Skip to main content
This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at [email protected] Learn more

pKEN GFP mut2
(Plasmid #20409)

Full plasmid sequence is not available for this item.

Ordering

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

  • 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
  • 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
  • (Common Sequencing Primers)

Resource Information

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.

How to cite this plasmid ( Back to top)

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