pLenti-DENV2A-GFP-puro
(Plasmid #140089)

• Purpose: Fluorescence activatable reporter of DENV-2 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-puro (Addgene Plasmid #39481)
• Backbone manufacturer: Invitrogen modified by Ie-Ming Shih
• Backbone size w/o insert (bp): 7057
• Total vector size (bp): 7912
• Modifications to backbone: Removal of SpeI, BamHI, EcoRI, PstI, and EcoRV restriction sites
• Vector type: Mammalian Expression, Lentiviral ; Low 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: DENV2A-GFP
• Insert Size (bp): 855
• Promoter: CMV
• Tags / Fusion Proteins:
  • GFP (N terminal on insert)
  • 6XHis tag (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SpeI (destroyed during cloning)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: CMV-fwd
• 3′ sequencing primer: BGH-Rev

Terms and Licenses

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

Depositor Comments

The DENV2A-GFP 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 - If the expression is too low you won’t detect enough signal over the background. Our recommendation is to use the pLenti-DENV2A-GFP-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 DENV-2 infection.