pKEN GFP mut2
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||20409||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Backbone manufacturerEzaz-Nikpay et al, 1994 Protein Science
- Backbone size w/o insert (bp) 3531
Vector typeBacterial Expression
Growth in Bacteria
Copy numberHigh Copy
Insert Size (bp)716
MutationS65A V68L S72A
- Cloning method Restriction Enzyme
- 5′ cloning site XbaI (not destroyed)
- 3′ cloning site PstI (not destroyed)
- 5′ sequencing primer T7
- 3′ sequencing primer T3 (Common Sequencing Primers)
Terms and Licenses
Article Citing this Plasmid
An XbaI site was introduced at the beginning of GFP, see attached map for more details.
The protein is expressed from IPTG inducible promoter in pKEN vector. GFP is cloned as XbaI-PstI fragment into the polylinker; there is also NdeI site at the AUG that can be used.
Mut 2 is probably best for expression in bacteria.
Overexpression of GFP renders the cells osmosensitive. In the absence of IPTG, DH12 carrying the enclosed plasmids will lyse in LB. We recommend growing this plasmid in low salt LB (<5g/L NaCl); alternatively use E.coli strain that grossly overproduces lacI and totally shuts expression down.
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:pKEN GFP mut2 was a gift from Stanley Falkow & Raphael Valdivia (Addgene plasmid # 20409 ; http://n2t.net/addgene:20409 ; RRID:Addgene_20409)
For your References section:FACS-optimized mutants of the green fluorescent protein (GFP). Cormack BP, Valdivia RH, Falkow S. Gene. 1996 . 173(1 Spec No):33-8. 10.1016/0378-1119(95)00685-0 PubMed 8707053
Map uploaded by the depositor.