LOCUS       sequence-430078-        8084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the loss-of-function version of the genetically encoded, 
            ratiometric hydrogen peroxide sensor oROS-Gr in mamalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8084)
  AUTHORS   Lee J.D., Won. W., Kimball K., Wang Y., Yeboah F., Evitts K.M., 
            Neiswanger C., Schattauer S., Rappleye M., Bremner S.B., Chun C., 
            Smith N., Mack D.L., Young J.E., Lee C.J., Chavkin C., Berndt A.
  TITLE     Structure-guided engineering of a fast genetically encoded sensor 
            for real-time H2O2 monitoring
  JOURNAL   bioRxiv 2024.01.31.578117
REFERENCE   2  (bases 1 to 8084)
  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: "bioRxiv 
            2024.01.31.578117"
COMMENT     Sequence Label: pC3.1_CAG_oROS-Gr_LF(C199S)
FEATURES             Location/Qualifiers
     source          1..8084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     453..472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(691..795)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    846..857
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     enhancer        861..1240
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1518..2534
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2458..2480
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2542..2561
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2595..2604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3215..3236
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3342..3363)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3603..3622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4041..4748
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4186..4204)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4454..4473
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     intron          4797..4929
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4912..4931
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4938..5526
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4991..5011)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5409..5420)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5553..5570)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5559..5670
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5766..5786)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5766..5784)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5805..5821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5927..5946)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5992..6047
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6046..6065)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6408..6424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6432..6448
                     /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(6456..6462,6463..6480,6481..6486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6501..6522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6581..6776
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6627..6762
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6782..6916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6819..6838)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6873..6892
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6984..7001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7155..7743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7235..7254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7914..8084,1..621,622..690))
                     /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"
ORIGIN
        1 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
       61 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      121 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      181 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      241 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      301 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      361 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      421 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      481 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      541 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      601 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
      661 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
      721 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      781 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcgacat tgattattga
      841 ctagagtcat cctcatcgtc gacattgatt attgactagt tattaatagt aatcaattac
      901 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      961 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     1021 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     1081 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     1141 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     1201 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
     1261 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
     1321 ttaattattt tgtgcagcga tgggggcggg gggggggggg gggcgcgccr gggggggggg
     1381 gggggggsgr ggggsggggs ggggsgrggc ggagaggtgc ggcggcagcc aatcagagcg
     1441 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1501 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1561 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1621 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     1681 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     1741 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     1801 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     1861 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     1921 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     1981 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     2041 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     2101 gggtgccggg cggggcgggg ccgcctcggg ccggggrggg ctcgggggrg gggcgcggcg
     2161 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2221 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     2281 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     2341 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     2401 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     2461 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     2521 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     2581 aagaattcgg taccgccacc atggagatgg cgagccagca gggcgagacg atgtccggac
     2641 cgctgcacat tggtttgatt cccacagttg gaccgtacct gctaccgcat attatcccta
     2701 tgctgcacca gacctttcca aagctggaaa tgtatctgca tgaagcacag acccaccagt
     2761 tactggcgca actggacagc ggcaaactcg attgcgtgat cctcgcactg gtgaaagaga
     2821 gcgaagcatt cattgaagtg ccgttgtttg atgagccaat gttgctggct atctatgaag
     2881 atcacccgtg ggcgaaccgc gaatgcgtac cgatggccga tctggcaggg gaaaaactgc
     2941 tgatgctgga agatggtcac agtttgcgcg atcaggcaat gggtttctgt tttgaagcct
     3001 cactcgagaa cgtctatatc aaggccgaca agcagaagaa cggcatcaag gcgaacttca
     3061 agatccgcca caacatcgag gacggcggcg tgcagctcgc ctaccactac cagcagaaca
     3121 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca
     3181 aactttcgaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3241 ccgccgggat cactctcggc atggacgagc tgtacaaggg cggtaccgga gggagcatgg
     3301 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     3361 acgtaaacgg ccacaagttc agcgtgtccg gcgagggtga gggcgatgcc acctacggca
     3421 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     3481 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     3541 acgacttctt caagtccgcc atgcccgaag gctacatcca ggagcgcacc atcttcttca
     3601 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     3661 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     3721 tggagtacaa cctgccggac caactggggg cggattacga tacacacttc cgcgcgacca
     3781 gcctggaaac tctgcgcaac atggtggcgg caggtagcgg gatcacttta ctgccagcgc
     3841 tggctgtgcc gccggagcgc aaacgcgatg gggttgttta tctgccgtgc attaagccgg
     3901 aaccacgccg cactattggt ctggtttatc gtcctggctc accgctgcgc agccgctatg
     3961 agcagctggc agaggccatc cgcgcaagaa tggatggcca tttcgataaa gttttaaaac
     4021 aggcggttgg tctccggccg atggtgagca agggcgagga ggataacatg gccatcatca
     4081 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     4141 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     4201 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     4261 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     4321 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     4381 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     4441 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     4501 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     4561 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     4621 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     4681 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     4741 tgtacaagtc cggaaactaa tgtacaagta aagcggcccc ggactcgagg ccgcaggtaa
     4801 gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga
     4861 gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc
     4921 tctccacagg tgtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     4981 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     5041 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     5101 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     5161 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5221 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5281 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     5341 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     5401 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     5461 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     5521 ccccgcctgg aattcgagct cggtacgatc agcctcgact gtgccttcta gttgccagcc
     5581 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     5641 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     5701 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcccagcttt
     5761 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     5821 gtgtgaaatt gttatccgct aattcactcc tcaggtgcag gctgcctatc agaaggtggt
     5881 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatcgatctt tttccctctg
     5941 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     6001 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     6061 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     6121 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga
     6181 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
     6241 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
     6301 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     6361 tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt
     6421 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     6481 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     6541 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa
     6601 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     6661 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     6721 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     6781 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6841 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6901 ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6961 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     7021 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     7081 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     7141 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     7201 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     7261 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     7321 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     7381 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     7441 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     7501 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     7561 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     7621 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     7681 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     7741 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     7801 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7861 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     7921 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7981 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     8041 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatc
//
