|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||26096||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerStructural Genomics Consortium
- Backbone size (bp) 7325
Vector typeBacterial Expression
- Promoter T7-lacO
/ Fusion Protein
- Hexahistidine tag with TEV cleavage (N terminal on backbone)
Growth in Bacteria
Copy numberHigh Copy
- 5′ sequencing primer T7
- 3′ sequencing primer T7-term (Common Sequencing Primers)
The pET28-MHL vector (GenBank accession EF456735) was derived from expression plasmid pET28a-LIC (SGC). It is used for T7 promoter driven expression of recombinant proteins with the addition of an 18 amino acid N-terminal fusion tag containing 6X His followed by a TEV cleavage site. The GSS residues after the Met start site were removed to reduce N-terminal gluconoylation via preventing N-terminal Met excision. 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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pET28-MHL was a gift from Cheryl Arrowsmith (Addgene plasmid # 26096 ; http://n2t.net/addgene:26096 ; RRID:Addgene_26096)