LOCUS       sequence-373875-        7195 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses OtUBD(V203D) mutant that is unable to bind ubiquitin with 
            an TEV-cleavable N-terminal 6xHis-MBP tag as well as an engineered 
            N-terminal cysteine in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7195)
  AUTHORS   Zhang M, Berk JM, Mehrtash AB, Kanyo J, Hochstrasser M
  TITLE     A versatile new tool derived from a bacterial deubiquitylase to 
            detect and purify ubiquitylated substrates and their interacting 
            proteins.
  JOURNAL   PLoS Biol. 2022 Jun 30;20(6):e3001501. doi: 
            10.1371/journal.pbio.3001501. eCollection 2022 Jun.
  PUBMED    35771886
REFERENCE   2  (bases 1 to 7195)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS 
            Biol."; date: "2022-06-30"; volume: "20(6):e3001501. doi"; pages: " 
            10.1371/journal.pbio.3001501. eCollection 2022 Jun"
COMMENT     Sequence Label: pRT498-OtUBD(V203D)
FEATURES             Location/Qualifiers
     source          1..7195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2335..2354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2495..2517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2511..2702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3274..4356)
                     /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     4318..4337
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4357..4434)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4640..4659
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4747..4766
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4747..4765
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4766..4790
                     /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             4805..4827
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4847..4864
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4874..5974
                     /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
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGIDAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     5948..5971
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6014..6034
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             7023..7046
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(7109..7127)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7123..7170
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     3181 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3241 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3301 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3361 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3421 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3481 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3541 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3601 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3661 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3721 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3781 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3841 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3901 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3961 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4021 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4081 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4141 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4201 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4261 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4321 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4381 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4441 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     4501 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     4561 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     4621 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     4681 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     4741 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     4801 taattttgtt taactttaag aaggagatat atccatgaac accattcatc accatcacca
     4861 tcacaacact agtatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa
     4921 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     4981 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     5041 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     5101 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     5161 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     5221 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     5281 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
     5341 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     5401 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     5461 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     5521 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     5581 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     5641 caaaccgttc gttggcgtgc tgagcgcagg tattgacgcc gccagtccga acaaagagct
     5701 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     5761 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     5821 acgtattgcc gccacaatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     5881 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     5941 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgacta gtggatctgg
     6001 tggtggtggc ggcgaaaacc tgtacttcca gggtggttct catatgtgtg gatgtggatc
     6061 aggagttaat ttcctgagcg aaaacagaat gattacctct gccgctgcag ccttggctga
     6121 tactttgctg aagaacaaca acagaatcac agagggtgtg ctggatgata gaatcttcga
     6181 caataagatt ttgtccgtgc aggaaaaaca gcaattgctg aacaatttgc tggacaacca
     6241 tattaaggaa aacaaatctc tgactaaaga gtccctgaca agaatgttgg cttctaccca
     6301 ctttgttcag caacaggcaa acgtgttgct gaatgagaag ttcaacaaat agtaaggatc
     6361 aggagtaaat tttctttctg aaaatagaat gataactagt gctgctgcag ctcttgcaga
     6421 tacattattg aagaataata acagaattac cgaaggtgtc ttagtagata ggatttttga
     6481 taataaaatt ttgtctgtac aagaaaagca acaactactt aataatttat tagataatca
     6541 tattaaagaa aataaatctt taactaaaga aagtttaaca agaatgttgg ctagtacgca
     6601 ttttgtgcag cagcaagcta atgttttgtt aaatgaaaaa tttaataaag gctctggtgt
     6661 aaattttttg tctgaaaatc gcatgattac ttctgccgca gcagctttgg cagatacctt
     6721 gctgaagaac aacaatcgca ttacagaagg cgtcttagtg gaccgtatct ttgacaataa
     6781 aatcttatcg gtgcaagaga agcaacagct tctgaacaat ctgttggaca accatattaa
     6841 agaaaacaaa agcctgacga aagagagcct gacgcgtatg ttggctagta cacatttcgt
     6901 gcagcagcag gcaaacgtgt tgctgaatga gaagttcaac aaatagtaag gatccgaatt
     6961 ctgtacaggc cttggcgcgc ctgcaggcga gctccgtcga caagcttgcg gccgcactcg
     7021 agcaccacca ccaccaccac caccactaat tgattaatac ctaggctgct aaacaaagcc
     7081 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     7141 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggat
//
