LOCUS       Exported                7335 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-Bio3 plasmid pNIC-Bio3, complete sequence.
ACCESSION   JN792439
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7335)
  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 29 (5), 515-525 (2012)
  PUBMED    22027370
REFERENCE   2  (bases 1 to 7335)
  AUTHORS   Burgess-Brown NA, Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-SEP-2011) Structural Genomics Consortium, SGC, 
            University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK
REFERENCE   3  (bases 1 to 7335)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7335
                     /organism="Expression vector pNIC-Bio3"
                     /plasmid="pNIC-Bio3"
                     /mol_type="other DNA"
                     /db_xref="taxon:1108600"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    42..110
                     /note="N-ternima tag; contains His6 and TEV-protease 
                     cleavage site"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        123..568
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             569..1990
                     /codon_start=1
                     /product="SacB"
                     /label=SacB
                     /note="levansucrase; negative selection marker; prevents 
                     growth on media containing 5% sucrose"
                     /protein_id="AER54653.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     misc_feature    2037..2102
                     /note="C-terminal biotin tag; biotin acceptor sequence"
     CDS             2052..2096
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2149..2166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2233..2280
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2317..2772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2865..3680)
                     /codon_start=1
                     /product="aminoglycoside 3-phosphotransferase"
                     /label=aminoglycoside 3-phosphotransferase
                     /note="kanamycin resistance"
                     /protein_id="AER54651.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3802..4390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4576..4718
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4820..5011)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5820..6902)
                     /codon_start=1
                     /gene="lacI"
                     /product="LacI"
                     /label=lacI
                     /note="Lac repressor"
                     /protein_id="AER54652.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(5820..6902)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(6903..6980)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        7289..7307
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7308..7332
                     /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)."