LOCUS       Exported                5940 bp ds-DNA    circular SYN 10-3-2014
DEFINITION  Compact bacterial vector encoding N-terminal MBP-TVMV and C-terminal
            TEV-6xHis, plus tRNA genes for rare Arg and Ile codons.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG71
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5940)
  AUTHORS   Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, 
            Jedrzejczak R, Joachimiak A.
  TITLE     New LIC vectors for production of proteins from genes containing 
            rare codons.
  JOURNAL   J. Struct. Funct. Genomics 2013;14:135-44.
  PUBMED    24057978
REFERENCE   2  (bases 1 to 5940)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
COMMENT     For ligation-independent cloning (LIC), linearize with SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5940
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(208..225)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(232..252)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /translation="ENLYFQS"
     CDS             complement(263..283)
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /note="TVMV site"
                     /translation="ETVRFQS"
     CDS             complement(320..1420)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /note="MBP"
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             1429..1434
                     /note="ribosome binding site"
     protein_bind    1465..1489
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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(1490..1508)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1783..1859)
                     /gene="argU"
                     /locus_tag="
                     "
                     /product="E. coli tRNA-Arg recognizing AGA and AGG"
                     /note="argU"
     tRNA            complement(2129..2204)
                     /gene="ileX"
                     /locus_tag="
                     "
                     /product="E. coli tRNA-Ile recognizing AUA"
                     /note="ileX"
     promoter        2344..2421
                     /gene="lacI"
                     /note="lacI promoter"
                     /note="
                     "
     CDS             2422..3504
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="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"
     rep_origin      complement(3933..4521)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4692..5552)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5553..5656)
                     /gene="bla"
                     /note="AmpR promoter"