pWPI
(Plasmid #12254)

• Purpose: (Empty Backbone) 2nd gen bicistronic lentiviral vector allows for simultaneous expression of a transgene and EGFP marker to facilitate tracking of transduced cells.
• Depositing Lab: Didier Trono
• Publication: Trono Lab Constitutive Lentiviral Plasmids (unpublished)

Backbone

• Vector backbone: pWPXL
• Backbone size (bp): 11101
• Vector type: Mammalian Expression, Lentiviral, Cre/Lox

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Use Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: None
• Tag / Fusion Protein:
  • EMCV IRES-EGFP cassette (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: MCS - see map (not destroyed)
• 3′ cloning site: MCS - see map (not destroyed)
• 5′ sequencing primer: See map

Resource Information

• Articles Citing this Plasmid:
  • 155 References

Terms and Licenses

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

Depositor Comments

This bicistronic vector allows for simultaneous expression of a transgene and EGFP marker to facilitate tracking of transduced cells. The EGFP marker cDNA has been inserted downstream of EMCV IRES.

A popular cloning strategy is to clone into the PmeI site upstream of the EMCV IRES. You can also clone into the PacI or SwaI site.

If you want to put another fluorescent protein after the IRES, note that you need to make sure that the open reading frame of the second gene is precisely fused to 11th ATG of IRES (i.e., 11th ATG of IRES is a start of your cDNA).

Please note that the full sequence for this plasmid is approximated and not fully verified. Please visit the Trono lab http://tronolab.epfl.ch for cloning strategies, protocols, publications, and more. See LentiWeb http://www.lentiweb.com for discussions on cloning strategies and protocols.

Updated sequence provided by Chang-Xin Shi, Mayo Clinic

Information for Cloning Grade DNA (Catalog # 12254-DNA.cg) ( Back to top)

Purpose

Cloning grade DNA is suitable for use in PCR, cloning reactions, or transformation into E. coli. The purity and amount is not suitable for direct transfections.

Delivery

• Amount: 2 µg
• Guaranteed Concentration: 100 ng/µl +/- 5 ng/µl
• Pricing: $105 USD
• Storage: DNA can be stored at 4℃ (short term) or -20℃ (long term).

Terms and Licenses

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

Quality Control

Addgene has verified this plasmid using Next Generation Sequencing. Results are available here