tttttccaaa agtgattttt gggcatacgc gatatctggc gatagcgctt atatcgttta
     7501 cgggggatgg cgatagacga ctttggtgac ttgggcgatt ctgtgtgtcg caaatatcgc
     7561 agtttcgata taggtgacag acgatatgag gctatatcgc cgatagaggc gacatcaagc
     7621 tggcacatgg ccaatgcata tcgatctata cattgaatca atattggcca ttagccatat
     7681 tatt
//
