p5A0-T
(Plasmid #92016)

• Purpose: Expresses TALE targeted to lac operator without TEV cut sites, anhydrotetracycline inducible TEV protease
• Depositing Lab: Brian Pfleger
• Publication: Copeland et al Nat Chem Biol. 2016 Apr;12(4):254-60. doi: 10.1038/nchembio.2021. Epub 2016 Feb 8.

Backbone

• Vector backbone: pBT-2
• Total vector size (bp): 6702
• Modifications to backbone: Encodes tetR.
• Vector type: TALEN

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: TALE targeting lac operator
• Alt name: TALElacO
• Alt name: TlacO
• Insert Size (bp): 2535
• Promoter: J23102 Promoter (from Anderson promoter library)
• Tags / Fusion Proteins:
  • FLAG tag (C terminal on insert)
  • 6x His Tag (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: gcgactcaactcatgaatc

Gene/Insert 2

• Gene/Insert name: Tobacco Etch Virus Protease
• Alt name: TEV protease
• Insert Size (bp): 750
• Mutation: S219V
• Promoter: tetA promoter
• Tags / Fusion Proteins:
  • 7x His Tag (N terminal on insert)
  • 5x Arginine Tag (C terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: cgagtttacgggttgtt

Resource Information

• Supplemental Documents:
  • p5A0-T.gb

Terms and Licenses

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

Depositor Comments

TEV protease induction will result in a growth defect if the culture is not outgrown to a suitable density prior to addition of anhydrotetracycline.