Actinin-M-stFRET
(Plasmid #61103)

• Purpose: Expression of α-actinin with internal stFRET inserted to measure mechanical stress in real time
• Depositing Lab: Fred Sachs
• Publication: Meng et al FEBS J. 2008 Jun;275(12):3072-87. doi: 10.1111/j.1742-4658.2008.06461.x. Epub 2008 May 10.

Backbone

• Vector backbone: pEYFP–C1
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 4700
• Total vector size (bp): 8432
• Modifications to backbone: Gene was subcloned into the pEYFP-C1 vector in which the yellow fluorescent protein (YFP) gene was deleted.
• 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-stFRET
• 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. Cerulean gene was sub-cloned from pECFP-C1 and EcoRI and ApaI restriction enzyme sites were introduced into 5 prime and 3 prime of Cerulean DNA fragment. This DNA fragment was inserted into multiple cloning site of pEYFP-C1 Venus by EcoRI and ApaI digestion then ligation. The resulting vector has Venus followed closely by Cerulean and between them there are two restriction enzyme sites BglII and EcoRI which then were employed to insert the alpha-helix linker. The alpha-helix linker DNA was amplified by PCR and BglII and EcoRI sites were introduced into 5 prime and 3 prime ends. The final construct with alpha-helix connecting Venus and Cerulean was named stFRET and ready for eukaryotic expression.

Terms and Licenses

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