LOCUS       sequence-430416-        5360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression HyPerFLEX targeted to the mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5360)
  AUTHORS   Potekhina ES, Bass DI, Ezerina D, Fleckenstein DD, Chebotarev AS, 
            Sysoeva VA, Maltsev DI, Pak VV, Moshchenko AA, Sokolov AI, 
            Myasnyanko IN, Baranov MS, Fedotov AB, Zheltikov AM, Lanin AA, 
            Messens J, Belousov VV
  TITLE     A color-tailored fluorogenic sensor for hydrogen peroxide.
  JOURNAL   Nat Chem Biol. 2025 Oct 16. doi: 10.1038/s41589-025-02036-6.
  PUBMED    41102408
REFERENCE   2  (bases 1 to 5360)
  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: "Nat Chem 
            Biol. 2025 Oct 16. doi: 10.1038/s41589-025-02036-6."
COMMENT     Sequence Label: pCS2+-tdmito-HyPerFLEX
FEATURES             Location/Qualifiers
     source          1..5360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             106..180
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             202..273
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     polyA_signal    1412..1546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1436..1455)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1490..1509
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1659..1679)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1659..1677)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1698..1714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1698..1714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1711..1733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1722..1738
                     /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(1746..1752,1753..1770,1771..1776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1791..1812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1929..1946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2100..2688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2180..2199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2859..3650,3651..3719))
                     /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     3482..3501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3720..3824)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3850..4305)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3987..4008)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4199..4218
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4376..5360
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattcg ccaccatgtc cgtcctgacg
      121 ccgctgctgc tgcggggctt gacaggctcg gcccggcggc tcccagtgcc gcgcgccaag
      181 atccattcgt tgggggatct gtccgtcctg acgccgctgc tgctgcgggg cttgacaggc
      241 tcggcccggc ggctcccagt gccgcgcgcc aagatccatt cgttggggga taccggtcac
      301 ctggctaatg aggagcaaaa cgaattggaa ggtgctttta agctgggctt gatctttacc
      361 gtcgccccgt acttgctgcc taagttgatc gtcagcctgc gccgcaccgc cccgaagatg
      421 cctttgatgt tggaggagaa ttacacccac acccttaccg agtcactgaa acgcggcgac
      481 gttgacgcca tcattgtggc agagccgttt caagagccgg gtattgtgac tgaacctttg
      541 tatgacgagc cgtttttcgt tatcgttccg aagggccata gcttcgagga gttggatgcc
      601 gttagccctc gcatgttggg cgaagaacaa gttctgcttc tgaccgaggg caactgtatg
      661 cgtgaccagg tgcttagcat ttttaccaag gtcaaggtgc acatgaagaa agccctttcc
      721 ggtgacagct attgggtctt tgtgaaacgg gtaggtggca ctggcggtga gcatgttgtc
      781 tttggcagtg aggacatcga gaacactatg gccaaaatgg acgacggaca actggatggg
      841 ttggcctttg gcgcaattca gctcgatggt gacgggaata tcctgcagta caatgctgct
      901 gaaggagaca tcacaggcag agatcccaaa caggtgattg ggaagaactt cttcaaggat
      961 gttgcacctg gaacggattc tcccgagttt tacggcaaat tcaaggaagg cgtagcgtca
     1021 gggaatctga acaccatgtt cgaatggatg ataccgactc ttaccgatca tagttgcagc
     1081 gagcttgccg ccaaacagcg cattcagggt ctcaccaaca cccttcaagg ctcaagcatc
     1141 gataccatcc gccacatggt tgccagtggc ttggcaatca gcgtcttgcc tgccactgcc
     1201 cttaccgaaa acgaccacat gctgtttagc attatcccgt ttgaaggcac cccgcctagc
     1261 cgccgcgttg tcctggccta tcgccgcaac tttgtccgcc ctaaggccct ttcagccatg
     1321 aaagccgcaa ttatgcaaag ccaactgcat ggtgttagct tcatctgcga ttgactcgag
     1381 cctctagaac tatagtgagt cgtattacgt agatccagac atgataagat acattgatga
     1441 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1501 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1561 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc
     1621 ggcgccaatg cattgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg
     1681 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1741 ccacacaaca tacgagccgg gagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1801 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1861 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1921 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1981 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2041 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2101 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2161 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2221 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2281 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2341 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2401 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2461 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2521 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2581 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2641 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2701 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2761 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2821 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2881 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2941 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3001 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3061 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3121 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3181 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3241 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3301 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3361 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3421 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3481 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3541 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3601 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3661 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3721 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3781 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3841 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3901 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3961 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     4021 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     4081 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     4141 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     4201 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     4261 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca
     4321 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga
     4381 ccgccaattc aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct
     4441 gtgccaattc aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc
     4501 cagccaattc aatatggcgg acttggcacc atgccaattc aatatggcgg acctggcact
     4561 gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc
     4621 tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat
     4681 atgccaggat caatatatag gcaatatcca atatggccct atgccaatat ggctattggc
     4741 caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta
     4801 ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat
     4861 accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg
     4921 acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg
     4981 taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc
     5041 aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac
     5101 ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt
     5161 actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa
     5221 tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc
     5281 aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa
     5341 gcaatgctcg tttagggaac
//
