pKAR1/Pur
(Plasmid #23105)

• Purpose: (Empty Backbone) Allows expression of shRNA from U6 promoter and a rescue cDNA from a doxycyclin inducible promoter in the same plasmid. Contains puromycin resistance gene
• Depositing Lab: Randy Poon
• Publication: Ma et al Nucleic Acids Res. 2007 . 35(4):e22.

Backbone

• Vector backbone: pUHD-P3
• Backbone size (bp): 5669
• Modifications to backbone: See associated publication for details of plasmid construction.
• Vector type: Mammalian Expression, RNAi
• Selectable markers: Puromycin

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
• Promoter: TRE for cDNA; U6 for shRNA
• Tag / Fusion Protein:
  • Flag (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: see paper (not destroyed)
• 3′ cloning site: see paper (not destroyed)
• 5′ sequencing primer: LNCX
• 3′ sequencing primer: EBV-rev

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

This vector is designed for dual expression of a shRNA and the respective rescue cDNA to allow a one-step validation of shRNAs and generation of stable shRNA-expressing cells. The shRNA is expressed under the control of a mouse U6 RNA promoter, while the rescue cDNA is expressed under the control of a doxycycline-inducible promoter. The effects of gene knockdown by the shRNA are effectively under conditional control as cDNA expression can be regulated by the inducible promoter.

The shRNA sequence must be cloned into this plasmid using BbsI and XbaI sites BEFORE cloning the cDNA, otherwise the NcoI, XhoI, EcoRI and BamHI sites will not be unique for subcloning of the cDNA. Note that the cloning of the shRNA sequence into the plasmid will replace the EGFP in this plasmid.

See pKAR1 (Addgene plasmid 23104; http://www.addgene.org/23104/) for sequence and map of this vector before the puromycin resistance gene was cloned in.