LOCUS       40924_16560        5207 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pEBDuet23A, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5207)
  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 5207)
  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 5207)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5207)
  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..5207
                     /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             117..1568
                     /codon_start=1
                     /product="A-IntN"
                     /label=A-IntN
                     /note="fusion protein of Azotobacter vinelandii mannuronan 
                     C5-epimerase AlgE4 A-module (A) and Nostoc punctiforme DnaE
                     intein (IntN) N-terminal fragment"
                     /protein_id="ADG44968.1"
                     /translation="MDYNVKDFGALGDGVSDDRASIQAAIDAAYAAGGGTVYLPAGEYR
                     VSAAGEPGDGCLMLKDGVYLAGAGMGETVIKLIDGSDQKITGMVRSAYGEETSNFGMRD
                     LTLDGNRDNTSGKVDGWFNGYIPGGDGADRDVTIERVEVREMSGYGFDPHEQTINLTIR
                     DSVAHDNGLDGFVADYLVDSVFENNVAYANDRHGFNVVTSTHDFVMTNNVAYGNGSSGL
                     VVQRGLEDLALPSNILIDGGAYYDNAREGVLLKMTSDITLQNADIHGNGSSGVRVYGAQ
                     DVQILDNQIHDNAQAAAVPEVLLQSFDDTAGASGTYYTTLNTRIEGNTISGSANSTYGI
                     QERNDGTDYSSLIDNDIAGVQQPIQLYGPHSTVSGEPGATKCLSYETEILTVEYGLLPI
                     GKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTV
                     DGQMLPIDEIFERELDLMRVDNLPN"
     promoter        1638..1656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1657..1681
                     /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             1790..1834
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     terminator      1886..1933
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2166..2978)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(2979..3070)
                     /label=AmpR promoter
     rep_origin      complement(3086..3835)
                     /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(3997..4018)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4034..5113)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5114..5191)
                     /label=lacI promoter
ORIGIN
        1 tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctgtag aaataatttt gtttaacttt aataaggaga tataccatgg
      121 attacaacgt caaggatttc ggtgcattgg gcgacggcgt cagcgacgac cgggcctcca
      181 tccaggcggc gatcgatgcc gcctacgccg ccggtggcgg taccgtctac ctgccggccg
      241 gcgagtaccg ggtcagcgcc gccggggagc cgggcgacgg ctgcctgatg ctcaaggacg
      301 gcgtctacct ggccggtgcc ggcatgggcg agacggtgat caagctgatc gacggctccg
      361 accagaagat caccggcatg gtccgctcgg cctacggcga ggaaaccagc aacttcggca
      421 tgcgcgacct gaccctcgac ggcaaccgcg acaacaccag cggcaaggtc gacggctggt
      481 tcaacggcta tatccccggc ggggacggcg ccgaccgcga cgtgaccatc gagcgggtgg
      541 aggtccgcga gatgtccggc tacggcttcg acccccacga gcagaccatc aacctgacga
      601 tccgcgacag cgtggcccac gacaacggcc tcgacggctt cgtcgccgac tacctggtcg
      661 acagcgtgtt cgagaacaac gtcgcctacg ccaacgaccg ccacggcttc aacgtggtca
      721 ccagcaccca cgatttcgtc atgaccaaca acgtcgccta cggcaacggc agcagcggcc
      781 tggtggtgca gcggggtctg gaggacctcg cgctgcccag caacatcctg atcgacggcg
      841 gcgcctacta cgacaacgcc cgcgaaggcg tgctgctcaa gatgaccagc gacatcaccc
      901 tgcagaacgc cgatatccac ggcaacggct cctccggggt gcgcgtctac ggcgcccagg
      961 acgtgcagat cctcgataac cagatccacg acaacgcgca ggcggccgcc gtgcccgagg
     1021 tcctgctgca gtccttcgac gataccgccg gggcgtccgg cacctactac acgaccctga
     1081 acacccggat cgagggcaac accatcagcg gctcggccaa ctccacctac ggcatccagg
     1141 agcgcaacga cggcaccgac tacagcagcc tgatcgacaa cgacatcgcc ggggtgcaac
     1201 agcccatcca actgtacgga cctcactcga cggtatccgg cgaacccggc gcgacaaaat
     1261 gtctaagcta tgaaacggaa atattgacag tagaatatgg attattaccg attggtaaaa
     1321 ttgtagaaaa gcgcatcgaa tgtactgttt atagcgttga taataatgga aatatttata
     1381 cacaacctgt agcacaatgg cacgatcgcg gagaacaaga ggtgtttgag tattgtttgg
     1441 aagatggttc attgattcgg gcaacaaaag accataagtt tatgactgtt gatggtcaaa
     1501 tgttgccaat tgatgaaata tttgaacgtg aattggattt gatgcgggtt gataatttgc
     1561 cgaattaagc ttgcggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac
     1621 ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1681 cccatcttag tatattagtt aagtataaga aggagatata catatggcag atctcaattg
     1741 gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta aagaaaccgc
     1801 tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta
     1861 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1921 gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt
     1981 agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga
     2041 accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga aatgtgcgcg
     2101 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2161 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2221 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2281 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2341 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2401 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2461 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2521 tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc
     2581 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2641 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2701 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2761 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2821 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2881 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2941 ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat
     3001 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3061 aataaacaaa taggcatgca gcgctcttcc gcttcctcgc tcactgactc gctacgctcg
     3121 gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg gttatccaca
     3181 gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc
     3241 aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3301 ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3361 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3421 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc tcagttcggt
     3481 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3541 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3601 ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc acctgttaag
     3661 gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta ccttggttca
     3721 aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg gtttttcttt
     3781 atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt ccgttactct
     3841 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     3901 agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3961 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4021 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4081 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4141 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4201 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4261 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     4321 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4381 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4441 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4501 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4561 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4621 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4681 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4741 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4801 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4861 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4921 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4981 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5041 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5101 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5161 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctc
//
