LOCUS       40924_16565        5258 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pEBDuet28A, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5258)
  AUTHORS   Buchinger E, Aachmann FL, Aranko AS, Valla S, Skjak-Braek G, Iwai H,
            Wimmer R.
  TITLE     Use of protein trans-splicing to produce active and segmentally 
            (2)H, (15)N labeled mannuronan C5-epimerase AlgE4
  JOURNAL   Protein Sci. 19 (8), 1534-1543 (2010)
  PUBMED    20552686
REFERENCE   2  (bases 1 to 5258)
  AUTHORS   Buchinger E, Aachmann FL, Aranko S, Valla S, Sjaak-Braek G, Iwai H, 
            Wimmer R.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-MAR-2010) Aalborg University, Sohngaardsholmsvej 49, 
            Aalborg 9000, Denmark
REFERENCE   3  (bases 1 to 5258)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5258)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Sci."; date: "2010"; volume: "19"; issue: "8"; pages: "1534-1543"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (23-MAR-2010) Aalborg University, Sohngaardsholmsvej 49, Aalborg 
            9000, Denmark"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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)."
     RBS             88..110
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             129..146
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             147..167
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     promoter        1689..1707
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1708..1732
                     /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)."
     CDS             1841..1885
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     terminator      1937..1984
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2217..3029)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(3030..3121)
                     /label=AmpR promoter
     rep_origin      complement(3137..3886)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     protein_bind    complement(4048..4069)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4085..5164)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5165..5242)
                     /label=lacI promoter
ORIGIN
        1 tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctgtag aaataatttt gtttaacttt aataaggaga tataccatgc
      121 ggggttctca tcatcatcat catcatgaga atttgtattt tcagtccatg gattacaacg
      181 tcaaggattt cggtgcattg ggcgacggcg tcagcgacga ccgggcctcc atccaggcgg
      241 cgatcgatgc cgcctacgcc gccggtggcg gtaccgtcta cctgccggcc ggcgagtacc
      301 gggtcagcgc cgccggggag ccgggcgacg gctgcctgat gctcaaggac ggcgtctacc
      361 tggccggtgc cggcatgggc gagacggtga tcaagctgat cgacggctcc gaccagaaga
      421 tcaccggcat ggtccgctcg gcctacggcg aggaaaccag caacttcggc atgcgcgacc
      481 tgaccctcga cggcaaccgc gacaacacca gcggcaaggt cgacggctgg ttcaacggct
      541 atatccccgg cggggacggc gccgaccgcg acgtgaccat cgagcgggtg gaggtccgcg
      601 agatgtccgg ctacggcttc gacccccacg agcagaccat caacctgacg atccgcgaca
      661 gcgtggccca cgacaacggc ctcgacggct tcgtcgccga ctacctggtc gacagcgtgt
      721 tcgagaacaa cgtcgcctac gccaacgacc gccacggctt caacgtggtc accagcaccc
      781 acgatttcgt catgaccaac aacgtcgcct acggcaacgg cagcagcggc ctggtggtgc
      841 agcggggtct ggaggacctc gcgctgccca gcaacatcct gatcgacggc ggcgcctact
      901 acgacaacgc ccgcgaaggc gtgctgctca agatgaccag cgacatcacc ctgcagaacg
      961 ccgatatcca cggcaacggc tcctccgggg tgcgcgtcta cggcgcccag gacgtgcaga
     1021 tcctcgataa ccagatccac gacaacgcgc aggcggccgc cgtgcccgag gtcctgctgc
     1081 agtccttcga cgataccgcc ggggcgtccg gcacctacta cacgaccctg aacacccgga
     1141 tcgagggcaa caccatcagc ggctcggcca actccaccta cggcatccag gagcgcaacg
     1201 acggcaccga ctacagcagc ctgatcgaca acgacatcgc cggggtgcaa cagcccatcc
     1261 aactgtacgg acctcactcg acggtatccg gcgaacccgg cgcgacaaaa tgtctaagct
     1321 atgaaacgga aatattgaca gtagaatatg gattattacc gattggtaaa attgtagaaa
     1381 agcgcatcga atgtactgtt tatagcgttg ataataatgg aaatatttat acacaacctg
     1441 tagcacaatg gcacgatcgc ggagaacaag aggtgtttga gtattgtttg gaagatggtt
     1501 cattgattcg ggcaacaaaa gaccataagt ttatgactgt tgatggtcaa atgttgccaa
     1561 ttgatgaaat atttgaacgt gaattggatt tgatgcgggt tgataatttg ccgaattaag
     1621 cttgcggccg cataatgctt aagtcgaaca gaaagtaatc gtattgtaca cggccgcata
     1681 atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt ccccatctta
     1741 gtatattagt taagtataag aaggagatat acatatggca gatctcaatt ggatatcggc
     1801 cggccacgcg atcgctgacg tcggtaccct cgagtctggt aaagaaaccg ctgctgcgaa
     1861 atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct aggctgctgc
     1921 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1981 tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa
     2041 accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac
     2101 aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta
     2161 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     2221 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     2281 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     2341 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2401 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2461 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     2521 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2581 gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2641 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2701 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     2761 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     2821 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     2881 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     2941 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     3001 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
     3061 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3121 ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     3181 ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac agaatcaggg
     3241 gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc caacgccgca
     3301 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagcca
     3361 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3421 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3481 gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt
     3541 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3601 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3661 cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa ggctaaactg
     3721 aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc aaagagttgg
     3781 tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt tatttatgag
     3841 atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc tagatttcag
     3901 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat
     3961 tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4021 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4081 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4141 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4201 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4261 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4321 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     4381 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4441 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4501 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4561 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4621 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     4681 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4741 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4801 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4861 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4921 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     4981 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5041 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5101 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5161 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5221 ttttgcgcca ttcgatggtg tccgggatct cgacgctc
//
