M50 Super 8x TOPFlash
(Plasmid #12456)

• Purpose: Beta-catenin reporter. TCF/LEF sites upstream of a luciferase reporter.
• Depositing Lab: Randall Moon
• Publication: Veeman et al Curr Biol. 2003 Apr 15. 13(8):680-5.

Backbone

• Vector backbone: pTA-Luc
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 4871
• Vector type: Luciferase

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: TCF/LEF binding sites
• Alt name: beta catenin reporter
• Insert Size (bp): 120
• Entrez Gene: Ctnnb1 (a.k.a. Bfc, Catnb, Mesc)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: MluI (unknown if destroyed)
• 3′ cloning site: MluI (unknown if destroyed)
• 5′ sequencing primer: RVPrimer3
• 3′ sequencing primer: LucNRev

Resource Information

• A portion of this plasmid was derived from a plasmid made by: The idea for the construct is from Hans Clevers lab, who designed the original TOPflash.
• Articles Citing this Plasmid:
  • 351 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This is a luciferase reporter of beta-catenin-mediated transcriptional activation. In HEK cells, maximal activation of this reporter is ~100-fold (activation by Wnt) up to ~1,000-fold (activation by phosphorylation mutants of beta-catenin). The appropriate control plasmid is clone M51, Super8XFOPflash, which has mutant TCF/LEF binding sites.

This construct was made by Ajamete Kaykas in the Moon lab. The backbone is the pTA-Luc vector of Clontech, which provides a minimal TA viral promoter driving expression of the firefly luciferase gene (see company publications for details). 7 TCF/LEF binding sites were cloned into the Mlu1 site of this vector (7 copies of: AGATCAAAGGgggta, with TCF/LEF binding site in CAP letters, and a spacer in lower case, separating each copy of the TCF/LEF site).

Note: This plasmid was published as M50 Super 8x TOPFlash, but the plasmid actually contains 7 TCF/LEF sites.