pCDH-puro-CMV-RC3AI
(Plasmid #78908)

• Purpose: Indicator of caspase-3-like protease activity
• Depositing Lab: Binghui Li
• Publication: Zhang et al Nat Commun. 2013;4:2157. doi: 10.1038/ncomms3157.

Backbone

• Vector backbone: pCDH
• Backbone size w/o insert (bp): 7378
• Vector type: Lentiviral
• Selectable markers: Puromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: RC3AI
• Species: Synthetic
• Insert Size (bp): 1285
• Mutation: S320A, deletion of AA1-200
• Promoter: CMV
• Tag / Fusion Protein:
  • HA (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: CMV-forward

Resource Information

• Supplemental Documents:
  • RC3AI.3 sequence information

Terms and Licenses

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

Depositor Comments

Usage information based on sensor VC3AI: VC3AI has no or little background fluorescence, and its activation depends on the cleavage of sensor sequence of DEVDG. In fact, the sensors containing DXXDG other than DEVDG do not work. However, cells expressing VC3AI may have “background” fluorescence in some conditions, and we can conform the so-called basal fluorescence still results from the cleaved VC3AI, because cells could have a low level of activated caspase3/7 in some conditions depending on cell line and culture condition.

To avoid the possible detectable basal fluorescence, we infect cells with different dilutions of lentivirus and use the stable cell line expressing sensor protein as high as possible but without basal fluorescence for experiments. Notably, the low level of sensor protein expression work poorly. During our investigation, the basal fluorescence often happens on GC3AI, if the sensor protein expression level is high. This is because GC3AI protein is more stable than other sensors.