pKK-mCherry-TEV
(Plasmid #105779)

• Purpose: (Empty Backbone) Expression of your protein of interest in fusion with red fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 15558047). mCherry descends from the first truly monomeric red FP mRFP1.
• 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:
  • mCherry-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:
  • mCherry-TEV (N terminal on backbone)

Resource Information

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

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• 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. mCherry descends from the first truly monomeric red fluorescent protein mRFP1, which was obtained by mutating DsRed form Discosoma sp. Note that EGFP and mCherry come from different organisms and their sequences differ greatly. However, GFP-type termini were introduced into mCherry to increase tolerance of N- and C-terminal fusions in mammalian cells (PMID: 15558047). Thus, terminal sequences of EGFP and mCherry are the same.