LOCUS       Exported                7321 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-CTB10H, complete sequence.
ACCESSION   KX139199
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  AUTHORS   Savitsky P.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-APR-2016) Structural Genomics Consortium, University 
            of Oxford, Old Road Campus Research Building, Roosevelt Drive, 
            Oxford, Oxfordshire OX3 7DQ, UK
REFERENCE   2  (bases 1 to 7321)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7321
                     /organism="Expression vector pNIC-CTB10H"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:1929939"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1..17
                     /regulatory_class="promoter"
     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)."
     misc_feature    70..85
                     /label=LIC5
                     /note="LIC5"
     protein_bind    113..146
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        161..606
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             607..2028
                     /codon_start=1
                     /gene="sacB"
                     /product="levansucrase"
                     /EC_number="2.4.1.10"
                     /label=sacB
                     /note="negative selection gene derived from Bacillus 
                     subtilis; converts sucrose to a toxic product; allows 
                     selection of recombinant plasmids on plates containing 5% 
                     sucrose"
                     /protein_id="APU50911.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            607..2028
                     /gene="sacB"
                     /label=sacB
     CDS             2098..2118
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     misc_feature    2098..2117
                     /label=TEV protease cleavage site
                     /note="TEV protease cleavage site"
     misc_feature    2101..2117
                     /label=LIC3
                     /note="LIC3"
     misc_feature    2125..2169
                     /note="AviTag; C-terminal biotin acceptor site"
     CDS             2125..2169
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     misc_feature    2170..2199
                     /label=His10
                     /note="His10"
     CDS             2170..2199
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2266..2313
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2350..2805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2898..3713)
                     /codon_start=1
                     /gene="aph"
                     /product="aminoglycoside 3-phosphotransferase"
                     /EC_number="2.7.1.95"
                     /label=aph
                     /note="kanamycin resistance protein derived from 
                     Corynebacterium diphtheriae"
                     /protein_id="APU50910.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(2898..3713)
                     /gene="aph"
                     /label=aph
     rep_origin      3835..4423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4609..4751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4853..5044)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5853..6935)
                     /codon_start=1
                     /gene="lacI"
                     /product="Lac repressor"
                     /label=lacI
                     /protein_id="APU50912.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(5853..6935)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(6936..7013)
                     /gene="lacI"
                     /label=lacI promoter