LOCUS       sequence-199007-        7141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  His6 tagged Keap1 for E.coli expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7141)
  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 7141)
  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-Keap1
FEATURES             Location/Qualifiers
     source          1..7141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    20..159
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     75..94
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(174..191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(345..933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(425..444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1044..1063)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1055..1870
                     /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             1055..1870
                     /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(1128..1147)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1962..2417)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2099..2120)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2311..2330
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2454..2501)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2497..2515
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4435..4455)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(4474..4491)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4510..4532
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4547..4571
                     /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(4571..4590)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4572..4590)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4674..4693)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4899..4976
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4977..6059
                     /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(4996..5015)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6868..7059
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(7053..7075)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
       61 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa
      121 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
      181 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      241 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      301 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      361 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      421 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      481 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
      541 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
      601 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
      661 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
      721 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
      781 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
      841 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
      901 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
      961 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact
     1021 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     1081 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     1141 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     1201 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     1261 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     1321 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     1381 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     1441 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     1501 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     1561 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     1621 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     1681 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     1741 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     1801 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     1861 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac
     1921 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     1981 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2041 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2101 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2161 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2221 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2281 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2341 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2401 gccgctacag ggcgcgtccc attcgccaat ccggatatag ttcctccttt cagcaaaaaa
     2461 cccctcaaga cccgtttaga ggccccgagg ggttatgcta gttattgctc agcggtggca
     2521 gcagccaact cagcttcctt tcgggctttg ttatcaacag gtacagttct gctggtcaat
     2581 ctgcttccgg cagggctcca tggtgacagc cacgcccacc ccactccggc ccgatgtcat
     2641 tcgggtcacc tcgctccagg tgtctgtatc tgggtcgtaa cactccacac tgtccaggaa
     2701 cgtgtgacca tcatagcctc caaggacgta gattctcccc tggtggacag tgatccccag
     2761 ggcacttcgc cggtgcttca tgggggctac gaaagtccac gtctctgttt ccacatcgta
     2821 gcgctccacg ctgttcagct ggtcctgacc atcatagccc ccagcagcat agatacagtt
     2881 gtgcaggacg cagacgcctg ccccgcttcg gatggtgttc attgctgtga tcattcgcca
     2941 ctcgttcctc tctgggtagt aacactcagc tgaattaagg cggtttgtcc cgtcaaagcc
     3001 ccccacggca taaaggagac gattgaggac agccacgccc accccgatcc ttcgtgtcag
     3061 cattggggcc accaagtgcc actcatcccg ctctggctca tacctctcca cactgttgtg
     3121 gtggatgcag ccgtgggagc cgccgacggc atagatgtgg ccatcgatga cccccacccc
     3181 gatgcggtta cggggcacgc tcatgggggc gcagggcgac cactgattgg tcatggggtt
     3241 gtaacagtcc agggcgctgg agtcggtgtt gccgtcgggc gagttgttcc tgccgcccac
     3301 ggcgtacaac agcccgccca ccacgcagcc ggccaggccg ctccgcggca cctgcaggtc
     3361 cgccaaccgg agccaggtgc cgtcactggg gttgtaagcc tccaggtagc tgagcgactg
     3421 tcggaagtag ccgcccgcgg tgtagatcag gcggcccacc ttgggcgccc ggcagggcat
     3481 cacctgcgtg ggcttgtgca gggtgagctc ctcgaagatc ttgaccaggt agtccttgca
     3541 gcgggagtcg gactgcagga tctcgcactt ctgcagctgc atctgcagga agttcggcgt
     3601 caacgagtgg cagcgcacgg cccgcagcag cgcctggacg tagaaccgtc gctgttcgca
     3661 gtcgtacttg acccagttga tgcaggcgtg gaagacctcg gactcgcagc gcacgttcag
     3721 gtcgtcccgg ctgatgaggg tcaccagttg gcagtgggac aggttgaaga actcctcttg
     3781 cttggccacc tccccaaaat gcatgtagat gtactcccgg gcacgctggt gcaactccac
     3841 acagccaatc tgctcagcga agttggcgat gccgatggca ttgctggggt ccagctgctg
     3901 caccaggaag tcactgcagg cacggacaac gctgtcgatc tggtacatga cagcaccgtt
     3961 catgacgtgg aggacacact tctcgcccat ggagatggag gccgtgtagg cgaattcaat
     4021 gaggcgctcc atgaccttgg ggtggatacc ctcaatggac accacctcca tgccctgctc
     4081 ccgcagcccg ttggtgaaca tggccttgaa gacagggctg gatgaggcca gcaccacctt
     4141 gtgggccatg aactgggcgg ccggtgcatc ctggtacttg acctgcagtg tgacgtcaca
     4201 cagctgctgg ctgagccgca gctcgttcat gatgccaaag gcctgcttgg tatgatcctc
     4261 cagggtgtag ctgaaggtgc ggttgccatg ctgggagggc gtcacctccg ccttgcactc
     4321 agtggaggcg tacatcaccg cgtcccctgc cccctcaggg cactgtgact gcaggggcag
     4381 gaatcggcag caggccccag ccccgctagg cctgggatct ggctgcatcc ctgagccctg
     4441 gaaatacaag ttttctccgg agccggagct gctgtgatga tgatgatgat ggctgctgcc
     4501 catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg
     4561 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta
     4621 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     4681 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     4741 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     4801 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     4861 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     4921 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     4981 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     5041 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     5101 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     5161 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     5221 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     5281 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     5341 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     5401 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     5461 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     5521 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     5581 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     5641 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     5701 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     5761 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     5821 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     5881 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     5941 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     6001 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6061 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     6121 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     6181 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     6241 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     6301 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     6361 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg
     6421 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     6481 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     6541 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     6601 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     6661 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     6721 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     6781 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     6841 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     6901 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     6961 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     7021 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     7081 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     7141 g
//
