LOCUS       sequence-324310-        5194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent biosensor for 2GSH/GSSG ratio with mitochondrial 
            localization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5194)
  AUTHORS   Shokhina AG, Kostyuk AI, Ermakova YG, Panova AS, Staroverov DB, 
            Egorov ES, Baranov MS, van Belle GJ, Katschinski DM, Belousov VV, 
            Bilan DS
  TITLE     Red fluorescent redox-sensitive biosensor Grx1-roCherry.
  JOURNAL   Redox Biol. 2019 Feb;21:101071. doi: 10.1016/j.redox.2018.101071. 
            Epub 2018 Dec 7.
  PUBMED    30576927
REFERENCE   2  (bases 1 to 5194)
  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"; journalName: "Redox 
            Biol."; date: "2019-02"; volume: "21:101071. doi"; pages: " 
            10.1016/j.redox.2018.101071. Epub 2018 Dec 7"
COMMENT     Sequence Label: pC1 Grx1-roCherry-mito
FEATURES             Location/Qualifiers
     source          1..5194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             597..671
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             693..764
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1161..1871
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWECTESERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNICLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1303..1321)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1571..1590
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1982..2103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2019..2038)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2073..2092
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2110..2565)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2247..2268)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2459..2478
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2592..2696
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2698..3055
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2698..2889
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2721..2741)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2906..3041
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3090..3884
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3090..3884
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3144..3163)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3754..3773
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4072..4091)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4116..4163
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4492..5080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4981..5000
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgt
      601 ccgtcctgac gccgctgctg ctgcggggct tgacaggctc ggcccggcgg ctcccagtgc
      661 cgcgcgccaa gatccattcg ttgggggatc tgtccgtcct gacgccgctg ctgctgcggg
      721 gcttgacagg ctcggcccgg cggctcccag tgccgcgcgc caagatccat tcgttggggg
      781 atccaccggt cgccaccgct caagagtttg tgaactgcaa aatccagcct gggaaggtgg
      841 ttgtgttcat caagcccacc tgcccgtact gcaggagggc ccaagagatc ctcagtcaat
      901 tgcccatcaa acaagggctt ctggaatttg tcgatatcac agccaccaac cacactaacg
      961 agattcaaga ttatttgcaa cagctcacgg gagcaagaac ggtgcctcga gtctttattg
     1021 gtaaagattg tataggcgga tgcagtgatc tagtctcttt gcaacagagt ggggaactgc
     1081 tgacgcggct aaagcagatt ggagctctgc agacttccgg aactgggggc aacgcctctg
     1141 acggtggtgg gtctggtggt gtgagcaagg gcgaggagga taacatggcc atcatcaagg
     1201 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     1261 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1321 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     1381 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     1441 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     1501 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     1561 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggagtgcacc gaatccgagc
     1621 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     1681 atggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1741 tgcccggcgc ctacaacgtc aacatctgct tggacatcac ctcccacaac gaggactaca
     1801 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     1861 tgtacaagta atctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     1921 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     1981 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2041 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2101 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2161 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2221 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2281 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2341 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2401 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2461 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2521 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2581 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2641 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     2701 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2761 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2821 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2881 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2941 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3001 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     3061 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3121 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3181 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3241 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     3301 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3361 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3421 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3481 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3541 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3601 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3661 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3721 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3781 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3841 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3901 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3961 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4021 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     4081 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     4141 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     4201 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     4261 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     4321 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     4381 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4441 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4501 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4561 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4621 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4681 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4741 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4801 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4861 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4921 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4981 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5041 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5101 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5161 tgattctgtg gataaccgta ttaccgccat gcat
//
