GgVcl 1-1066 FRET tail probe 1
(Plasmid #46276)

• Depositing Lab: Susan Craig
• Publication: Chen et al J Cell Biol. 2005 May 9;169(3):459-70.

Backbone

• Vector backbone: pECFP-N3
• Backbone manufacturer: Craig lab
• Modifications to backbone: pECFP-N3 vector was constructed as an intermediate vector for generating tail probe. ECFP was PCR amplified with 5-GGTACCatggtgagcaagggc-3 and 5-GCGGCCGCTttacttgtacagctc-3 to generate 5' KpnI and 3' NotI. The product was subcloned into TOPO pCRII and sequenced. The KpnI and NotI fragment of pCRII/ECFP was used to replace the EGFP fragment of pEGFP-N3 to generate pECFP-N3.
• 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: Vcl 1-1066 + EYFP
• Alt name: Vinculin
• Alt name: Vcl
• Species: G. gallus (chicken)
• Mutation: addition of 9 bp linker and EYFP at aa 883
• Entrez Gene: VCL (a.k.a. VINC1)
• Promoter: CMV
• Tag / Fusion Protein:
  • ECFP (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (not destroyed)
• 3′ cloning site: SalI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: ?

Terms and Licenses

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

Depositor Comments

A 9-bp fragment encoding a NotI site was introduced by mutagenesis into pEGFP-C1/vinculin cDNA (chicken) immediately after the codon for aa 883 (Coutu and Craig, 1988) using
the Quick Change kit (Stratagene). The cDNA of EYFP (CLONTECH Laboratories, Inc.) minus the stop codon and flanked by NotI was inserted into NotI-digested vinculin.