AIMTOR A757
(Plasmid #140829)

• Purpose: AIMTOR BRET Biosensor containing a mutated non-phosphorylable ULK1 peptide to use as a control constuct in parrallel with AIMTOR T757
• Depositing Lab: Anne Bonnieu
• Publication: Bouquier et al BMC Biol. 2020 Jul 3;18(1):81. doi: 10.1186/s12915-020-00790-8.

Backbone

• Vector backbone: YEN
• Backbone manufacturer: Goyet et al 2016, Scientific Reports, 6:28231. DOI: 10.1038/srep28231 and Komatsu, N et al.Molecular biology of the cell 22, 464
• Backbone size w/o insert (bp): 6529
• Total vector size (bp): 6583
• Modifications to backbone: The plasmid coding for YEN contains a cytoplasmic Extracellular signal-regulated Kinase Activity Reporter (EKAR) boxed between BRET-compatible entities (Ypet and Nanoluciferase ), pEevee-Ypet-EKARcyto-Nluc. It was designed from the optimized backbone structure of a FRET biosensor with the 116-amino-acid (SAGG)29-repeats linker (pEKAREV(3560)NES pEevee) constructed by Komatsu and colleagues (Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Molecular biology of the cell 22, 4647–4656, doi: 10.1091/mbc.E11-01-0072, 2011). The ECFP-coding sequence, removed by NotI/XbaI digestion, was replaced by the Nanoluciferase-coding sequence, amplified by PCR (from pNanoLuc3.2 NFKB RE) and inserted into the NotI/XbaI sites by an Infusion reaction (Clontech), The YEN plasmid served then as a backbone plasmid to construct the plasmid AIMTOR A757. To this end, we removed the sequence coding for the ERK sensor site by a BspeI/NotI restriction digest. Following a ligation with a cohesive double strand pre-annealed oligonucleotides encoding the A757 mutated sensor peptide derived from human UlK1 protein, we obtained the plasmid AIMTOR A757
• Vector type: Mammalian Expression

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: AIMTOR A757
• Species: H. sapiens (human)
• Insert Size (bp): 34
• Mutation: The insert comprises only a small sequence of human Ulk1 encoding for a 9 aminoacids peptide, conserved 100% in mouse Ulk1. The original serine residue phosphorylable by mTOR in this peptide (position 757 in mouse Ulk1) has been mutated to a non-phosphoylable Alanine residue to serve as a negative control for BRET experiments.
• GenBank ID: NM_003565.4
• Entrez Gene: ULK1 (a.k.a. ATG1, ATG1A, UNC51, Unc51.1, hATG1)
• Tag / Fusion Protein:
  • NES Nuclear export signal (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BspEI (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: CCACTACCTGAGCTACCAG
• 3′ sequencing primer: GCCAGAAGTCAGATGCTCA

Terms and Licenses

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