LOCUS       sequence-205720-        7667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid to target the rmRuby1-roGFP1 sensor to 
            the mitochondrial matrix..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7667)
  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 7667)
  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-mRuby2-roGFP1
FEATURES             Location/Qualifiers
     source          1..7667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        205..584
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        585..788
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     739..759
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     785..809
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          924..1750
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             1956..2030
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2052..2126
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2148..2222
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2244..2318
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2358..3068
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             join(3090..3092,3093..3806)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FISTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3132..3153)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3393..3412)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3740..3761
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3927..4061
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3964..3983)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4018..4037
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4084..4100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4084..4100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4097..4119)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4108..4124
                     /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(4132..4138,4139..4156,4157..4162))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4314..4331)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4485..5073)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4565..4584)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5244..6035,6036..6104))
                     /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     5867..5886
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6161..6674)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6356..6377)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6568..6587
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6846..6864
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6902..6924)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7024..7043
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7275..7297
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7289..7306
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7290..7306
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gctatatcgc cgatagaggc gacatcaagc tggcacatgg ccaatgcata tcgatctata
       61 cattgaatca atattggcca ttagccatat tattcattgg ttatatagca taaatcaata
      121 ttggctattg gccattgcat acgttgtatc catatcataa tatgtacatt tatattggct
      181 catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa
      241 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      301 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      361 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      421 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      481 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      541 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      601 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      661 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      721 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      781 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      841 tccatagaag acaccgggac cgatccagcc tccgcggccg ggaacggtgc attggaacgc
      901 ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg cccaccccct
      961 tggcttctta tgcatgctat actgtttttg gcttggggtc tatacacccc cgcttcctca
     1021 tgttataggt gatggtatag cttagcctat aggtgtgggt tattgaccat tattgaccac
     1081 tcccctattg gtgacgatac tttccattac taatccataa catggctctt tgccacaact
     1141 ctctttattg gctatatgcc aatacactgt ccttcagaga ctgacacgga ctctgtattt
     1201 ttacaggatg gggtctcatt tattatttac aaattcacat atacaacacc accgtcccca
     1261 gtgcccgcag tttttattaa acataacgtg ggatctccac gcgaatctcg ggtacgtgtt
     1321 ccggacatgg gctcttctcc ggtagcggcg gagcttctac atccgagccc tgctcccatg
     1381 cctccagcga ctcatggtcg ctcggcagct ccttgctcct aacagtggag gccagactta
     1441 ggcacagcac gatgcccacc accaccagtg tgccgcacaa ggccgtggcg gtagggtatg
     1501 tgtctgaaaa tgagctcggg gagcgggctt gcaccgctga cgcatttgga agacttaagg
     1561 cagcggcaga agaagatgca ggcagctgag ttgttgtgtt ctgataagag tcagaggtaa
     1621 ctcccgttgc ggtgctgtta acggtggagg gcagtgtagt ctgagcagta ctcgttgctg
     1681 ccgcgcgcgc caccagacat aatagctgac agactaacag actgttcctt tccatgggtc
     1741 ttttctgcag tcaccgtcct tgacacgaag cttcccggcc acagcaatcg atccatgaca
     1801 caaccagtga caagcagtcc cccagtgaaa ccgcatccgc atccgagtcc gataccgata
     1861 aagattctga atcggagtga gtgccgcgtc cgagagcgtt ccctgtccac gtccaccatc
     1921 ggcggagcag gtgtgcctga ggcctctaga ccaccatgag cgtgctgaca ccattgttgc
     1981 tcagaggcct cactggttca gccaggcggc tgccagtccc aagagctaag atccatagtc
     2041 tgggagatcc aatgtcagtt ctcactcccc ttttgcttag gggactgacc ggaagcgctc
     2101 ggcggttgcc tgtcccacgc gccaagattc actccctggg cgaccctatg agtgtcctta
     2161 ctcctctttt gctgagaggc ttgacaggga gcgctcggag gctcccagtg cctcgcgcaa
     2221 agatccattc tctcggagat cccatgtccg tcctcactcc tctgctgctg cgcgggctta
     2281 caggttccgc tcgccgcctg cctgttcctc gggcaaagat acacagtctg ggcgatcctc
     2341 accggcgggc tgctgctatg gtgtctaagg gcgaagagct gatcaaggaa aatatgcgta
     2401 tgaaggtggt catggaaggt tcggtcaacg gccaccaatt caaatgcaca ggtgaaggag
     2461 aaggcaatcc gtacatggga actcaaacca tgaggatcaa agtcatcgag ggaggacccc
     2521 tgccatttgc ctttgacatt cttgccacgt cgttcatgta tggcagccgt acttttatca
     2581 agtacccgaa aggcattcct gatttcttta aacagtcctt tcctgagggt tttacttggg
     2641 aaagagttac gagatacgaa gatggtggag tcgtcaccgt catgcaggac accagccttg
     2701 aggatggctg tctcgtttac cacgtccaag tcagaggggt aaactttccc tccaatggtc
     2761 ccgtgatgca gaagaagacc aagggttggg agcctaatac agagatgatg tatccagcag
     2821 atggtggtct gaggggatac actcatatgg cactgaaagt tgatggtggt ggccatctgt
     2881 cttgctcttt cgtaacaact tacaggtcaa aaaagaccgt cgggaacatc aagatgcccg
     2941 gtatccatgc cgttgatcac cgcctggaaa ggttagagga aagtgacaat gaaatgttcg
     3001 tagtacaacg cgaacacgca gttgccaagt tcgccgggct tggtggtggg atggacgagc
     3061 tgtacaaggg aggttcagga ggtcgatccg tgagcaaggg cgaggagctg ttcaccgggg
     3121 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     3181 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tccaccaccg
     3241 gcaagctgcc cgtgccctgg cccaccctcg tgaccactct gtcttacggc gtgcagtgct
     3301 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3361 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     3421 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3481 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaactgc cacaacgtct
     3541 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     3601 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3661 gccccgtgct gctgcccgac aaccactacc tgagcacctg ctccgccctg agcaaagacc
     3721 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3781 tcggcatgga cgagctgtac aagtaatcga cgaattcatt gatcataatc agccatacca
     3841 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3901 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3961 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4021 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgtaccgag ctcgcgtaat
     4081 catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4141 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tacattaatt
     4201 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4261 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4321 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4381 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4441 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4501 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4561 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4621 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4681 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4741 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4801 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4861 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4921 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4981 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5041 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5101 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5161 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5221 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5281 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5341 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5401 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5461 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5521 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5581 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5641 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5701 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5761 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5821 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5881 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5941 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6001 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6061 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6121 taagcggccg cggccatgcc ggccactagt ctcgagggcc accccggttg ataatcagaa
     6181 aagccccaaa aacaggaaga ttgtataagc aaatatttaa attgtaagcg ttaatatttt
     6241 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6301 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6361 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6421 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6481 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6541 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6601 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6661 gctacagggc gcgtggggat ccctcgagtt attattgaag catttatcag ggttattgtc
     6721 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6781 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6841 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     6901 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     6961 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     7021 atgcggcatc agagcagatt gtactgagag tgcaccatat gaagacgtcg cctcctcact
     7081 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta
     7141 gtcagggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     7201 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     7261 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgccc gtgtcaagga
     7321 cggtgactgc agtgaataat aaaatgtgtg tttgtccgaa atacgcgttt tgagatttct
     7381 gtcgccgact aaattcatgt cgcgcgatag tggtgtttat cgccgataga gatggcgata
     7441 ttggaaaaat cgatatttga aaatatggca tattgaaaat gtcgccgatg tgagtttctg
     7501 tgtaactgat atcgccattt ttccaaaagt gatttttggg catacgcgat atctggcgat
     7561 agcgcttata tcgtttacgg gggatggcga tagacgactt tggtgacttg ggcgattctg
     7621 tgtgtcgcaa atatcgcagt ttcgatatag gtgacagacg atatgag
//
