pcDNA5/FRT/TO-Venus-Flag-Gateway (1124)
(Plasmid #40999)

• Purpose: (Empty Backbone)
• Depositing Lab: Jonathon Pines
• Publication: Di Fiore et al J Cell Biol. 2010 Aug 23;190(4):501-9.

Backbone

• Vector backbone: pcDNA5/FRT/TO
• Backbone manufacturer: Invitrogen
• Modifications to backbone: Venus-3xFlag added (C-terminal), converted to Gateway destination vector (see notes below)
• Vector type: Mammalian Expression
• Promoter: CMV
• Selectable markers: Hygromycin
• Tags / Fusion Proteins:
  • Venus YFP (C terminal on insert)
  • 3xFLAG (DYKDHDGDYKDHDIDYKDDDDK) (C terminal on insert)

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol and Ampicillin, 25 & 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): ccdB Survival
• Copy number: Unknown

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: LNCX
• 3′ sequencing primer: EGFP-N, BGH-rev

Resource Information

• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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

Depositor Comments

Venus YFP-3xFlag was inserted via BstXI-XhoI sites (to create pcDNA5/FRT/TO-Venus-Flag, Addgene Plasmid# 40998).

The plasmid was then converted to a Gateway destination vector by inserting Gateway cassette C1, containing recombination sites attR1 and attR2, CmR, and ccdB, into the Afl II site (blunt ended).

This plasmid can be used to place genes into the pcDNA5/FRT/TO-Venus-Flag construct by Gateway reaction, to make tetracycline-inducible stable cell lines.

For subcloning, the following restriction sites are present:
Between the CMV promoter and the Gateway cassette: PmeI (non-unique)
Between the Gateway cassette and Venus: HindIII, Asp718I, KpnI, BamHI (non-unique, also in GW casette), and BstXI
Between Venus and BGHpA: XhoI, Eco0109I, ApaI, and PmeI (non-unique)