pKEN GFP mut2
(Plasmid #20409)

• Depositing Labs: Stanley Falkow, Raphael Valdivia
• Publication: Cormack et al Gene. 1996 . 173(1 Spec No):33-8.

Backbone

• Vector backbone: pKEN
• Backbone manufacturer: Ezaz-Nikpay et al, 1994 Protein Science
• Backbone size w/o insert (bp): 3531
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: GFP
• Species: Aequorea victoria
• Insert Size (bp): 716
• Mutation: S65A V68L S72A

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XbaI (not destroyed)
• 3′ cloning site: PstI (not destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: T3

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.