pTriEx-mVenus-PA-Rac1
(Plasmid #22007)

• Purpose: Expression of photoactivatable Rac1
• Depositing Lab: Klaus Hahn
• Publication: Wu et al Nature. 2009 Sep 3. 461(7260):104-8.

Backbone

• Vector backbone: pTriEx
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 6000
• Vector type: Mammalian Expression, Bacterial Expression, Insect 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: PA-Rac1
• Alt name: LOV2-Rac1
• Species: H. sapiens (human); Avena sativa (oat)
• Insert Size (bp): 1100
• Mutation: LOV(Leu404-Leu546), Rac1 starts at I4 and contains mutations Q61L, E91H and N92H.
• Entrez Gene: RAC1 (a.k.a. MIG5, MRD48, Rac-1, TC-25, p21-Rac1)
• Promoter: CMV, p10
• Tags / Fusion Proteins:
  • 6X His (N terminal on backbone)
  • mVenus (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Bam HI (not destroyed)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: pTriExUP

Resource Information

• Supplemental Documents:
  • Annotated sequence from Hahn lab
• Articles Citing this Plasmid:
  • 7 References

Terms and Licenses

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

Depositor Comments

Protocols for production and use of Hahn lab biosensors and photoactivatable proteins can be found on their web page, at the URL below. If you have suggestions for the protocols or have any questions, please feel free to contact the Hahn lab. Good luck with your experiments!

Hahn lab biosensor protocol: http://www.hahnlab.com/tools/index.html

Please note, Addgene has not confirmed all the mutations associated with this construct. We suggest sequence verifying this plasmid upon receipt.

Further remarks from the Hahn lab regarding some mutations in the fluorescent marker: "the C-S mutation was introduced by us to allow site specific labeling of the protein and we have not detected any effect.
The F-L mutation reverts the Venus sequence back to the YFP sequence at this site. It will have a very slight detrimental effect on the maturation speed of the chromophore, but will not affect the stability or wavelength of the chromophore.
Neither of these 2 mutations will have any effect on the function of the Lov-Rac construct , and the Venus will still function as it should, i.e. as a reporter for the construct."