AIMTOR A757 | Addgene 140829 product information

This webpage contains legacy information. The product is either no longer available from the supplier or has been delisted at Labome.

product summary

company name :

Addgene

product type :

cDNA

product name :

AIMTOR A757

catalog :

140829

citations: 1

Reference
Bouquier N, Moutin E, Tintignac L, Reverbel A, Jublanc E, Sinnreich M, et al. AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions. BMC Biol. 2020;18:81 pubmed publisher

product information

Catalog Number :

140829

Product Name :

AIMTOR A757

article :

doi	10.1186/s12915-020-00790-8
id	28211018
pubmed_id	32620110

bacterial resistance :

Ampicillin

cloning :

backbone

YEN

backbone_mutation

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

backbone_origin

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

6529

promoter

sequencing_primer_3

sequencing_primer_5

vector_types

Mammalian Expression

growth strain :

AIMTOR BRET Biosensor containing a mutated non-phosphorylable ULK1 peptide to use as a control constuct in parrallel with AIMTOR T757

origin :

pi :

alt_names

cloning

clone_method	Restriction Enzyme
cloning_site_3	NotI
cloning_site_5	BspEI
promoter
sequencing_primer_3	GCCAGAAGTCAGATGCTCA
sequencing_primer_5	CCACTACCTGAGCTACCAG
site_3_destroyed
site_5_destroyed

entrez_gene

aliases	ATG1, ATG1A, UNC51, Unc51.1, hATG1
gene	ULK1
id	8408

genbank_ids

NM_003565.4

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.

name

AIMTOR A757

shRNA_sequence

size

species

9606

Homo sapiens