LOCUS       sequence-174258-        8050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  His6-halo tagged human Keap1 protein for E.coli expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8050)
  AUTHORS   Parvez S, Fu Y, Li J, Long MJ, Lin H, Lee DK, Hu GS, Aye Y
  TITLE     Substoichiometric Hydroxynonenylation of a Single Protein 
            Recapitulates Whole-Cell-Stimulated Antioxidant Response.
  JOURNAL   J Am Chem Soc. 2014 Dec 31.
  PUBMED    25544059
REFERENCE   2  (bases 1 to 8050)
  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: "J Am Chem 
            Soc. 2014 Dec 31."
COMMENT     Sequence Label: pet28a-His6-Halo-Tev-Keap1
FEATURES             Location/Qualifiers
     source          1..8050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1212..1403
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1397..1419)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1505..1644
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1560..1579
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1659..1676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1830..2418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1910..1929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2529..2548)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2540..3355
                     /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             2540..3355
                     /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     complement(2613..2632)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3447..3902)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3584..3605)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3796..3815
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3939..3986)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3982..4000
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5920..5940)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5947..6837)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             complement(6841..6858)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(6868..6885)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6904..6926
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6941..6965
                     /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)."
     primer_bind     complement(6965..6984)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6966..6984)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7068..7087)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7293..7370
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(7371..8050,1..403)
                     /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     complement(7390..7409)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
       61 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
      121 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
      181 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
      241 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
      301 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      361 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      421 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
      481 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
      541 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
      601 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
      661 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
      721 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
      781 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
      841 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
      901 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
      961 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     1021 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     1081 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     1141 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     1201 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     1261 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     1321 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     1381 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     1441 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     1501 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     1561 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     1621 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1681 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1741 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1801 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1861 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1921 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1981 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2041 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2101 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2161 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2221 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     2281 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2341 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2401 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2461 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     2521 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     2581 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     2641 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     2701 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     2761 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     2821 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     2881 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     2941 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     3001 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     3061 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     3121 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     3181 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     3241 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     3301 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     3361 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3421 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     3481 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3541 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3601 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3661 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3721 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3781 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3841 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3901 gtcccattcg ccaatccgga tatagttcct cctttcagca aaaaacccct caagacccgt
     3961 ttagaggccc caaggggtta tgctagttat tgctcagcgg tggcagcagc caactcagct
     4021 tcctttcggg ctttgttatc aacaggtaca gttctgctgg tcaatctgct tccggcaggg
     4081 ctccatggtg acagccacgc ccaccccact ccggcccgat gtcattcggg tcacctcgct
     4141 ccaggtgtct gtatctgggt cgtaacactc cacactgtcc aggaacgtgt gaccatcata
     4201 gcctccaagg acgtagattc tcccctggtg gacagtgatc cccagggcac ttcgccggtg
     4261 cttcatgggg gctacgaaag tccacgtctc tgtttccaca tcgtagcgct ccacgctgtt
     4321 cagctggtcc tgaccatcat agcccccagc agcatagata cagttgtgca ggacgcagac
     4381 gcctgccccg cttcggatgg tgttcattgc tgtgatcatt cgccactcgt tcctctctgg
     4441 gtagtaacac tcagctgaat taaggcggtt tgtcccgtca aagcccccca cggcataaag
     4501 gagacgattg aggacagcca cgcccacccc gatccttcgt gtcagcattg gggccaccaa
     4561 gtgccactca tcccgctctg gctcatacct ctccacactg ttgtggtgga tgcagccgtg
     4621 ggagccgccg acggcataga tgtggccatc gatgaccccc accccgatgc ggttacgggg
     4681 cacgctcatg ggggcgcagg gcgaccactg attggtcatg gggttgtaac agtccagggc
     4741 gctggagtcg gtgttgccgt cgggcgagtt gttcctgccg cccacggcgt acaacagccc
     4801 gcccaccacg cagccggcca ggccgctccg cggcacctgc aggtccgcca accggagcca
     4861 ggtgccgtca ctggggttgt aagcctccag gtagctgagc gactgtcgga agtagccgcc
     4921 cgcggtgtag atcaggcggc ccaccttggg cgcccggcag ggcatcacct gcgtgggctt
     4981 gtgcagggtg agctcctcga agatcttgac caggtagtcc ttgcagcggg agtcggactg
     5041 caggatctcg cacttctgca gctgcatctg caggaagttc ggcgtcaacg agtggcagcg
     5101 cactgcccgc agcagcgcct ggacgtagaa ccgtcgctgt tcgcagtcgt acttgaccca
     5161 gttgatgcag gcgtggaaga cctcggactc gcagcgcacg ttcaggtcgt cccggctgat
     5221 gagggtcacc agttggcagt gggacaggtt gaagaactcc tcttgcttgg ccacctcccc
     5281 aaaatgcatg tagatgtact cccgggcacg ctggtgcaac tccacacagc caatctgctc
     5341 agcgaagttg gcgatgccga tggcattgct ggggtccagc tgctgcacca ggaagtcact
     5401 gcaggcacgg acaacgctgt cgatctggta catgacagca ccgttcatga cgtggaggac
     5461 acacttctcg cccatggaga tggaggccgt gtaggcgaat tcaatgaggc gctccatgac
     5521 cttggggtgg ataccctcaa tggacaccac ctccatgccc tgctcccgca gcccgttggt
     5581 gaacatggcc ttgaagacag ggctggatga ggccagcacc accttgtggg ccatgaactg
     5641 ggcggccggt gcatcctggt acttgacctg cagtgtgacg tcacacagct gctggctgag
     5701 ccgcagctcg ttcatgatgc caaaggcctg cttggtatga tcctccaggg tgtagctgaa
     5761 ggtgcggttg ccatgctggg agggcgtcac ctccgccttg cactcagtgg aggcgtacat
     5821 caccgcgtcc cctgccccct cagggcactg tgactgcagg ggcaggaatc ggcagcaggc
     5881 cccagccccg ctaggcctgg gatctggctg catccctgag ccctggaaat acaagttttc
     5941 tccggagccg gaaatctcga gcgtcgacag ccagcgcgcg atctcgctgc cgatcaggtc
     6001 cgggttgtct tcttgcagca gattcagacc cgggccgatg tccacagcct tgcagttagg
     6061 caggcttttg gccaggcgag cggcttcggc cggtgggatc agaacgcctg gggtgcccca
     6121 gaacagcagc ttcgggacag gggactggtg cagccagtcc atgtattctt cgaccagcgc
     6181 gacgatgttc gctggctcac cggcgattgg cagctcgttt gggaagcgcc acagtggctc
     6241 gcggtcaaca gggttcagga acggctcgcg gtaatggtcc atctcgactt cagtcagcgg
     6301 gcggacgaca cccatcggca gcgtaccctc gataaaaacg ttctgatcga tgatcagctt
     6361 gcggccgacg tcggtggtgc ggaaggcctg gaaggtctcg cgggcaaatt ctggccattc
     6421 gtcccaggtc gggatagggc ggatgaactc cataaatgca atacctttga cgcgctctgg
     6481 attgcgcttg gcccagtgga aacccagagc ggagccccag tcgtgaatga ccaggacgac
     6541 ctcttccaga cccagggctt cgatgaaggc atccatgaag cggacgtggt cgtcgaagaa
     6601 ataacccagg tctggtttgt cggatttgcc cataccgatc aggtctggag caatgcagcg
     6661 atgggtcggt gcaacatgcg ggatgatgtt gcgccacacg taggaggagg tcgggttacc
     6721 gtgcaggaac agcacagggg tgccatcgcg cggaccaaca tcgacgtagt gcatgcgctc
     6781 gcccaggact tccacataat gggggtcgaa tggaaagcca gtaccgattt ctgccatatg
     6841 gctaccacga ggcaccaggc cgctgctgtg atgatgatga tgatggctgc tgcccatggt
     6901 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     6961 attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg
     7021 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     7081 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     7141 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     7201 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     7261 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     7321 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     7381 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     7441 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     7501 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     7561 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     7621 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     7681 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     7741 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     7801 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     7861 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     7921 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     7981 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     8041 ccggttttca
//
