LOCUS       Exported                5258 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pEBDuet28A, complete sequence.
ACCESSION   HM070249
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5258
                     /organism="Expression vector pEBDuet28A"
                     /mol_type="other DNA"
                     /db_xref="taxon:762606"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             117..1619
                     /codon_start=1
                     /product="H6-A-IntN"
                     /label=H6-A-IntN
                     /note="fusion protein of Azotobacter vinelandii mannuronan 
                     C5-epimerase AlgE4 A-module (A) and Nostoc punctiforme DnaE
                     intein (IntN) N-terminal fragment plus His-tag"
                     /protein_id="ADG44969.1"
                     /translation="MRGSHHHHHHENLYFQSMDYNVKDFGALGDGVSDDRASIQAAIDA
                     AYAAGGGTVYLPAGEYRVSAAGEPGDGCLMLKDGVYLAGAGMGETVIKLIDGSDQKITG
                     MVRSAYGEETSNFGMRDLTLDGNRDNTSGKVDGWFNGYIPGGDGADRDVTIERVEVREM
                     SGYGFDPHEQTINLTIRDSVAHDNGLDGFVADYLVDSVFENNVAYANDRHGFNVVTSTH
                     DFVMTNNVAYGNGSSGLVVQRGLEDLALPSNILIDGGAYYDNAREGVLLKMTSDITLQN
                     ADIHGNGSSGVRVYGAQDVQILDNQIHDNAQAAAVPEVLLQSFDDTAGASGTYYTTLNT
                     RIEGNTISGSANSTYGIQERNDGTDYSSLIDNDIAGVQQPIQLYGPHSTVSGEPGATKC
                     LSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCL
                     EDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN"
     CDS             129..146
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             147..167
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV 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
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      1937..1984
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2214..3029)
                     /codon_start=1
                     /gene="aph(3)-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(3030..3121)
                     /gene="bla"
                     /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."
     CDS             complement(4082..5164)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5165..5242)
                     /gene="lacI"
                     /label=lacI promoter