M72 Super(16X)TOPFLASH
(Plasmid #17165)

• Depositing Lab: Randall Moon
• Publication: DasGupta et al Science. 2005 May 6;308(5723):826-33. doi: 10.1126/science.1109374. Epub 2005 Apr 7.

Backbone

• Vector backbone: pTA-Luc
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 4800
• Vector type: Luciferase

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Stbl3
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: 16x Tcf/Lef sites
• Tag / Fusion Protein:
  • luciferase (C terminal on backbone)

Cloning Information

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

Terms and Licenses

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

Depositor Comments

Description: This is a luciferase reporter of beta-catenin-mediated transcriptional activation. The idea for the construct is from Hans Clevers lab, who designed the original TOPflash. However, this version has a much higher signal/noise ratio. 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). 16 TCF/LEF binding sites were cloned into the Mlu1 site of this vector (16 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).

Reference: Made by Ajamete Kaykas, citation Science 308: 826-833 (2005)