LOCUS       62056_18365        7935 bp DNA     circular SYN 21-SEP-2020
DEFINITION  Shuttle-expression vector pPBEx2, complete sequence.
ACCESSION   MT670349
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7935)
  AUTHORS   Bakkes PJ, Ramp P, Bida A, Dohmen-Olma D, Bott M, Freudl R.
  TITLE     Improved pEKEx2-derived expression vectors for tightly controlled 
            production of recombinant proteins in Corynebacterium glutamicum
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7935)
  AUTHORS   Bakkes PJ, Ramp P, Bida A, Dohmen-Olma D, Bott M, Freudl R.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JUN-2020) Institute of Bio- and Geosciences; IBG-1: 
            Biotechnology, Forschungszentrum Juelich GmbH, Wilhelm-Johnen 
            Strasse, Juelich, NRW 52425, Germany
REFERENCE   3  (bases 1 to 7935)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (25-JUN-2020) Institute of Bio- and Geosciences"; volume: " IBG-1"; 
            pages: " Biotechnology, Forschungszentrum Juelich GmbH, 
            Wilhelm-Johnen Strasse, Juelich, NRW 52425, German"
FEATURES             Location/Qualifiers
     source          1..7935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(1..56)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(58..74)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      287..373
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      465..492
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        511..602
                     /label=AmpR promoter
     rep_origin      complement(1114..1702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             1935..2747
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     CDS             4366..5649
                     /codon_start=1
                     /transl_table=11
                     /product="replicase"
                     /label=replicase
                     /note="Orf-1 from Corynebacterium glutamicum pBL1"
                     /protein_id="QNT09622.1"
                     /translation="MNTSKEPQVNEGSKVTRARAWRRQNVMYKITNSKALAGCHRWRRD
                     EAVAVSWSSNGASQFEGLQNSHSRWGSPLAELEVMGERRIELAIATKNHLAAGGALMMF
                     VGTVRHNRSQSFAQVEAGIKTAYSSMVKTSQWKKERARYGVEHTYSDYEVTDSWANGWH
                     LHRNMLLFLDRPLSDDELKAFEDSMFSRWSAGVVKAGMDAPLREHGVKLDQVSTWGGDA
                     AKMATYLAKGMSQELTGSATKTASKGSYTPFQMLDMLADQSDAGEDMDAVLVARWREYE
                     VGSKNLRSSWSRGAKRALGIDYIDADVRREMEEELYKLAGLEAPERVESTRVAVALVKP
                     DDWKLIQSDFAVRQYVLDCVDKAKDVAAAQRVANEVLASLGVDSTPCMIVMDDVDLDAV
                     LPTHGDATKRDLNAAVFAGNEQTILRTH"
     promoter        complement(6370..6398)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             complement(6447..7526)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(7527..7604)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        7838..7866
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    7874..7890
                     /label=lac operator
                     /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)."
ORIGIN
        1 aagcttgcat gcctgcaggt cgactctaga ggatccccgg gtaccgagct cgaatacact
       61 ggccgtcgtt ttacagccaa gcttggctgt tttggcggat gagagaagat tttcagcctg
      121 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
      181 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
      241 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
      301 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
      361 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
      421 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
      481 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
      541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      601 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      661 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      721 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
      781 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
      841 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
      901 tggttgagta attcgtaatc atgtcatagg ctcttccgct tcctcgctca ctgactcgct
      961 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1021 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1081 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1141 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1201 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1261 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1321 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1381 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1441 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1501 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1561 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1621 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1681 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1741 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1801 ctagatcctt ttgggggggg ggggaaagcc acgttgtgtc tcaaaatctc tgatgttaca
     1861 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     1921 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctcgagg ccgcgattaa
     1981 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     2041 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     2101 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     2161 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     2221 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     2281 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     2341 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     2401 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     2461 aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc
     2521 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     2581 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     2641 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     2701 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     2761 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc ggctttgttg
     2821 aataaatcga acttttgctg agttgaagga tcagatcacg catcttcccg acaacgcaga
     2881 ccgttccgtg gcaaagcaaa agttcaaaat caccaactgg tccacctaca acaaagctct
     2941 catcaaccgt ggctccctca ctttctggct ggatgatggg gcgattcagg cctggtatga
     3001 gtcagcaaca ccttcttcac gaggcagacc tcagcgcccc ccccccccta gcttgtctac
     3061 gtctgatgct ttgaatcgga cggacttgcc gatcttgtat gcggtgattt ttccctcgtt
     3121 tgcccacttt ttaatggtgg ccggggtgag agctacgcgg gcggcgacct gctgcgctgt
     3181 gatccaatat tcggggtcgt tcactggttc ccctttctga tttctggcat agaagaaccc
     3241 ccgtgaactg tgtggttccg ggggttgctg atttttgcga gacttctcgc gcaattccct
     3301 agcttaggtg aaaacaccat gaaacactag ggaaacaccc atgaaacacc cattagggca
     3361 gtagggcggc ttcttcgtct agggcttgca tttgggcggt gatctggtct ttagcgtgtg
     3421 aaagtgtgtc gtaggtggcg tgctcaatgc actcgaacgt cacgtcattt accgggtcac
     3481 ggtgggcaaa gagaactagt gggttagaca ttgttttcct cgttgtcggt ggtggtgagc
     3541 ttttctagcc gctcggtaaa cgcggcgatc atgaactctt ggaggttttc accgttctgc
     3601 atgcctgcgc gcttcatgtc ctcacgtagt gccaaaggaa cgcgtgcggt gaccacgacg
     3661 ggcttagcct ttgcctgcgc ttctagtgct tcgatggtgg cttgtgcctg cgcttgctgc
     3721 gcctgtagtg cctgttgagc ttcttgtagt tgctgttcta gctgtgcctt ggttgccatg
     3781 ctttaagact ctagtagctt tcctgcgata tgtcatgcgc atgcgtagca aacattgtcc
     3841 tgcaactcat tcattatgtg cagtgctcct gttactagtc gtacatactc atatttacct
     3901 agtctgcatg cagtgcatgc acatgcagtc atgtcgtgct aatgtgtaaa acatgtacat
     3961 gcagattgct gggggtgcag ggggcggagc caccctgtcc atgcggggtg tggggcttgc
     4021 cccgccggta cagacagtga gcaccggggc acctagtcgc ggataccccc cctaggtatc
     4081 ggacacgtaa ccctcccatg tcgatgcaaa tctttaacat tgagtacggg taagctggca
     4141 cgcatagcca agctaggcgg ccaccaaaca ccactaaaaa ttaatagttc ctagacaaga
     4201 caaacccccg tgcgagctac caactcatat gcacgggggc cacataaccc gaaggggttt
     4261 caattgacaa ccatagcact agctaagaca acgggcacaa cacccgcaca aactcgcact
     4321 gcgcaacccc gcacaacatc gggtctaggt aacactgaaa tagaagtgaa cacctctaag
     4381 gaaccgcagg tcaatgaggg ttctaaggtc actcgcgcta gggcgtggcg taggcaaaac
     4441 gtcatgtaca agatcaccaa tagtaaggct ctggcggggt gccataggtg gcgcagggac
     4501 gaagctgttg cggtgtcctg gtcgtctaac ggtgcttcgc agtttgaggg tctgcaaaac
     4561 tctcactctc gctgggggtc acctctggct gaattggaag tcatgggcga acgccgcatt
     4621 gagctggcta ttgctactaa gaatcacttg gcggcgggtg gcgcgctcat gatgtttgtg
     4681 ggcactgttc gacacaaccg ctcacagtca tttgcgcagg ttgaagcggg tattaagact
     4741 gcgtactctt cgatggtgaa aacatctcag tggaagaaag aacgtgcacg gtacggggtg
     4801 gagcacacct atagtgacta tgaggtcaca gactcttggg cgaacggttg gcacttgcac
     4861 cgcaacatgc tgttgttctt ggatcgtcca ctgtctgacg atgaactcaa ggcgtttgag
     4921 gattccatgt tttcccgctg gtctgctggt gtggttaagg ccggtatgga cgcgccactg
     4981 cgtgagcacg gggtcaaact tgatcaggtg tctacctggg gtggagacgc tgcgaaaatg
     5041 gcaacctacc tcgctaaggg catgtctcag gaactgactg gctccgctac taaaaccgcg
     5101 tctaaggggt cgtacacgcc gtttcagatg ttggatatgt tggccgatca aagcgacgcc
     5161 ggcgaggata tggacgctgt tttggtggct cggtggcgtg agtatgaggt tggttctaaa
     5221 aacctgcgtt cgtcctggtc acgtggggct aagcgtgctt tgggcattga ttacatagac
     5281 gctgatgtac gtcgtgaaat ggaagaagaa ctgtacaagc tcgccggtct ggaagcaccg
     5341 gaacgggtcg aatcaacccg cgttgctgtt gctttggtga agcccgatga ttggaaactg
     5401 attcagtctg atttcgcggt taggcagtac gttctagatt gcgtggataa ggctaaggac
     5461 gtggccgctg cgcaacgtgt cgctaatgag gtgctggcaa gtctgggtgt ggattccacc
     5521 ccgtgcatga tcgttatgga tgatgtggac ttggacgcgg ttctgcctac tcatggggac
     5581 gctactaagc gtgatctgaa tgcggcggtg ttcgcgggta atgagcagac tattcttcgc
     5641 acccactaaa agcggcataa accccgttcg atattttgtg cgatgaattt atggtcaatg
     5701 tcgcgggggc aaactatgat gggtcttgtt gttgacaatg gctgatttca tcaggaatgg
     5761 aactgtcatg ctgttatgtg cctggctcct aatcaaagct ggggacaatg ggttgccccg
     5821 ttgatctgat ctagttcgga ttggcggggc ttcactgtat ctgggggtgg catcgtgaat
     5881 agattgcaca ccgtagtggg cagtgtgcac accatagtgg ccatgagcac caccaccccc
     5941 agggacgccg acggcgcgaa gctctgcgcc tggtgcggct cggagatcaa gcaatccggc
     6001 gtcggccgga gccgggacta ctgccgccgc tcctgccgcc agcgggcgta cgaggcccgg
     6061 cgccagcgcg aggcgatcgt gtccgccgtg gcgtcggcag tcgctcgccg agatacgtca
     6121 cgtgacgaaa tgcagcagcc ttccattccg tcacgtgacg aaactcgggc cgcaggtcag
     6181 agcacggttc cgcccgctcc ggccctgccg gacccccggc atcccgcaag aggcccggca
     6241 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     6301 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     6361 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atggcctgtc gcttgcggta
     6421 ttcggaatct tgcacgccct cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6481 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     6541 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     6601 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     6661 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     6721 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     6781 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     6841 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     6901 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     6961 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     7021 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     7081 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     7141 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     7201 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     7261 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     7321 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     7381 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     7441 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     7501 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     7561 tatcatgcca taccgcgaaa ggttttgcac cattcgatgg tgtcaacgta aatgcatgcc
     7621 gcttcgcctt cgcgcgcgaa ttgcaagctg atccgggctt atcgactgca cggtgcacca
     7681 atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca
     7741 ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac
     7801 atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc atcggctcgt
     7861 ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagaat taaaagatat
     7921 gaccatgatt acgcc
//
