LOCUS       sequence-492978-        7520 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  bacterial expression of cpCys203 FRET-based cysteine sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  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 Chem Biol. 2025 Nov 10:S2451-9456(25)00343-5. doi: 
            10.1016/j.chembiol.2025.10.006.
  PUBMED    41218609
REFERENCE   2  (bases 1 to 7520)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Chem 
            Biol. 2025 Nov 10:S2451-9456(25)00343-5. doi: 
            10.1016/j.chembiol.2025.10.006."
COMMENT     Sequence Label: pET29b_cpCys203
FEATURES             Location/Qualifiers
     source          1..7520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2058..2075
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2128..2146)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2142..2189
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2226..2681
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2313..2332)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2523..2544
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2773..3588)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2773..3588)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3496..3515
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3580..3599
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3710..4298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4199..4218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4452..4469
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4484..4623
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4549..4568)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4709..4731
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4725..4916)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5725..6807)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6769..6788
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6808..6885)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7091..7110
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7198..7217
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7198..7216
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7217..7241
                     /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             7256..7278
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7304..7321
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7322..7342
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
ORIGIN
        1 tggttactac cttgacatgg ggcgttcaat gtttttcgcg ctaccccgat cacatgaaac
       61 aacacgattt ttttaaaagt gcgatgccag agggctatgt ccaggagcgt accattttct
      121 ttaaggatga tgggaactat aaaacacgcg cagaagtgaa attcgagggt gatacattag
      181 ttaaccgcat cgaattgaag ggaattgatt ttaaggaaga tggcaatatt ctgggtcata
      241 agttagaata caattatatt agccacaatg tttatattac cgcggacaag caaaagaacg
      301 gcatcaaagc taacttcaag atccgccaca acattgaaga cggaagcgtg cagttggcag
      361 atcactacca acaaaatact cctattggtg atgggccggt gcttctgcct gataatcatt
      421 acttgtctac acagtccgcc ttgtcaaaag atccaaatga aaagcgcgat catatggttc
      481 tgttagaatt cgtaacggcg gctgggatca ctttgggaat ggacgagctg tacaaagtaa
      541 tcgctcccgc ggtaaaaaag ggggataagg agcttaagga attcatcgat aatcttatta
      601 caaaacttgg ggaagagcag ttcttccaca aagcctatga cgagacgtta aaatcccatt
      661 tcggtgacga tgtcaaggcg gatgatgtgg tcattgaggg cggcaaaatt ggcggatcgg
      721 gtggttcggg aggaagtgga gggtctggag gtaactccga ctcaggtgct agtaacagtt
      781 tggagcgcat taaacaagac ggtgtcgtcc gcattggtgt ctttggtgat aagccaccgt
      841 ttggctatgt agacgaaaag ggcgtcaatc agggttatga tatcgttctg gctaaacgta
      901 tcgcgaaaga attactgggg gatgaaaaca aagtacaatt cgttttagtc gaagccgcta
      961 accgcgtcga gtttcttaaa tcaaacaagg tggatatcat cttagcgaat tttactcaaa
     1021 ctcctgagcg cgcagagcaa gtggactttt gtcttcctta catgaaggtc gcactgggtg
     1081 tggccgtacc ccaggactcg aacatctcga gtattgagga cctgaaggac aaaaccttat
     1141 tacttaataa aggaaccaca gctgacgctt actttaccaa agagtatcca gacattaaga
     1201 cgttgaaata tgatcaaaac actgaaacgt tcgcggcatt gattgatcaa cgtggcgacg
     1261 cgttatcgca cgacaacacg ttactgttcg cgtgggtgaa agaacaccca gaattcaaaa
     1321 tggcgattat ggtatcaaag ggtgaagagt tattcactgg tgtcgtacct atccttgttg
     1381 aacttgacgg tgatgtgaac ggccacaaat tcagtgtgag cggcgagggc gagggcgatg
     1441 ccacatatgg taagctgaca ttgaagttaa tctgcacgac gggaaagttg cccgtgccat
     1501 ggccaacact tgtgacaaca ttgggttacg gattgcagtg tttcgcccgc tatcccgatc
     1561 atatgaagca gcacgacttt ttcaaaagcg ccatgccaga aggctacgta caagaacgta
     1621 cgattttttt caaggacgac gggaattata aaacgcgtgc ggaagtcaaa ttcgaaggtg
     1681 acaccttagt taatcgtatc gaattgaaag gaatcgactt taaggaggac gggaatatcc
     1741 tgggtcataa attggaatat aactacaact cacataatgt atatattacc gctgataaac
     1801 agaaaaacgg catcaaggcc aattttaaga ttcgccataa cattgaagat ggcggagtcc
     1861 agttggcgga tcactatcag cagaatacgc ccatcggaga tgggccagtg cttttaccgg
     1921 acaaccacta tttaagctat cagtcagcgc tttccaaaga ccccaacgaa aaacgcgatc
     1981 atatggtact tttggaattc gtaacggctg ctggtatcac tcttgggatg gacgagttgt
     2041 ataaatgata actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     2101 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2161 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2221 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2281 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2341 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2401 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2461 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2521 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2581 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2641 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2701 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2761 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     2821 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     2881 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     2941 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3001 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3061 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3121 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     3181 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3241 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3301 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     3361 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     3421 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     3481 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     3541 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     3601 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     3661 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3721 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3781 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3841 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3901 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3961 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4021 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4081 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4141 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4201 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4261 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4321 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4381 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4441 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4501 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4561 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4621 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4681 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4741 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     4801 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     4861 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4921 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4981 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5041 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5101 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5161 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5221 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5281 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5341 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     5401 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5461 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5521 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5581 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5641 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5701 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5761 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5821 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5881 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5941 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     6001 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6061 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6121 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6181 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6241 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6301 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     6361 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6421 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     6481 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6541 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6601 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6661 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6721 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     6781 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     6841 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     6901 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6961 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7021 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     7081 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     7141 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
     7201 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     7261 ttaactttaa gaaggagata tacatatggg atctgataaa attcatcatc atcatcatca
     7321 cgaaaacctg tacttccaga tggtatctaa gggggaagaa ttatttacgg gcgtcgttcc
     7381 cattcttgtc gaattggacg gtgacgtaaa tggccacaaa ttctctgtgt ccggcgaggg
     7441 tgagggagat gcgacctatg gcaaattaac cttgaagttc atctgtacta ctggaaaact
     7501 tccggtaccg tggccaacgc
//
