pLenti-CMV-FlaviA-mNeptune-puro
(Plasmid #140091)

• Purpose: Fluorescence activatable reporter of flavivirus NS2B-NS3 protease activity
• Depositing Lab: Jorge Luis Arias-Arias
• Publication: Arias-Arias et al J Biol Chem. 2020 Jan 9. pii: RA119.011319. doi: 10.1074/jbc.RA119.011319.

Backbone

• Vector backbone: pLenti CMV GFP Puro (658-5) (Addgene Plasmid #17448)
• Backbone manufacturer: Eric Campeau, Paul Kaufman
• Backbone size w/o insert (bp): 7871
• Total vector size (bp): 8681
• Modifications to backbone: Removal of EGFP coding sequence
• Vector type: Mammalian Expression, Lentiviral ; High expression
• Selectable markers: Puromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: FlaviA-mNeptune
• Insert Size (bp): 813
• Promoter: CMV
• Tag / Fusion Protein:
  • mNeptune (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XbaI (not destroyed)
• 3′ cloning site: SalI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: WPRE-R

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

The FlaviA-mNeptune reporter is not suitable for transient transfection as the reporter has a basal background that makes it sensitive to the levels of expression: If the expression is too high the background will mask the signal produced by the reporter and/or generate OSER structures - If the expression is too low you won’t detect enough signal over the background. Our recommendation is to use the pLenti-CMV-FlaviA-mNeptune-puro construct to pack lentiviral particles with the psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) plasmids, generate stable cell lines and sort cell subpopulations with different levels of the reporter's basal background before testing them. The best reporter cells will be those with the highest signal-to-noise ratio upon flavivirus infection.