pET28GST- LIC

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  • Model: PVT10579
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pET28GST-LIC


PVT10579          2ug

 

pET28GST-LIC Information

Promoter: T7/Laco promoter

Replicon: pBR322 ori, F1 ori

Terminator: T7 Terminator

Plasmid size: 7994bp

Plasmid tagging: N-GST, N-6 x HIS, N-thrombin site, SacB

Prokaryotic resistance: kanamycin Kan

Screening marker: SacB/ sucrose screening

Clone strain: Escherichia coli DH5 alpha

Culture conditions: 37 C, aerobic, LB

Expression vector: Escherichia coli BL21 (DE3)

Culture conditions: 37 C, aerobic, LB

Induction: IPTG and lactose and its analogues.

5'sequencing primers: T7 (TAATACGACTCACTATAGGG)

3'sequencing primers: T7-ter (TGCTAGTTATTGCTCAGCGG)

 

 

pET28GST-LIC Description

pET28GST-LIC is an E.coli expression vector. T7 promoter drives GST to express with fusion expression. SacB lethal gene is on the vector and positive clones can be screened by 5% sucrose.

pET28GST-LIC vector was derived from expression plasmid pET28a-LIC (SGC) by inserting the GST-tag from pET41a (Novagen) into the XbaI and NcoI sites. It is used for T7 promoter driven expression of recombinant proteins with the addition of a 242 amino acid N-terminal fusion tag containing the 217 amino acid GST-tag protein followed by a 6X His followed by a thrombin cleavage site. Two stop codons are included in the vector at the C-terminal cloning site.Insertion of DNA sequence into the cloning/expression region is preformed using BD-Biosciences Infusion enzyme mediated directional recombination between complementary 15 nucleotide DNA sequences at the ends of the insert (PCR product) and BseRI linearized vector. Insertion of target sequence involves replacement of a SacB gene stuffer sequence, which provides for negative selection of the original plasmid on 5% sucrose.

 

pET28GST-LIC Multiple cloning site

pET28GST-LIC-vector

 

pET28GST-LIC Sequence

LOCUS       Exported                7998 bp ds-DNA     circular SYN 17-NOV-2017
DEFINITION  synthetic circular DNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7998)
  AUTHORS   .
  TITLE     Direct Submission
           
FEATURES             Location/Qualifiers
     source          1..7998
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /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(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /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             5042..5064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5072..5725
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             5753..5770
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5780..5797
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    5825..5858
                     /label=loxP
                     /bound_moiety="Cre recombinase"
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