PEG-Actinin-M-sstFRET
(Plasmid #61100)

• Purpose: Expression of α-actinin with internal sstFRET inserted to measure cytoskeletal stress in vivo
• Depositing Lab: Fred Sachs
• Publication: Meng et al J Cell Sci. 2011 Jan 15;124(Pt 2):261-9. doi: 10.1242/jcs.071928. Epub 2010 Dec 20.

Backbone

• Vector backbone: pEYFP–C1
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 4700
• Total vector size (bp): 8432
• Modifications to backbone: To create chimeric gene constructs of α-actinin, we subcloned the actinin gene into pEYFP–C1 vector, where the YFP gene was removed beforehand. The restriction enzyme sites introduced into PCR products for subcloning were NheI-Actinin-KpnI.
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: α-actinin-sstFRET
• Mutation: sstFRET inserted at position 300
• Promoter: CMV

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: KpnI (not destroyed)
• 5′ sequencing primer: CMV Forward
• 3′ sequencing primer: SV40 poly A Reverse

Resource Information

• A portion of this plasmid was derived from a plasmid made by: pEYFP–C1 Venus and pECFP–C1 Cerulean plasmids were generous gifts from David W. Piston. sstFRET were inserted into actinin at position 300, which was located in the linker domain between first and second spectrin repeat domains by restriction sites AgeI and NotI. The restriction enzyme sites were introduced into the host proteins using the site-directed mutagenesis kit from Stratagene (La Jolla, CA) with the host protein amino acid unchanged. An 8-histidine tag followed by TAA stop codon was inserted in front of the NotI site to make sure that the tag was located in the C-terminal and well-exposed to solution.
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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