LOCUS       sequence-342928-        7358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promotes expression of disulfide-rich protein in E. coli T7 
            expression system. The plasmid encodes expression of the folding 
            factors Erv1p and hPDI controlled by the Ptac promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7358)
  AUTHORS   Bertelsen AB, Hackney CM, Bayer CN, Kjelgaard LD, Rennig M, 
            Christensen B, Sorensen ES, Safavi-Hemami H, Wulff T, Ellgaard L, 
            Norholm MHH
  TITLE     DisCoTune: versatile auxiliary plasmids for the production of 
            disulphide-containing proteins and peptides in the E. coli T7 
            system.
  JOURNAL   Microb Biotechnol. 2021 Nov;14(6):2566-2580. doi: 
            10.1111/1751-7915.13895. Epub 2021 Aug 18.
  PUBMED    34405535
REFERENCE   2  (bases 1 to 7358)
  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"; doi: 
            "10.1111/1751-7915.13895"; journalName: "Microb Biotechnol"; date: 
            "2021-11"; volume: "14"; issue: "6"; pages: "2566-2580"
COMMENT     Sequence Label: pDisCoTune
FEATURES             Location/Qualifiers
     source          1..7358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             85..540
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPYACPSFDLKRWWEKNELVTSDRG"
     terminator      610..657
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        join(693..698,699..714,715..721)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    729..745
                     /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     734..756
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     RBS             1431..1453
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2993..3010
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3233..3892)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3807..3826
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3893..3995)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4387..4404)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4521..5066)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4601..4620)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5397..6245)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(6319..7155)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITCGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        join(7301..7358,1..61)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
ORIGIN
        1 gcccattttc ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt
       61 aatgaaattc aggaggtggt cgacatggct cgtgtacagt ttaaacaacg tgaatctact
      121 gacgcaatct ttgttcactg ctcggctacc aagccaagtc agaatgttgg tgtccgtgag
      181 attcgccagt ggcacaaaga gcagggttgg ctcgatgtgg gataccactt tatcatcaag
      241 cgagacggta ctgtggaggc aggacgagat gagatggctg taggctctca cgctaagggt
      301 tacaaccaca actctatcgg cgtctgcctt gttggtggta tcgacgataa aggtaagttc
      361 gacgctaact ttacgccagc ccaaatgcaa tcccttcgct cactgcttgt cacactgctg
      421 gctaagtacg aaggcgctgt gcttcgcgcc catcatgagg tggcgccgta cgcttgccct
      481 tcgttcgacc ttaagcgttg gtgggagaag aacgaactgg tcacttctga ccgtggataa
      541 ggatccccgc gccctcatcc gaaagggcgt attcatatgc cctaggctgc tgccaccgct
      601 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgcat
      661 aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat
      721 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agccagtccg tttaggtgtt
      781 ttcacgagct ctagaaataa ttatcagcag gacgcactga ccaggaggta catatgaaag
      841 ccatcgataa aatgaccgac aatccgcctc aagagggtct gagtggtcgt aaaatcatct
      901 atgatgagga cggcaaacca tgtcgcagct gcaataccct gctggacttc cagtatgtta
      961 ccgggaaaat ctcaaatggc ctgaaaaacc tgagcagcaa tggtaaactg gcgggtacag
     1021 gtgctctgac tggggaagca tctgaactga tgccgggttc tcgtacctat cgtaaagtcg
     1081 atcctccgga tgttgaacag ctgggacgtt cttcatggac actgctgcat agcgtagcag
     1141 cctcttatcc agctcaacca accgatcagc aaaaaggcga aatgaaacag ttcctgaaca
     1201 tcttcagcca catctatccg tgtaactggt gcgctaaaga cttcgaaaaa tatatccgtg
     1261 agaatgcccc tcaagttgaa tcacgtgagg agctgggtcg ctggatgtgt gaggcccata
     1321 acaaagtgaa caaaaagctg cgcaaaccga agttcgactg taacttctgg gaaaaacgct
     1381 ggaaagatgg ttgggatgag taataaggat ccgaattcac tagaaataat tttgtttaac
     1441 tttaagaagg agatacatat ggatgcccca gaggaggaag atcacgtcct ggttctgcgt
     1501 aaaagcaact tcgctgaagc actggcagct cacaaatatc tgctggtcga gttctatgct
     1561 ccgtggtgtg gtcattgcaa agccctggct ccggaatatg ctaaagcagc cggcaaactg
     1621 aaagctgagg gcagtgaaat tcgtctggcc aaagtggacg ctaccgaaga atcagatctg
     1681 gcacaacagt atggtgttcg tggttatccg actatcaaat ttttccgtaa cggcgataca
     1741 gcaagcccta aagagtatac cgctggccgt gaagctgatg atatcgtgaa ctggctgaaa
     1801 aaacgtacag gtccggcggc aacgactctg cctgatggtg ccgctgccga gtcactggta
     1861 gaatcatccg aagtggccgt gattggcttc tttaaagacg tggagagcga ttcagcaaaa
     1921 cagttcctgc aagcagctga agcgattgat gacatcccgt ttggtattac gagcaatagc
     1981 gacgtgttct ccaaatatca actggacaaa gacggtgtgg ttctgttcaa aaaattcgac
     2041 gaaggccgta acaactttga aggtgaggtg accaaagaaa acctgctgga ctttatcaaa
     2101 cacaatcaac tgccgctggt gattgagttc accgaacaga cagctccgaa aatctttggc
     2161 ggcgagatca aaacccacat tctgctgttt ctgcctaaaa gtgtgtccga ctatgacggc
     2221 aaactgagca acttcaaaac cgccgctgag agctttaaag gcaaaatcct gttcatcttc
     2281 atcgacagcg atcataccga caaccagcgt attctggagt tttttggcct gaaaaaagag
     2341 gaatgtccgg ccgttcgcct gattacactg gaagaggaga tgacgaaata taaaccagaa
     2401 agcgaggagc tgacagctga acgtattacc gagttctgcc accgttttct ggaaggcaaa
     2461 atcaaacctc atctgatgtc ccaagaactg ccagaagatt gggataaaca gcctgtgaaa
     2521 gtgctggtag gcaaaaactt cgaggatgtg gcgttcgacg agaaaaaaaa cgtgtttgtg
     2581 gagttttatg ccccttggtg tggacactgc aaacagctgg caccgatttg ggataaactg
     2641 ggcgaaacct ataaagatca tgaaaacatt gttattgcca aaatggacag caccgccaat
     2701 gaagtcgaag ccgtgaaagt tcattcgttt ccgaccctga aattctttcc tgccagcgcc
     2761 gatcgtactg tgatcgatta taacggggag cgtaccctgg atggttttaa aaaattcctg
     2821 gagagcggtg gtcaagatgg tgccggtgat gatgacgatc tggaggatct ggaagaggct
     2881 gaagaaccgg atatggagga ggatgacgat cagaaagcag tcaaagacga gctgtgataa
     2941 ggatccgaat tcactagtga gctccgtcga caagcttgcg gccgcactcg agcaccacca
     3001 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3061 caccgctgag caataactag cagataaaaa aaatccttag ctttcgctaa ggatgatttc
     3121 tactggttat ggtcgaattt gctttcgaat ttctgccatt catccgctta ttatcactta
     3181 ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc
     3241 cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc
     3301 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg
     3361 tataatattt gcccatagtg aaaacggggg cgaagaagtt gtccatattg gccacgttta
     3421 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa
     3481 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt
     3541 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt
     3601 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt
     3661 tcattgccat acggaactcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg
     3721 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct
     3781 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac
     3841 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt
     3901 ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat
     3961 tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg
     4021 cccagggctt cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt
     4081 ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt
     4141 agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag ctgtccctcc
     4201 tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag
     4261 cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg
     4321 tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc
     4381 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     4441 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     4501 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     4561 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4621 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     4681 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     4741 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     4801 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     4861 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     4921 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     4981 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     5041 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     5101 aggtttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     5161 agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5221 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5281 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5341 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgcccgataa gcttaattaa
     5401 tctttctgcg aattgagatg acgccactgg ctgggcgtca tcccggtttc ccgggtaaac
     5461 accaccgaaa aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaac
     5521 aggcggctat gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga
     5581 ttgatggtca ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc actgcacgat
     5641 gcctcatcac aaaatttatc cagcgcaaag ggacttttca ggctagccgc cagccgggta
     5701 atcagcttat ccagcaacgt ttcgctggat gttggcggca acgaatcact ggtgtaacga
     5761 tggcgattca gcaacatcac caactgcccg aacagcaact cagccatttc gttagcaaac
     5821 ggcacatgct gactactttc atgctcaagc tgaccaataa cctgccgcgc ctgcgccatc
     5881 cccatgctac ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc
     5941 ccctgccagt caagattcag cttcagacgc tccgggcaat aaataatatt ctgcaaaacc
     6001 agatcgttaa cggaagcgta ggagtgttta tcatcagcat gaatgtaaaa gagatcgcca
     6061 cgggtaatgc gataagggcg atcgttgagt acatgcaggc cattaccgcg ccagacaatc
     6121 accagctcac aaaaatcatg tgtatgttca gcaaagacat cttgcggata acggtcagcc
     6181 acagcgactg cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgc
     6241 gccaccgtgg ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaat
     6301 acgttgagaa gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa tatcacgcgg
     6361 tgaccagtta aactctcggc gaaaaagcgt cgaaaagtgg ttactgtcgc tgaatccaca
     6421 gcgataggcg atgtcagtaa cgctggcctc gctgtggcgt agcagatgtc gggctttcat
     6481 cagtcgcagg cggttcaggt atcgctgagg cgtcagtccc gtttgctgct taagctgccg
     6541 atgtagcgta cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatc
     6601 ggcaaaatgg tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccag
     6661 gttctcctgc aaactgcttt tacgcagcaa gagcagtaat tgcataaaca agatctcgcg
     6721 actggcggtc gagggtaaat cattttcccc ttcctgctgt tccatctgtg caaccagctg
     6781 tcgcacctgc tgcaatacgc tgtggttaac gcgccagtga gacggatact gcccatccag
     6841 ctcttgtggc agcaactgat tcagcccggc gagaaactga aatcgatccg gcgagcgata
     6901 cagcacattg gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcg
     6961 tacgaaacag accgtgccac aggtgatggt atagggctgc ccattaaaca catgaatacc
     7021 cgtgccatgt tcgacaatca caatttcatg aaaatcatga tgatgttcag gaaaatccgc
     7081 ctgcgggagc cggggttcta tcgccacgga cgcgttacca gacggaaaaa aatccacact
     7141 atgtaatacg gtcatactgg cctcctgatg tcgtcaacac ggcgaaatag taatcacgag
     7201 gtcaggttct taccttaaat tttcgacgga aaaccacgta aaaaacgtcg atttttcaag
     7261 atacagcgtg aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg
     7321 tgaacatcat cacgttcatc tttccctggt tgccaatg
//
