LOCUS       Exported                7284 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC28-Bsa4, complete sequence.
ACCESSION   EF198106
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  AUTHORS   Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI.
  TITLE     A new vector for high-throughput, ligation-independent cloning 
            encoding a tobacco etch virus protease cleavage site
  JOURNAL   Protein Expr. Purif. 25 (1), 8-15 (2002)
  PUBMED    12071693
REFERENCE   2  (bases 1 to 7284)
  AUTHORS   Keates T, Cooper CD, Savitsky P, Allerston CK, Phillips C, 
            Hammarstrom M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA, 
            Muller S, Graslund S, Gileadi O.
  TITLE     Expressing the human proteome for affinity proteomics: optimising 
            expression of soluble protein domains and in vivo biotinylation
  JOURNAL   N Biotechnol (2011) In press
  PUBMED    22027370
REFERENCE   3  (bases 1 to 7284)
  AUTHORS   Gileadi O, Burgess-Brown N, Loppnau P.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-DEC-2006) Structural Genomics Consortium, University 
            of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK
REFERENCE   4  (bases 1 to 7284)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7284
                     /organism="Expression vector pNIC28-Bsa4"
                     /mol_type="other DNA"
                     /label=derived from pET28a (Novagen)
                     /note="derived from pET28a (Novagen)"
                     /db_xref="taxon:420517"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1..17
                     /regulatory_class="promoter"
                     /note="T7"
     misc_feature    20..44
                     /label=lac operator
                     /note="lac operator"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             89..154
                     /codon_start=1
                     /product="N-terminal tag"
                     /label=N-terminal tag
                     /note="N-tag"
                     /protein_id="ABM88724.1"
                     /translation="MHHHHHHSSGVDLGTENLYFQS"
     misc_feature    92..109
                     /label=hexahistidine tag
                     /note="hexahistidine tag"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    134..154
                     /label=TEV protease cleavage site (cut after Q)
                     /note="TEV protease cleavage site (cut after Q)"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     misc_feature    143..157
                     /note="5-LIC sequence; add to upstream PCR primer"
     promoter        170..615
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             616..2037
                     /codon_start=1
                     /gene="sacB"
                     /product="levansucrase"
                     /label=sacB
                     /note="negative selection gene from Bacillus subtilis; 
                     converts sucrose to a toxic product; allows selection of 
                     recombinant plasmids on plates containing 5% sucrose"
                     /protein_id="ABM88723.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            616..2037
                     /gene="sacB"
                     /label=sacB
     misc_feature    complement(2083..2097)
                     /note="3-LIC sequence; add complement to downstream PCR 
                     primer"
     CDS             2145..2162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2229..2276
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2313..2768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2861..3676)
                     /codon_start=1
                     /gene="aph"
                     /product="aminoglycoside 3-phosphotransferase"
                     /label=aph
                     /note="kanamycin resistance protein from Corynebacterium 
                     diphtheriae"
                     /protein_id="ABM88721.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(2861..3676)
                     /gene="aph"
                     /label=aph
     rep_origin      3798..4386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      4385
                     /label=colE1
                     /note="colE1"
     misc_feature    4572..4714
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4816..5007)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5816..6898)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="from Escherichia coli"
                     /protein_id="ABM88722.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(5816..6898)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(6899..6976)
                     /gene="lacI"
                     /label=lacI promoter