LOCUS       Exported                5445 bp ds-DNA    circular SYN 12-3-2014
DEFINITION  Bacterial vector with a p15A origin encoding tRNA genes for rare Arg
            and Ile codons, for expressing a protein with a 6xHis-TEV leader 
            plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG77
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5445)
  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 5445)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
COMMENT     There are two sites for ligation-independent cloning (LIC).
            
            To express a 6xHis-TEV-tagged protein, linearize with SspI and treat
            with T4 DNA polymerase plus dGTP.
            
            To express a second untagged protein, linearize with SmaI and treat 
            with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5445
                     /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"
     RBS             243..248
                     /note="ribosome binding site"
     CDS             complement(279..299)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /translation="ENLYFQS"
     CDS             complement(324..341)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(342..344)
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     RBS             353..358
                     /note="ribosome binding site"
     protein_bind    389..413
                     /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(414..432)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(707..783)
                     /gene="argU"
                     /locus_tag="
                     "
                     /product="E. coli tRNA-Arg recognizing AGA and AGG"
                     /note="argU"
     tRNA            complement(1053..1128)
                     /gene="ileX"
                     /locus_tag="
                     "
                     /product="E. coli tRNA-Ile recognizing AUA"
                     /note="ileX"
     promoter        1245..1322
                     /gene="lacI (mutant)"
                     /note="lacIq promoter"
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1323..2405
                     /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"
     CDS             complement(3350..4165)
                     /codon_start=1
                     /gene="aph(3)-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPRK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4760..5305)
                     /direction=LEFT
                     /note="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."