Resource Summary Report  New Search

URL: http://www.addgene.org/110914

Proper Citation: RRID:Addgene_110914

Insert Name: rab-3 promoter

Organism: Caenorhabditis elegans

Bacterial Resistance: Ampicillin

Defining Citation: PMID:

Vector Backbone Description: Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin

Comments: We used Q5 polymerase and primers engineered with restriction sites to amplify the 1885 bp TIR1 sequence from pLZ31 and cloned it into Nhe I/ Kpn I cut KG#59 (4.9 Kb). Transformed into XL1-Blue electrocompetent cells. Did Qiagen preps on 2 clones to find one with correct size insert for sequence verification. Features of the construct: The rab-3:: promoter drives expression pan-neuronally. AAAA provides a consensus ribosome binding site. TAA AT provides a consensus stop translation site. This is the Arabidopsis thaliana (mustard weed) TIR1 sequence published in the Zhang et al., 2015 Auxin Inducible Degradation paper from the Dernburg lab. It has been codon optimized for C. elegans and contains 2 synthetic introns to boost expression. It also contains 2 point mutations (D170E and M473L) shown to increase the affinity of the Arabidopsis TIR1 protein for its substrates and to increase auxin sensitivity without causing auxin-independent activity. There are also introns in the 5' and 3' UTR in this vector.

Expand All Collapse