LOCUS       sequence-492566-        7605 bp    DNA     circular SYN 11-NOV-2025
DEFINITION  mammalian expression of the cpCys203 FRET-based cysteine sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7605)
  AUTHORS   Abrams RPM, Donahue RG, Ma J, Mao Y, Diolaiti ME, Ashworth A
  TITLE     An engineered cysteine sensor optimized for high-throughput 
            screening identifies regulators of intracellular thiol levels
  JOURNAL   Cell Chemical Biology, 2025
REFERENCE   2  (bases 1 to 7605)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell 
            Chemical Biology, 2025"
COMMENT     Sequence Label: pcDNA3.1_cpCys203
FEATURES             Location/Qualifiers
     source          1..7605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1229..1248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2743..2754
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(3196..3213)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3202..3426
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3472..3900
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3559..3578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3769..3790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3909..3929)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3914..4243
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4094..4229
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4156..4175
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4310..5104
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4364..4383)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4974..4993
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5278..5399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5315..5334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5369..5388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5448..5464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5448..5464)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5461..5483)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5472..5488
                     /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(5496..5502,5503..5520,5521..5526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5541..5562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5679..5696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5850..6438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5930..5949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6609..7400,7401..7469))
                     /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     7232..7251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7470..7574)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggtttc caagggcgaa gaactgttca ccggcgtggt
      961 gcccattctg gtggaactgg acggggatgt gaacggccac aagtttagcg ttagcggcga
     1021 aggcgaaggg gatgccacat acggaaagct gaccctgaag ttcatctgca ccaccggcaa
     1081 gctgcctgtg ccttggccta cactggtcac cacactgaca tggggcgtgc agtgcttcag
     1141 cagatacccc gaccatatga agcagcacga cttcttcaag agcgccatgc ctgagggcta
     1201 cgtgcaagag cggaccatct tctttaagga cgacggcaac tacaagacca gggccgaagt
     1261 gaagttcgag ggcgacaccc tggtcaaccg gatcgagctg aagggcatcg acttcaaaga
     1321 ggacggcaac atcctgggcc acaagctcga gtacaactac atcagccaca acgtgtacat
     1381 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccggc acaacatcga
     1441 ggacggctct gtgcagctgg ccgatcacta ccagcagaac acacccatcg gagatggccc
     1501 tgtgctgctg cccgataacc actacctgag cacacagagc gccctgagca aggaccccaa
     1561 cgagaagagg gatcacatgg tgctgctgga attcgtgacc gccgctggca tcacactcgg
     1621 catggacgaa ctgtacaaag taatagcgcc agccgtcaaa aagggggata aggaactgaa
     1681 ggagtttatc gacaatctca tcacaaaatt gggggaggag caattctttc ataaggctta
     1741 cgatgagacc ctcaagagtc atttcgggga tgatgtgaag gccgacgacg tagtaattga
     1801 aggtgggaag atcggtgggt ccggtggcag tgggggctct ggcggtagcg gtgggaatag
     1861 tgatagtggt gcgagtaact ctcttgagcg aataaagcaa gatggagtgg ttcgaattgg
     1921 tgtttttggc gataagccgc cgtttggata cgttgacgag aaaggcgtaa atcaaggata
     1981 cgacatcgtt ctggcgaaga gaatagcgaa agaacttctg ggggacgaga ataaggttca
     2041 gtttgtcttg gtggaggcag ccaaccgggt cgagttcctc aagtcaaaca aagtggatat
     2101 aatacttgcc aattttactc agacacctga aagagcagag caggtagatt tttgtcttcc
     2161 ttacatgaag gtcgctctcg gagtggccgt acctcaagac tctaacatat ctagtattga
     2221 agatttgaag gataagactc tgctgctcaa caaaggcacc acagcggacg catatttcac
     2281 aaaggaatac cctgacatca agactcttaa gtacgatcag aacacggaaa catttgccgc
     2341 gctcatcgat cagaggggag atgcgctttc ccacgataat accctcttgt tcgcgtgggt
     2401 gaaggaacac cctgaattta aaatggcaat catggtgtct aaaggcgagg aactctttac
     2461 aggcgtggtc cctatcctcg tcgagctgga tggcgacgtg aacggacata agttctccgt
     2521 gtctggcgaa ggcgagggcg acgctaccta tggcaagctg acactcaagc tgatctgtac
     2581 aacagggaaa ctgcccgtgc catggccaac tctcgtgaca acactcggct acggcctgca
     2641 gtgtttcgcc agatatcctg atcatatgaa gcaacatgat ttctttaagt ccgctatgcc
     2701 cgagggatat gtccaagaga gaacgatctt tttcaaggat gatgggaatt acaaaacccg
     2761 cgctgaagtc aagtttgaag gcgatacact cgtgaaccgc attgagctga aagggattga
     2821 tttcaaagag gatgggaata ttctcggaca caaacttgag tacaattata actcccacaa
     2881 tgtctatatc acggctgata agcagaaaaa tgggatcaaa gctaatttca agatcagaca
     2941 taatattgag gatggcggcg tccagctggc tgaccattat cagcaaaaca cccctatcgg
     3001 cgacggaccc gttctgctgc ctgacaatca ctatctgtcc taccagtccg ctctgtccaa
     3061 ggatcctaat gagaaacgcg accacatggt ccttctcgag tttgtgacag ccgccggaat
     3121 taccctcgga atggatgagc tttacaaatg agcggccgct cgagtctaga gggcccgttt
     3181 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3241 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3301 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3361 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3421 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3481 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3541 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3601 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3661 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3721 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3781 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3841 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3901 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3961 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4021 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4081 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4141 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     4201 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4261 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4321 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4381 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4441 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4501 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4561 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4621 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4681 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4741 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4801 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4861 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4921 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4981 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5041 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5101 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5161 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5221 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5281 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5341 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5401 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5461 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5521 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5581 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5641 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5701 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5761 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5821 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5881 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5941 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6001 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6061 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6121 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6181 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6241 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6301 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6361 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6421 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6481 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6541 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6601 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6661 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6721 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6781 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6841 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6901 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6961 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7021 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7081 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7141 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7201 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7261 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7321 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7381 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7441 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7501 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7561 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
