LOCUS       sequence-202530-        7120 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 7120)
  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 7120)
  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..7120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             189..263
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             285..359
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             381..455
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             477..551
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             591..1301
                     /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(1323..1325,1326..2039)
                     /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(1365..1386)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1626..1645)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1973..1994
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2160..2294
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2197..2216)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2251..2270
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2318..2334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2318..2334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2331..2353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2342..2358
                     /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(2366..2372,2373..2390,2391..2396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2411..2432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2549..2566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2720..3308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2800..2819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3479..4270,4271..4339))
                     /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     4102..4121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     4549..4567
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4605..4627)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4727..4746
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4978..5000
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4992..5009
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4993..5009
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        5558..5937
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5938..6141
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6092..6112
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6138..6162
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          6277..7103
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
ORIGIN
        1 aagcttcccg gccacagcaa tcgatccatg acacaaccag tgacaagcag tcccccagtg
       61 aaaccgcatc cgcatccgag tccgataccg ataaagattc tgaatcggag tgagtgccgc
      121 gtccgagagc gttccctgtc cacgtccacc atcggcggag caggtgtgcc tgaggcctct
      181 agaccaccat gagcgtgctg acaccattgt tgctcagagg cctcactggt tcagccaggc
      241 ggctgccagt cccaagagct aagatccata gtctgggaga tccaatgtca gttctcactc
      301 cccttttgct taggggactg accggaagcg ctcggcggtt gcctgtccca cgcgccaaga
      361 ttcactccct gggcgaccct atgagtgtcc ttactcctct tttgctgaga ggcttgacag
      421 ggagcgctcg gaggctccca gtgcctcgcg caaagatcca ttctctcgga gatcccatgt
      481 ccgtcctcac tcctctgctg ctgcgcgggc ttacaggttc cgctcgccgc ctgcctgttc
      541 ctcgggcaaa gatacacagt ctgggcgatc ctcaccggcg ggctgctgct atggtgtcta
      601 agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca
      661 acggccacca attcaaatgc acaggtgaag gagaaggcaa tccgtacatg ggaactcaaa
      721 ccatgaggat caaagtcatc gagggaggac ccctgccatt tgcctttgac attcttgcca
      781 cgtcgttcat gtatggcagc cgtactttta tcaagtaccc gaaaggcatt cctgatttct
      841 ttaaacagtc ctttcctgag ggttttactt gggaaagagt tacgagatac gaagatggtg
      901 gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg ctgtctcgtt taccacgtcc
      961 aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag accaagggtt
     1021 gggagcctaa tacagagatg atgtatccag cagatggtgg tctgagggga tacactcata
     1081 tggcactgaa agttgatggt ggtggccatc tgtcttgctc tttcgtaaca acttacaggt
     1141 caaaaaagac cgtcgggaac atcaagatgc ccggtatcca tgccgttgat caccgcctgg
     1201 aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca acgcgaacac gcagttgcca
     1261 agttcgccgg gcttggtggt gggatggacg agctgtacaa gggaggttca ggaggtcgat
     1321 ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1381 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1441 gcaagctgac cctgaagttc atctccacca ccggcaagct gcccgtgccc tggcccaccc
     1501 tcgtgaccac tctgtcttac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1561 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1621 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1681 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1741 agctggagta caactacaac tgccacaacg tctatatcat ggccgacaag cagaagaacg
     1801 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1861 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1921 acctgagcac ctgctccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1981 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaat
     2041 cgacgaattc attgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2101 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2161 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2221 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2281 atcatgtctg gatccgtacc gagctcgcgt aatcatggtc atagctgttt cctgtgtgaa
     2341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2881 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     2941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3121 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     3181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4321 ggaaatgttg aatactcata ctcttccttt ttcaataagc ggccgcggcc atgccggcca
     4381 ctagtctcga gttattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4441 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4501 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4561 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4621 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4681 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     4741 attgtactga gagtgcacca tatgaagacg tcgcctcctc actacttctg gaatagctca
     4801 gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcg cgccattcgc
     4861 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     4921 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     4981 agtcacgacg ttgtaaaacg acggccagtg ccatctgcag tgaataataa aatgtgtgtt
     5041 tgtccgaaat acgcgtttga gatttctgtc ccgactaaat tcatgtcgcg cgatagtggt
     5101 gtttatcgcc gatagagatg gcgatattgg aaaaatcgat atttgaaaat atggcatatt
     5161 gaaaatgtcg ccgatgtgag tttctgtgta actgatatcg ccatttttcc aaaagttgat
     5221 ttttgggcat acgcgatatc tggcgatacg cttatatcgt ttacggggga tggcgataga
     5281 cgcctttggt gacttgggcg attctgtgtg tcgcaaatat cgcagtttcg atataggtga
     5341 cagacgatat gaggctatat cgccgataga ggcgacatca agctggcaca tggccaatgc
     5401 atatcgatct atacattgaa tcaatattgg ccattagcca tattattcat tggttatata
     5461 gcataaatca atattggcta ttggccattg catacgttgt atccatatca taatatgtac
     5521 atttatattg gctcatgtcc aacattaccg ccatgttgac attgattatt gactagttat
     5581 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     5641 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     5701 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     5761 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     5821 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     5881 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     5941 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     6001 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     6061 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     6121 gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
     6181 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg
     6241 tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta tagagtctat
     6301 aggcccaccc ccttggcttc ttatgcatgc tatactgttt ttggcttggg gtctatacac
     6361 ccccgcttcc tcatgttata ggtgatggta tagcttagcc tataggtgtg ggttattgac
     6421 cattattgac cactccccta ttggtgacga tactttccat tactaatcca taacatggct
     6481 ctttgccaca actctcttta ttggctatat gccaatacac tgtccttcag agactgacac
     6541 ggactctgta tttttacagg atggggtctc atttattatt tacaaattca catatacaac
     6601 accaccgtcc ccagtgcccg cagtttttat taaacataac gtgggatctc cacgcgaatc
     6661 tcgggtacgt gttccggaca tgggctcttc tccggtagcg gcggagcttc tacatccgag
     6721 ccctgctccc atgcctccag cgactcatgg tcgctcggca gctccttgct cctaacagtg
     6781 gaggccagac ttaggcacag cacgatgccc accaccacca gtgtgccgca caaggccgtg
     6841 gcggtagggt atgtgtctga aaatgagctc ggggagcggg cttgcaccgc tgacgcattt
     6901 ggaagactta aggcagcggc agaagaagat gcaggcagct gagttgttgt gttctgataa
     6961 gagtcagagg taactcccgt tgcggtgctg ttaacggtgg agggcagtgt agtctgagca
     7021 gtactcgttg ctgccgcgcg cgccaccaga cataatagct gacagactaa cagactgttc
     7081 ctttccatgg gtcttttctg cagtcaccgt ccttgacacg
//
