LOCUS       sequence-357282-        7339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human domain with His-tag and MBP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7339)
  AUTHORS   Benz C, Ali M, Krystkowiak I, Simonetti L, Sayadi A, Mihalic F, 
            Kliche J, Andersson E, Jemth P, Davey NE, Ivarsson Y
  TITLE     Proteome-scale mapping of binding sites in the unstructured regions 
            of the human proteome.
  JOURNAL   Mol Syst Biol. 2022 Jan;18(1):e10584. doi: 10.15252/msb.202110584.
  PUBMED    35044719
REFERENCE   2  (bases 1 to 7339)
  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: "Mol Syst 
            Biol."; date: "2022-01"; volume: "18(1):e10584. doi"; pages: " 
            10.15252/msb.202110584"
COMMENT     Sequence Label: pETM41_KEAP1_KELCH
FEATURES             Location/Qualifiers
     source          1..7339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3578..3595
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3599..4699
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     4673..4696
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4751..4771
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5688..5705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5758..5776)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5772..5819
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5856..6311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5943..5962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6153..6174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6403..7218)
                     /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(6403..7218)
                     /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     7126..7145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7210..7229
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgaaacat caccatcacc atcaccccat
     3601 gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct
     3661 cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc
     3721 ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat
     3781 cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac
     3841 cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa
     3901 cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga
     3961 tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa
     4021 agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct
     4081 gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga
     4141 cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa
     4201 aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg
     4261 cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt
     4321 gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg
     4381 cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct
     4441 cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg
     4501 tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac
     4561 tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg
     4621 gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc
     4681 cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca ataacaacaa
     4741 ccccatgagc gagaatcttt attttcaggg cgccatggcg ccgaaagtgg gtcgtctgat
     4801 ctacaccgcg ggtggctatt tccgtcagag cctgagctac ctggaggcgt ataacccgag
     4861 cgatggtacc tggctgcgtc tggcggacct gcaagttccg cgtagcggtc tggcgggttg
     4921 cgtggttggt ggcctgctgt acgcggttgg tggccgtaac aacagcccgg atggtaacac
     4981 cgacagcagc gcgctggatt gctataaccc gatgaccaac caatggagcc cgtgcgcgcc
     5041 gatgagcgtt ccgcgtaacc gtatcggtgt gggcgttatc gatggtcaca tttacgcggt
     5101 gggtggcagc cacggctgca ttcaccacaa cagcgttgag cgttatgagc cggaacgtga
     5161 cgaatggcat ctggtggcgc cgatgctgac ccgtcgtatt ggcgtgggcg ttgcggtgct
     5221 gaaccgtctg ctgtatgcgg ttggtggctt tgatggtacc aaccgtctga acagcgcgga
     5281 gtgctactat ccggagcgta acgaatggcg tatgatcacc gcgatgaaca ccattcgtag
     5341 cggtgcgggc gtttgcgtgc tgcacaactg catttacgcg gcgggtggct atgatggtca
     5401 ggaccaactg aacagcgtgg aacgttacga cgttgagacc gaaacctgga ccttcgtggc
     5461 gccgatgaaa caccgtcgta gcgcgctggg tatcaccgtg caccaaggcc gtatttacgt
     5521 tctgggtggc tatgacggcc acacctttct ggacagcgtg gagtgctatg acccggatac
     5581 cgacacctgg agcgaagtta cccgtatgac cagcggccgt agcggtgttg gcgtggcggt
     5641 tacctaagaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     5701 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     5761 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     5821 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     5881 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5941 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     6001 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     6061 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     6121 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6181 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     6241 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     6301 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6361 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     6421 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     6481 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     6541 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     6601 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     6661 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     6721 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     6781 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     6841 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     6901 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     6961 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     7021 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     7081 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     7141 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     7201 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     7261 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7321 aaaagatcaa aggatcttc
//
