LOCUS       Exported                7317 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-NHT-CF, complete sequence.
ACCESSION   EF199845
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7317)
  AUTHORS   Gileadi O, Burgess-Brown N, Loppnau P.
  TITLE     Vectors for Ligation-independent Cloning (LIC)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7317)
  AUTHORS   Gileadi O, Burgess-Brown N.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-DEC-2006) Structural Genomics Consortium, University 
            of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK
REFERENCE   3  (bases 1 to 7317)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7317
                     /organism="Expression vector pNIC-NHT-CF"
                     /mol_type="other DNA"
                     /db_xref="taxon:420521"
     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="ABM88739.1"
                     /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="sequence used for ligation-independent cloning; add 
                     to upstream PCR primer; the same sequence appears in a 
                     vector fragment created by BsaI cleavage"
     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="levansucrase converts sucrose to a toxic product; 
                     allows selection of recombinant plasmids on plates 
                     containing 5% sucrose"
                     /protein_id="ABM88736.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            616..2037
                     /gene="sacB"
                     /label=sacB
     misc_feature    2081..2097
                     /note="sequence used for ligation-independent cloning; add 
                     to downstream primer; the same sequence appears in a vector
                     fragment created by BsaI cleavage"
     CDS             2084..2131
                     /codon_start=1
                     /product="C-terminal FLAG epitope tag"
                     /label=C-terminal FLAG epitope tag
                     /note="C-tag"
                     /protein_id="ABM88740.1"
                     /translation="SKGGYGSDYKDDDDK"
     CDS             2105..2128
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2178..2195
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2262..2309
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2346..2801
                     /label=f1
                     /note="f1"
     CDS             complement(2894..3709)
                     /codon_start=1
                     /gene="aph"
                     /product="kanamycin resistance protein"
                     /label=aph
                     /protein_id="ABM88737.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(2894..3709)
                     /gene="aph"
                     /label=aph
     rep_origin      3831..4419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      4418
                     /label=colE1
                     /note="colE1"
     misc_feature    4605..4747
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4849..5040)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5849..6931)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="ABM88738.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(5849..6931)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(6932..7009)
                     /gene="lacI"
                     /label=lacI promoter