Spectrin-M-cpstFRET
(Plasmid #61109)

• Purpose: Expression of Spectrin-cpstFRET to measure mechanical stress and report multidimensional stress fields
• Depositing Lab: Fred Sachs
• Publication: Meng et al J Cell Sci. 2012 Feb 1;125(Pt 3):743-50. doi: 10.1242/jcs.093104.

Backbone

• Vector backbone: pEGFP-C1
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 4700
• Total vector size (bp): 12863
• Modifications to backbone: Based on Venus and Cerulean, we constructed circularly permutated cpVenus and cpCerulean. For cpVenus, we first subcloned the gene fragments of 175Gly–239Lys from Venus into pET-52b (+) vector with BamHI and EcoRI. Then we subcloned the gene fragments of 1Met–174Asp and connected them to the C-terminal of the first fragment (supplementary material Fig. S1). Five glycines were added between them to give flexibility with EcoRI and SacI. We generated cpCerulean similarly except that a different primer was used for fragment 175Gly–239Lys, which was 59- GGTACCAGGATCCATGGGCAGCGTGCAGC-39 with BamHI. For eukaryotic cell expression, we subcoloned cpVenus and cpCerulean into pEGFP vector with EGFP removed beforehand, and with Nhe1 for cpCerulean and with NheI for cpVenus. pET-cpstFRET was created by connecting cpVenus to the C-terminal of cpCerulean in pET-52b(+) vector with BglII and SacI. Fore eukaryotic cell expression of cpstFRET, we subcloned it into pEGFP vector with NheI and ApaI. To create chimeric gene constructs of α-spectrin, we subcloned the gene encoding spectrin into pEGFP-C1 vector with AgeI and SacII from which the EGFP gene was removed beforehand. cpst-FRET was inserted into spectrin at amino acid position 1200, which located in the linker domain between the 10th and 11th spectrin repeat domains by restriction sites SalI and NotI.
• 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: spectrin-cpstFRET
• Promoter: CMV

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AgeI (not destroyed)
• 3′ cloning site: SacII (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, Vanderbilt University, Nashville, TN.
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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