RANLS-DEVD-BNES
(Plasmid #50840)

• Purpose: Expresses a tandem ddFP heterodimer in mammalian cells, plasmid 1 for a translocation-based red fluorescent caspase-3 biosensor, used together with plasmid 2 (BNLS).
• Depositing Lab: Robert Campbell
• Publication: Ding et al Nat Methods. 2015 Jan 26. doi: 10.1038/nmeth.3261.

Backbone

• Vector backbone: pcDNA3.1
• Backbone size w/o insert (bp): 5500
• Total vector size (bp): 7024
• Modifications to backbone: Multi cloning sites has been replaced by a customized sequence, which is compatible to the reading frame of vector pBAD hisB.
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH10B
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ddRFP A and ddRFP B
• Alt name: dimerization dependent red fluorescent protein (ddRFP)
• Species: Synthetic; synthetic construct
• Insert Size (bp): 1524
• GenBank ID: KF976776
• Promoter: CMV
• Tags / Fusion Proteins:
  • triplicated NLS sequence DPKKKRKV placed after ddRFP A
  • NES sequence LALKLAGLDIGS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XhoI (unknown if destroyed)
• 3′ cloning site: HindIII (unknown if destroyed)
• 5′ sequencing primer: TAATACGACTCACTATAGGG
• 3′ sequencing primer: TAGAAGGCACAGTCGAGG

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

There is a small discrepancy between the reference sequence and Addgene's quality control sequence, this causes an A to G mutation in the linker region between NLS and ddRFP B. This change should not affect plasmid function.