LOCUS       sequence-430076-        8085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 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 8085)
  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 8085)
  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
FEATURES             Location/Qualifiers
     source          1..8085
                     /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          1519..2535
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2459..2481
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2543..2562
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2596..2605
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3216..3237
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3343..3364)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3604..3623)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4042..4749
                     /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(4187..4205)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4455..4474
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     intron          4798..4930
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4913..4932
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4939..5527
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4992..5012)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5410..5421)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5554..5571)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5560..5671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5767..5787)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5767..5785)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5806..5822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5928..5947)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5993..6048
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6047..6066)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6409..6425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6433..6449
                     /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(6457..6463,6464..6481,6482..6487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6502..6523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6582..6777
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6628..6763
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6783..6917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6820..6839)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6874..6893
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6985..7002)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7156..7744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7236..7255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7915..8085,1..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 gnsgcgcgcc rggsggggsg
     1381 gggsggggsg rggggsgggg sggggsgagg cggagaggtg cggcggcagc caatcagagc
     1441 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
     1501 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
     1561 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
     1621 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
     1681 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
     1741 ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg
     1801 cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc
     1861 gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa
     1921 ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg
     1981 ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
     2041 gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
     2101 ggggtgccgg gcggggcggg gccgcctcgg gccggggrgg gctcggggga ggggcgcggc
     2161 ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg
     2221 taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct
     2281 gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg
     2341 aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc
     2401 cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg
     2461 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc
     2521 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc
     2581 aaagaattcg gtaccgccac catggagatg gcgagccagc agggcgagac gatgtccgga
     2641 ccgctgcaca ttggtttgat tcccacagtt ggaccgtacc tgctaccgca tattatccct
     2701 atgctgcacc agacctttcc aaagctggaa atgtatctgc atgaagcaca gacccaccag
     2761 ttactggcgc aactggacag cggcaaactc gattgcgtga tcctcgcact ggtgaaagag
     2821 agcgaagcat tcattgaagt gccgttgttt gatgagccaa tgttgctggc tatctatgaa
     2881 gatcacccgt gggcgaaccg cgaatgcgta ccgatggccg atctggcagg ggaaaaactg
     2941 ctgatgctgg aagatggtca ctgtttgcgc gatcaggcaa tgggtttctg ttttgaagcc
     3001 tcactcgaga acgtctatat caaggccgac aagcagaaga acggcatcaa ggcgaacttc
     3061 aagatccgcc acaacatcga ggacggcggc gtgcagctcg cctaccacta ccagcagaac
     3121 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cgtgcagtcc
     3181 aaactttcga aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     3241 gccgccggga tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg
     3301 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3361 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc
     3421 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3481 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3541 cacgacttct tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc
     3601 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3661 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3721 ctggagtaca acctgccgga ccaactgggg gcggattacg atacacactt ccgcgcgacc
     3781 agcctggaaa ctctgcgcaa catggtggcg gcaggtagcg ggatcacttt actgccagcg
     3841 ctggctgtgc cgccggagcg caaacgcgat ggggttgttt atctgccgtg cattaagccg
     3901 gaaccacgcc gcactattgg tctggtttat cgtcctggct caccgctgcg cagccgctat
     3961 gagcagctgg cagaggccat ccgcgcaaga atggatggcc atttcgataa agttttaaaa
     4021 caggcggttg gtctccggcc gatggtgagc aagggcgagg aggataacat ggccatcatc
     4081 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     4141 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     4201 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     4261 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     4321 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     4381 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     4441 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     4501 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     4561 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     4621 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     4681 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     4741 ctgtacaagt ccggaaacta atgtacaagt aaagcggccc cggactcgag gccgcaggta
     4801 agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag
     4861 agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt
     4921 ctctccacag gtgtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     4981 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     5041 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     5101 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     5161 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     5221 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     5281 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     5341 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     5401 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     5461 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     5521 tccccgcctg gaattcgagc tcggtacgat cagcctcgac tgtgccttct agttgccagc
     5581 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5641 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5701 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcccagctt
     5761 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     5821 tgtgtgaaat tgttatccgc taattcactc ctcaggtgca ggctgcctat cagaaggtgg
     5881 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct ttttccctct
     5941 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     6001 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     6061 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     6121 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     6181 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     6241 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     6301 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     6361 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     6421 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6481 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6541 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     6601 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     6661 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     6721 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     6781 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     6841 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     6901 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6961 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     7021 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     7081 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     7141 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     7201 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     7261 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     7321 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     7381 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     7441 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     7501 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     7561 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     7621 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7681 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7741 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7801 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7861 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7921 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7981 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     8041 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatc
//
