PurposeExpression of α-actinin with C-term sstFRET inserted to measure cytoskeletal stress in vivo
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||61101||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4700
- Total vector size (bp) 8432
Modifications to backboneTo 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 typeMammalian Expression
Selectable markersNeomycin (select with G418)
Growth in Bacteria
- Promoter CMV
/ Fusion Protein
- sstFRET (C terminal on insert)
- 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 (Common Sequencing Primers)
A portion of this plasmid was derived from a plasmid made bypEYFP–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.
Terms and Licenses
- Not Available to Industry
Article Citing this Plasmid
sstFRET is present in this plasmid at the C-terminus of actinin
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:PEG-Actinin-C-sstFRET was a gift from Fred Sachs (Addgene plasmid # 61101 ; http://n2t.net/addgene:61101 ; RRID:Addgene_61101)
For your References section:Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor. Meng F, Sachs F. J Cell Sci. 2011 Jan 15;124(Pt 2):261-9. doi: 10.1242/jcs.071928. Epub 2010 Dec 20. 10.1242/jcs.071928 PubMed 21172803