pKK-mAmber-TEV
(Plasmid #105775)

• Purpose: (Empty Backbone) Expression of your protein of interest in fusion with non-absorbing, non-emitting Venus mutant at the N-terminus (cleavable by TEV). Negative control for FRET measurements (PMID: 17040988, 22264545).
• Depositing Lab: Andrzej Dziembowski
• Publication: Szczesny et al PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018.

Backbone

• Vector backbone: pcDNA5/FRT/TO
• Backbone manufacturer: Invitrogen
• Vector type: Mammalian Expression ; Flp-In competent
• Selectable markers: Hygromycin
• Tag / Fusion Protein:
  • mAmber-TEV (N terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: None
• Tag / Fusion Protein:
  • mAmber-TEV (N terminal on backbone)

Resource Information

• Supplemental Documents:
  • pKK-mAmber-TEV.txt

Terms and Licenses

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

Depositor Comments

This vector is a member of the pKK vector family. Its cloning site was optimized for the SLIC approach, however, a traditional restriction and ligation based approach is also possible (see Szczesny RJ et al. for detailed protocols; bioRxiv, doi: https://doi.org/10.1101/160101) This vector is a derivate of the pcDNA5/FRT/TO plasmid (Invitrogen) and is suitable for stable cell line generation using the Flp-In system. Transcription of the gene of interest is driven by a tetracycline responsive CMV promoter. A given coding sequence can be cloned into all pKK vectors using just three universal PCR primers and a ligation-independent DNA cloning method. Amber is a Venus mutant (Y67C) that neither absorbs nor emits light but retains full tertiary structure of Venus. Used as negative control for FRET measurements. Amber coding sequence was cloned from Addgene ID: 27798.