LOCUS       sequence-196837-        4859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded H2O2 probe for ratiometric and fluorescence 
            lifetime imaging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4859)
  AUTHORS   Bilan DS, Pase L, Joosen L, Gorokhovatsky AY, Ermakova YG, Gadella 
            TW, Grabher C, Schultz C, Lukyanov S, Belousov VV
  TITLE     HyPer-3: a genetically encoded H2O2 probe with improved performance 
            for ratiometric and fluorescence lifetime imaging.
  JOURNAL   ACS Chem Biol. 2012 Dec 20.
  PUBMED    23256573
REFERENCE   2  (bases 1 to 4859)
  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: "ACS Chem 
            Biol. 2012 Dec 20."
COMMENT     Sequence Label: pQE-30-HyPer3
FEATURES             Location/Qualifiers
     source          1..4859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             151..1584
                     /codon_start=1
                     /product="improved fluorescent indicator for intracellular 
                     H2O2 (Bilan et al., 2013)"
                     /label=HyPer-3
                     /translation="EMASQQGETMSGPLHIGLIPTVGPYLLPHIIPMLYQTFPKLEMYL
                     HEAQTHQLLAQLDSGKLDCVILALVKESEAFIEVPLFDEPMLLAIYEDHPWANRECVPM
                     ADLAGEKLLMLEDGHCLRDQAMSAGYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQL
                     ADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELY
                     NVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTT
                     GKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTR
                     AEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGTGFCFEAGADEDTHFRATSLETL
                     RNMVAAGSGITLLPALAVPPERKRDGVVYLPCIKPEPRRTIGLVYRPGSPLRSRYEQLA
                     EAIRARMDGHFDKVLKQAV"
     primer_bind     756..777
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(886..907)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1147..1166)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     terminator      1606..1700
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1744..2403
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1810..1829)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2468..2554
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2601..2623)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2709..2849
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2764..2783
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2864..2881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3035..3623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3115..3134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3794..4585,4586..4654))
                     /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     4417..4436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4655..4759)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4827..4845
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc gagatggcaa gccagcaggg cgagacgatg
      181 tccggaccgc tgcacattgg tttgattccc acagttggac cgtacctgct accgcatatt
      241 atccctatgc tgtaccagac ctttccaaag ctggaaatgt atctgcatga agcacagacc
      301 caccagttac tggcgcaact ggacagcggc aaactcgatt gcgtgatcct cgcactggtg
      361 aaagagagcg aagcattcat tgaagtgccg ttgtttgatg agccaatgtt gctggctatc
      421 tatgaagatc acccgtgggc gaaccgcgaa tgcgtaccga tggccgatct ggcaggggaa
      481 aaactgctga tgctggaaga tggtcactgt ttgcgcgatc aggcaatgtc cgccggctac
      541 aacagcgaca acgtctatat catggccgac aagcagaaga acggcatcaa ggccaacttc
      601 aagatccgcc acaacgtcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
      661 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cttccagtcc
      721 gtcctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
      781 gccgccggga tcactctcgg catggacgag ctgtacaacg tggatggcgg tagcggtggc
      841 accggcagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      901 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      961 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1021 ctcgtgacca ccctcggcta cggcctgaag tgcttcgccc gctaccccga ccacatgaag
     1081 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1141 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1201 gtgaaccgca tcgagctgaa gggcatcggc ttcaaggagg acggcaacat cctggggcac
     1261 aagctggagt acaacggcac cggtttctgt tttgaagccg gggcggatga agatacacac
     1321 ttccgcgcga ccagcctgga aactctgcgc aacatggtgg cggcaggtag cgggatcact
     1381 ttactgccag cgctggctgt gccgccggag cgcaaacgcg atggggttgt ttatctgccg
     1441 tgcattaagc cggaaccacg ccgcactatt ggtctggttt atcgtcctgg ctcaccgctg
     1501 cgcagccgct atgagcagct ggcagaggcc atccgcgcaa gaatggatgg ccatttcgat
     1561 aaagttttaa aacaggcggt ttaaaagctt aattagctga gcttggactc ctgttgatag
     1621 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     1681 cgttttttat tggtgagaat ccaagctagc ttggcgagat tttcaggagc taaggaagct
     1741 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     1801 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     1861 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     1921 attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac
     1981 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     2041 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     2101 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     2161 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     2221 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     2281 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2341 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg
     2401 taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt
     2461 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     2521 tttgtcggtg aacgctctcc tgagtaggac aaatccgccc tctagagctg cctcgcgcgt
     2581 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     2641 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     2701 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     2761 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     2821 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     2881 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2941 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3001 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3061 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3121 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3181 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3241 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3301 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3361 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3421 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3481 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3541 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3601 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3661 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3721 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3781 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3841 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3901 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3961 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4021 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4081 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4141 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4201 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4261 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4321 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4381 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4441 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4501 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4561 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4621 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4681 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4741 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4801 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
