Spectrin-C-cpstFRET
(Plasmid #61111)

• Purpose: Expression of Spectrin-C-term 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): 11312
• Modifications to backbone: 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: NotI (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. 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.

Terms and Licenses

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