pB6-Cdk6-T2A-TagRFPT-dre-PBD*-FNF-TK
(Plasmid #141452)

• Purpose: B6J Cdk6-T2A-TagRFPT-dre-PBD* allele targeting vector, low copy number
• Depositing Lab: Mario Capecchi
• Publication: Miscellaneous gene targeting vector plasmids (unpublished)

Backbone

• Vector backbone: pWS-TK6
• Backbone manufacturer: Sen Wu, Capecchi laboratory
• Vector type: Mammalian Expression, Mouse Targeting ; dre / rox
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): DH10B
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: Cdk6
• Species: M. musculus (mouse)
• Entrez Gene: Cdk6 (a.k.a. AI504062, Crk2)
• Tag / Fusion Protein:
  • C-terminal fusion of T2A-TagRFPT-dre-PBD* (C terminal on insert)

Resource Information

• Supplemental Documents:
  • pCdk6-mT2A-TagRFP-T-drePBD*-TK.gb
• A portion of this plasmid was derived from a plasmid made by: TagRFP-T is from Roger Tsien. dre-PBD* is from Francis Stewart. FRT-Neo-FRT is from pFNF (#22687).

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Linker between TagRFP-T and dre-PBD* is a mutated P2A sequence where 2 conserved amino acids are changed to Alanines. This mutated sequence does not skip.

The TagRFP-T fluorescence from the TagRFP-T-dre-PBD* is dim, presumably for the same reason as the sfGFP-iCre-ERT2 is dim, as described in Nam and Capecchi, 2020 (https://doi.org/10.1186/s13064-020-00139-5) and a Figshare page (https://doi.org/10.6084/m9.figshare.13218437.v35) Thus, the TagRFP-T fluorescence should be detectable only in cells in which the marker gene expression level is high.

Homology arms subcloned by recombineering from a clone of RPCI-24 C57BL/6J mouse genomic DNA library.

Please make sure the plasmid DNA preparation is pure by gel electrophoresis before linearizing for an electroporation.