LOCUS       Exported                7602 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNH-TrxT, complete sequence.
ACCESSION   GU269914
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7602)
  AUTHORS   Cooper CDO., Gileadi O.
  TITLE     A 6xHis & thioredoxin-tagged vector for LIC cloning and 
            high-throughput protein expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7602)
  AUTHORS   Cooper CDO., Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-DEC-2009) Structural Genomics Consortium, University 
            of Oxford, ORCRB, Old Road Campus, off Roosevelt Drive, Oxford, 
            Oxfordshire OX3 7DQ, United Kingdom
REFERENCE   3  (bases 1 to 7602)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7602
                     /organism="Expression vector pNH-TrxT"
                     /mol_type="other DNA"
                     /db_xref="taxon:706476"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      27..33
                     /regulatory_class="ribosome_binding_site"
     misc_feature    42..398
                     /note="6x His thioredoxin; Escherichia coli thioredoxin 
                     N-terminal tag"
     misc_feature    42..62
                     /label=hexahistidine tag
                     /note="hexahistidine tag"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             72..398
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             405..425
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     misc_feature    405..422
                     /label=TEV protease cleavage site (cut after Q)
                     /note="TEV protease cleavage site (cut after Q)"
     misc_feature    414..428
                     /note="5-LIC sequence; add to upstream PCR primer"
     promoter        441..886
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             887..2308
                     /codon_start=1
                     /gene="sacB"
                     /product="SacB"
                     /label=sacB
                     /note="levansucrase; negative selection gene from Bacillus 
                     subtilis; converts sucrose to a toxic product; allows 
                     selection of recombinant plasmids on plates containing 5% 
                     sucrose"
                     /protein_id="ADB27020.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            887..2308
                     /gene="sacB"
                     /label=sacB
     misc_feature    2354..2368
                     /note="3-LIC sequence; add complement to downstream PCR 
                     primer"
     CDS             2416..2433
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2500..2547
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2584..3039
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(3132..3947)
                     /codon_start=1
                     /product="Aph"
                     /label=Aph
                     /note="kanamycin resistance protein from Corynebacterium 
                     diphtheriae"
                     /protein_id="ADB27021.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      4069..4657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      4656
     misc_feature    4843..4985
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5087..5278)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     gene            complement(6087..7187)
                     /gene="lacI"
                     /label=lacI
     CDS             complement(6087..7178)
                     /codon_start=1
                     /gene="lacI"
                     /product="LacI"
                     /label=lacI
                     /protein_id="ADB27022.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     CDS             complement(6087..7169)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7170..7247)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        7556..7574
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      7556..7572
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     misc_feature    7575..7599
                     /label=lac operator
                     /note="lac operator"
     protein_bind    7575..7599
                     /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)."