PA-DAD-dark
(Plasmid #53096)

• Purpose: Expresses dark mutant PA-DAD in mammalian cells, an optogenetic tool to activate endogenous diaphanous related formins in live cells using light with LOV2 in the closed conformation
• Depositing Labs: Klaus Hahn, Ronen Zaidel-Bar
• Publication: Rao et al Cytoskeleton (Hoboken). 2013 Jul;70(7):394-407. doi: 10.1002/cm.21115. Epub 2013 May 24.

Backbone

• Vector backbone: pTriEx4
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5238
• 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: LOV(dark)-6aa-DAD
• Alt name: mVenus - 3x GS linker - LOV2-Jα (404-546) - 3x GS linker - DAD (1177-1222)
• Alt name: LOV2 "dark state” (closed conformation) mutant
• Alt name: pTriEx-mVenus-LOV(dark)-6aa-DAD
• Species: M. musculus (mouse); Avena sativa
• Mutation: L47F and C49S in Venus; C450M mutation in LOV2
• Tags / Fusion Proteins:
  • 6x His (N terminal on backbone)
  • mVenus (N terminal on backbone)

Cloning Information

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

Resource Information

• Supplemental Documents:
  • Annotated GenBank file

Terms and Licenses

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

Depositor Comments

Expresses photoactivatable (PA) Diaphanous auto-regulatory domain (DAD) consisting of a fusion of mVenus, dark LOV2 Jα protein domain of Avena sativa Phototropin1, and Diaphanous auto-regulatory domain of mouse Diaphanous-related formin-1 (mDia1)

Please note that the C450M mutation in LOV2 creates a "dark state" mutant. See Yi et al., ACS Synth Biol. 2014 Nov 21;3(11):788-95 for more information.