LOCUS       sequence-357286-        7933 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 7933)
  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 7933)
  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_KPNB1_HEAT
FEATURES             Location/Qualifiers
     source          1..7933
                     /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             6282..6299
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6352..6370)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6366..6413
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6450..6905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6537..6556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6747..6768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6997..7812)
                     /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(6997..7812)
                     /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     7720..7739
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7804..7823
                     /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 cgccatggac atggagctga tcaccattct
     4801 ggaaaagacc gttagcccgg atcgtctgga gctggaagcg gcgcagaaat tcctggagcg
     4861 tgcggcggtg gaaaacctgc cgacctttct ggttgagctg agccgtgtgc tggcgaaccc
     4921 gggtaacagc caagttgcgc gtgtggcggc gggcctgcaa atcaagaaca gcctgaccag
     4981 caaggacccg gacattaaag cgcaatacca gcaacgttgg ctggcgattg atgcgaacgc
     5041 gcgtcgtgag gttaaaaact acgtgctgca gaccctgggc accgaaacct atcgtccgag
     5101 cagcgcgagc cagtgcgttg cgggtattgc gtgcgcggag attccggtga accagtggcc
     5161 ggaactgatc ccgcaactgg ttgcgaacgt gaccaacccg aacagcaccg aacacatgaa
     5221 ggagagcacc ctggaagcga tcggttatat ttgccaggat attgacccgg agcagctgca
     5281 agataaaagc aacgaaatcc tgaccgcgat cattcaaggc atgcgtaaag aggagccgag
     5341 caacaacgtt aaactggcgg cgaccaacgc gctgctgaac agcctggagt tcaccaaggc
     5401 gaactttgat aaagagagcg aacgtcactt cattatgcag gtggtttgcg aagcgaccca
     5461 atgcccggac acccgtgttc gtgtggcggc gctgcagaac ctggtgaaga tcatgagcct
     5521 gtactatcaa tacatggaaa cctatatggg tccggcgctg ttcgcgatca ccattgaagc
     5581 gatgaaaagc gatatcgacg aggttgcgct gcagggcatt gaattttgga gcaacgtgtg
     5641 cgatgaggaa atggacctgg cgattgaggc gagcgaagcg gcggagcagg gtcgtccgcc
     5701 ggagcacacc agcaagtttt acgcgaaagg cgcgctgcaa tatctggttc cgatcctgac
     5761 ccagaccctg accaagcaag acgaaaacga cgatgacgat gactggaacc cgtgcaaagc
     5821 ggcgggtgtg tgcctgatgc tgctggcgac ctgctgcgag gatgacattg ttccgcacgt
     5881 gctgccgttc atcaaggaac acattaaaaa cccggattgg cgttaccgtg acgcggcggt
     5941 tatggcgttt ggttgcatcc tggaaggtcc ggagccgagc cagctgaagc cgctggtgat
     6001 ccaagcgatg ccgaccctga ttgaactgat gaaagatccg agcgtggttg ttcgtgacac
     6061 cgcggcgtgg accgttggtc gtatctgcga gctgctgccg gaagcggcga ttaacgatgt
     6121 gtacctggcg ccgctgctgc agtgcctgat tgaaggcctg agcgcggaac cgcgtgttgc
     6181 gagcaacgtg tgctgggcgt tcagcagcct ggcggaggcg gcgtatgaag cggcggacta
     6241 agaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     6301 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     6361 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     6421 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     6481 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     6541 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     6601 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     6661 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     6721 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     6781 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     6841 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     6901 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     6961 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     7021 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     7081 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     7141 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     7201 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     7261 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     7321 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     7381 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     7441 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     7501 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     7561 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     7621 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     7681 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     7741 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     7801 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     7861 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7921 tcaaaggatc ttc
//
