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URL: http://www.addgene.org/110878

Proper Citation: RRID:Addgene_110878

Insert Name: unc-17beta promoter

Organism: Caenorhabditis elegans

Bacterial Resistance: Ampicillin

Defining Citation: PMID:15489510

Vector Backbone Description: Backbone Size:2617; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin

Comments: Used Age I/ Xho I to cut out the 3800 bp acy-1(P260S) cDNA from KG#81 and cloned into the like-digested unc-17b expression vector KG#65 (4200 bp). Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones to find one with correct size insert and vector and made glycerol stock. The insert was previously checked by sequencing and contains only the P260S mutation. Features of the construct: This expression construct has a promoter (unc-17beta) that will drive strong expression of the acy-1(P260S) cDNA in the A and B classes of ventral cord motor neurons + AS's, but not VC's or cholinergic neurons in the head or tail. The vector contains a "decoy" (see 1995 vector kit documentation) upstream of the promoter to reduce background expression in the gut and pharynx from read-through transcription. An unc-54 3' end containing a poly A addition signal is downstream of the MCS to stop transcription and provide a 3' UTR for the transcript. Three introns in the UTR's are included to give stable and uniform expression (1 in the 5' UTR, 1 just upstream of the unc-54 3' end, and 1 in the unc-54 3' end sequence). This construct includes only the coding region. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG. An AT immediately follows the TAA stop codon for a consensus stop site. Note: the acy-1 cDNA from which this clone was produced corresponds to the single acy-1 cDNA predicted by wormbase, which is F17C8.1.

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