LOCUS       Exported                7515 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Bacterial expression vector pBEN1-SGC, complete sequence.
ACCESSION   GU725055
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Savitsky P, Bray J, Cooper CDO., Marsden BD, Mahajan P, 
            Burgess-Brown NA, Gileadi O.
  TITLE     High-throughput production of human proteins for crystallization: 
            the SGC experience
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7515)
  AUTHORS   Gileadi O, Savitsky P, Burgess-Brown NA, Oppermann U, Ng S.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-FEB-2010) SGC, University of Oxford, ORCRB, Roosevelt 
            Drive, Oxford OX3 7DQ, UK
REFERENCE   3  (bases 1 to 7515)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7515
                     /organism="Bacterial expression vector pBEN1-SGC"
                     /mol_type="other DNA"
                     /db_xref="taxon:747347"
     CDS             8..307
                     /codon_start=1
                     /product="pBEN-SGC tag"
                     /label=pBEN-SGC tag
                     /note="N-terminal tag, includes SET and SBP regions and TEV
                     protease cleavage site"
                     /protein_id="ADE27957.1"
                     /translation="MDPEEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQTS
                     DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPSGGCKLGLGTENLYFQSM"
     misc_feature    8..136
                     /note="SET; derived from plasmid pBEn-SBP-SET1A 
                     (Stratagene); Region: solubility enhancement tag"
     CDS             11..136
                     /codon_start=1
                     /product="solubility-enhancement tag derived from T7 phage 
                     gene 10B"
                     /label=SET1
                     /translation="DPEEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQ"
     misc_feature    143..274
                     /note="streptavidin-binding peptide; derived from plasmid 
                     pBEn-SBP-SET1A (Stratagene); Region: SBP"
     CDS             143..253
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     misc_feature    284..304
                     /note="protein cleaved after Q; Region: TEV protease 
                     cleavage site"
     CDS             284..304
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        320..765
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             766..2187
                     /codon_start=1
                     /product="SacB"
                     /label=SacB
                     /note="levansucrase; negative selection marker; prevents 
                     growth on media containing 5% sucrose"
                     /protein_id="ADE27955.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             2295..2312
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2379..2426
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2463..2918
                     /label=F1 origin
                     /note="F1 origin"
     CDS             complement(3011..3826)
                     /codon_start=1
                     /product="aminoglycoside 3-phosphotransferase"
                     /label=aminoglycoside 3-phosphotransferase
                     /note="kanamycin-resistance protein"
                     /protein_id="ADE27954.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3948..4536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4722..4864
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4966..5157)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5966..7048)
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /note="lac repressor"
                     /protein_id="ADE27956.1"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7049..7126)
                     /gene="lacI"
                     /label=lacI promoter
     regulatory      7435..7478
                     /regulatory_class="promoter"
                     /note="regulatory region: T7 promoter and Lac operator"
     promoter        7435..7453
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7454..7478
                     /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)."
     RBS             7493..7515
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"