LOCUS       Exported                7947 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pGTVL2, complete sequence.
ACCESSION   JF522100
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7947)
  AUTHORS   Savitsky P, Burgess-Brown N, Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-MAR-2011) Structural Genomics Consortium, University 
            of Oxford, Old Road Campus Research Building, Roosevelt Drive, 
            Oxford, Oxfordshire OX3 7DQ, UK
REFERENCE   2  (bases 1 to 7947)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7947
                     /organism="Cloning vector pGTVL2"
                     /mol_type="other DNA"
                     /db_xref="taxon:1079932"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    42..773
                     /label=N-tag
                     /note="N-tag"
     misc_feature    45..62
                     /label=hexahistidine tag
                     /note="hexahistidine tag"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..722
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     misc_feature    70..725
                     /label=glutathione S-transferase
                     /note="glutathione S-transferase"
     misc_feature    750..773
                     /label=TEV protease cleavage site
                     /note="TEV protease cleavage site"
     CDS             750..770
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     misc_feature    759..773
                     /label=5-LIC
                     /note="5-LIC"
     promoter        786..1231
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             1232..2653
                     /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="AEN74907.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            1232..2653
                     /gene="sacB"
                     /label=sacB
     misc_feature    2699..2713
                     /label=3-LIC
                     /note="3-LIC"
     CDS             2761..2778
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2845..2892
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    2929..3384
                     /label=f1 origin
                     /note="f1 origin"
     rep_origin      2929..3384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(3477..4292)
                     /codon_start=1
                     /gene="aph"
                     /product="aminoglycoside 3-phosphotransferase"
                     /label=aph
                     /note="kanamycin resistance protein from Corynebacterium 
                     diphtheriae"
                     /protein_id="AEN74908.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(3477..4292)
                     /gene="aph"
                     /label=aph
     rep_origin      4414..5002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      5001
                     /label=colE1
                     /note="colE1"
     misc_feature    5188..5330
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5432..5623)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6432..7514)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="AEN74909.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(6432..7514)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(7515..7592)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        7901..7919
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      7901..7917
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     misc_feature    7920..7944
                     /label=lac operator
                     /note="lac operator"
     protein_bind    7920..7944
                     /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)."