LOCUS       40924_31045        8061 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle-expression vector pMKEx2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8061)
  AUTHORS   Kortmann M, Kuhl V, Klaffl S, Bott M.
  TITLE     A chromosomally encoded T7 RNA polymerase-dependent gene expression 
            system for Corynebacterium glutamicum: construction and comparative 
            evaluation at the single-cell level
  JOURNAL   Microb Biotechnol (2014) In press
  PUBMED    25488698
REFERENCE   2  (bases 1 to 8061)
  AUTHORS   Kortmann M, Kuhl V, Klaffl S, Bott M.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-SEP-2014) IBG-1: Biotechnology, Forschungszentrum 
            Juelich GmbH, Wilhelm-Johnen-Strasse, Juelich 52425, Germany
REFERENCE   3  (bases 1 to 8061)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8061)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Microb 
            Biotechnol (2014) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (25-SEP-2014) IBG-1: Biotechnology, Forschungszentrum Juelich GmbH, 
            Wilhelm-Johnen-Strasse, Juelich 52425, Germany"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(73..120)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(160..186)
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             complement(193..210)
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             complement(256..279)
                     /codon_start=1
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
                     /translation="LEVLFQGP"
     CDS             complement(286..309)
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     RBS             complement(325..347)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(362..386)
                     /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)."
     promoter        complement(387..405)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        718..795
                     /label=lacI promoter
     CDS             796..1875
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1891..1912
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      2451..2996
                     /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."
     CDS             3591..4403
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5380..6837)
                     /codon_start=1
                     /gene="repA"
                     /product="replicase"
                     /label=repA
                     /protein_id="AIZ68604.1"
                     /translation="MTLADSRDIDTPAWKFSADLFDTHPELALRSRGWTSEDRREFLAH
                     LGRENFQGSKTRDFASAWIKDPDTGETQPKLYRVGSKSLARCQYVALTHAQHAAVLVLD
                     IDVPSHQAGGKIEHVNPEVYAILERWARLEKAPAWIGVNPLSGKCQLIWLIDPVYAAAG
                     MSSPNMRLLAATTEEMTRVFGADQAFSHRLSRWPLHVSDDPTAYRWHAQHNRVDRLADL
                     MEVARMISGTEKPKKRYEQEFSSGRARIEAARKATAEAKALATLEASLPSAAEASGELI
                     DGVRVLWTAPGRAARDETAFRHALTVGYQLKAAGERLKDTKIIDAYERAYTVAQAVGAD
                     GREPDLPPMRDRQTMARRVRGYVAKGQPVVPARQTETQSSRGRKALATMGRRGGKKAAE
                     RWKDPNSEYARAQREKLAKSSQRQARKAKGNRLTIAGWFMTVEGETGSWPTINEAMSEF
                     SVSRQTVNRALKSAGIELPRGRRKASQ"
     gene            complement(5380..6837)
                     /gene="repA"
                     /label=repA
ORIGIN
        1 tcgacctgca gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt
       61 tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag
      121 ttattgctca gcggtggcag cagcctaggt taattagtgg tggtgatggt gatgatggtg
      181 gtgatgagcg gaactaccgc gtggcaccag agcgagctct gcggccgcgt cgacttgtac
      241 aggatcctgg tacccgggtc cctgaaagag gacttcaagt gcaccctttt cgaactgcgg
      301 gtggctccag cttgccatgg tatatctcct tcttaaagtt aaacaaaatt atttctagag
      361 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
      421 gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
      481 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
      541 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
      601 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
      661 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
      721 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
      781 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
      841 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
      901 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
      961 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     1021 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     1081 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     1141 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     1201 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     1261 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     1321 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     1381 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     1441 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     1501 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     1561 gccattaccg agtccgggct gcgcgttggt gcggacatct cggtagtggg atacgacgat
     1621 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     1681 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     1741 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     1801 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     1861 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     1921 gggatctcga ccgatgccct tgagagcctt caacccagtc agctccctgc agcaatggca
     1981 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2041 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2101 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2161 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2221 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2281 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2341 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2401 cgtgagtttt cgttccactg agcgtcagac cccttaataa gatgatcttc ttgagatcgt
     2461 tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt
     2521 tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg aggagcgcag
     2581 tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa gactaactcc
     2641 tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt ccgggttgga
     2701 ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg gttcgtgcat
     2761 acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca
     2821 aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca
     2881 cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2941 actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg
     3001 gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt ccaggaaatc
     3061 tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc
     3121 agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca ccggtgcagc
     3181 cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc aacatagtaa
     3241 gccagtatac actccgctag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat
     3301 accaggcctg aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag
     3361 ctttgttgta ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg
     3421 cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac
     3481 aaagccacgt tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     3541 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     3601 ttcaacggga aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat
     3661 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     3721 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     3781 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     3841 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     3901 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     3961 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     4021 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     4081 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     4141 agcttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     4201 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     4261 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     4321 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     4381 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     4441 gcattacgct gacttgacgg gacggcggct ttgttgaata aatcgaactt ttgctgagtt
     4501 gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt
     4561 caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt
     4621 ctggctggat gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg
     4681 cagacctcag cgctcaaaga tgcaggggta aaagctaacc gcatctttac cgacaaggca
     4741 tccggcagtt caacagatcg ggaagggctg gatttgctga ggatgaaggt ggaggaaggt
     4801 gatgtcattc tggtgaagaa gctcgaccgt cttggccgcg acaccgccga catgatccaa
     4861 ctgataaaag agtttgatgc tcagggtgta gcggttcggt ttattgacga cgggatcagt
     4921 accgacggtg atatggggca aatggtggtc accatcctgt cggctgtggc acaggctgaa
     4981 cgccggagga tcaagtcggt caagccaagc gcaaccagcg gcaccgccgc gagcaacgtc
     5041 gcaagggcga tcaggggacg atttttgcga agaatttcca cggtaagaat ccaatctctc
     5101 gaatttaggg tgaaagaagc ttggcatagg ggtgtgcacg aactcggtgg aggaaatttc
     5161 cgcggggcaa ggcttcgcga agcggagtcg cggcagtggc tttgaagatc tttgggagca
     5221 gtccttgtgc gcttacgagg tgagccggtg gggaaccgtt atctgcctat ggtgtgagcc
     5281 cccctagaga gcttcaagag caatcagccc gacctagaaa ggaggccaag agagagaccc
     5341 ctacgggggg aaccgttttc tgcctacgag atggcacatt tactgggaag ctttacggcg
     5401 tcctcgtgga agttcaatgc ccgcagactt aagtgctcta ttcacggtct gacgtgacac
     5461 gctaaattca gacatagctt cattgattgt cggccacgag ccagtctctc cctcaacagt
     5521 cataaaccaa cctgcaatgg tcaagcgatt tcctttagct ttcctagctt gtcgttgact
     5581 ggacttagct agtttttctc gctgtgctcg ggcgtactca ctgtttgggt ctttccagcg
     5641 ttctgcggcc tttttaccgc cacgtcttcc catagtggcc agagcttttc gccctcggct
     5701 gctctgcgtc tctgtctgac gagcagggac gactggctgg cctttagcga cgtagccgcg
     5761 cacacgtcgc gccatcgtct ggcggtcacg catcggcggc agatcaggct cacggccgtc
     5821 tgctccgacc gcctgagcga cggtgtaggc acgctcgtag gcgtcgatga tcttggtgtc
     5881 ttttaggcgc tcaccagccg cttttaactg gtatcccaca gtcaaagcgt ggcgaaaagc
     5941 cgtctcatca cgggcggcac gccctggagc agtccagagg acacggacgc cgtcgatcag
     6001 ctctccagac gcttcagcgg cgctcggcag gcttgcttca agcgtggcaa gtgcttttgc
     6061 ttccgcagtg gcttttcttg ccgcttcgat acgtgcccgt ccgctagaaa actcctgctc
     6121 atagcgtttt ttaggttttt ctgtgcctga gatcatgcga gcaacctcca taagatcagc
     6181 taggcgatcc acgcgattgt gctgggcatg ccagcggtac gcggtgggat cgtcggagac
     6241 gtgcagtggc caccggctca gcctatgtga aaaagcctgg tcagcgccga aaacgcgggt
     6301 catttcctcg gtcgttgcag ccagcaggcg catattcggg ctgctcatgc ctgctgcggc
     6361 atacaccgga tcaatgagcc agatgagctg gcatttcccg ctcagtggat tcacgccgat
     6421 ccaagctggc gctttttcca ggcgtgccca gcgctccaaa atcgcgtaga cctcggggtt
     6481 tacgtgctcg attttcccgc cggcctggtg gctcggcaca tcaatgtcca ggacaagcac
     6541 ggctgcgtgc tgcgcgtgcg tcagagcaac atactggcac cgggcaagcg attttgaacc
     6601 aactcggtat aacttcggct gtgtttctcc cgtgtccggg tctttgatcc aagcgctggc
     6661 gaagtcgcgg gtcttgctgc cctggaaatt ttctctgccc aggtgagcga ggaattcgcg
     6721 gcggtcttcg ctcgtccagc cacgtgatcg cagcgcgagc tcgggatggg tgtcgaacag
     6781 atcagcggaa aatttccagg ccggtgtgtc aatgtctcgt gaatccgcta gagtcatttt
     6841 tgagcgcttt ctcccaggtt tggactgggg gttagccgac gccctgtgag ttaccgctca
     6901 cggggcgttc aacatttttc aggtattcgt gcagcttatc gcttcttgcc gcctgtgcgc
     6961 tttttcgacg cgcgacgctg ctgccgattc ggtgcaggtg gtggcggcgc tgacacgtcc
     7021 tgggcggcca cggccacacg aaacgcggca tttacgatgt ttgtcatgcc tgcgggcacc
     7081 gcgccacgat cgcggataat tctcgctgcc gcttccagct ctgtgacgac catagccaaa
     7141 atttcgctcg ggggacgcac ttccagcgcc atttgcgacc tagccgcctc cagctcctcg
     7201 gcgtggcgtt tgttggcgcg ctcgcggctg gctgcggcac gacacgcatc tgagcaatat
     7261 tttgcgcgcc gtcctcgcgg gtcaggccgg ggaggaatca ggccaccgca gtaggcgcaa
     7321 ctgattcgat cctccactac tgtgcgtcct cctggcgctg ccgagcacgc agctcgtcag
     7381 ccagctcctc aagatccgcc acgagagttt ctaggtcgct cgcggcactg gcccagtctc
     7441 gtgatgctgg cgcgtccgtc gtatcgagag ctcggaaaaa tccgatcacc gtttttaaat
     7501 cgacggcagc atcgagcgcg tcggactcca gcgcgacatc agagagatcc atagctgatg
     7561 attcgggcca attttggtac ttcgtcgtga aggtcatgac accattataa cgaacgttcg
     7621 ttaaagtttt tggcggaaaa tcacgcggca cgaaaatttt cacgaagcgg gactttgcgc
     7681 agctcagggg tgctaaaaat tttgtatcgc acttgatttt tccgaaagac agattatctg
     7741 caaacggtgt gtcgtatttc tggcttggtt tttaaaaaat ctggaatcga aaatttgcgg
     7801 ggcgaccgag aagtttttta caaaaggcaa aaactttttc gggatcgaca gaaataaaac
     7861 gatcgacggt acgcaacaaa aaagcgtcag gatcgccgta gagcgattga agaccgtcaa
     7921 ccaaagggga agcctccaat cgacgcgacg cgcgctctac ggcgatcctg acgcagattt
     7981 ttagctatct gtcgcagcgc cctcagggac aagccacccg cacaacgtcg cgagggcgat
     8041 cagcgacgcc gcagggggat c
//
