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URL: http://www.addgene.org/110874

Proper Citation: RRID:Addgene_110874

Insert Name: rab-3 promoter

Organism: Caenorhabditis elegans

Bacterial Resistance: Ampicillin

Defining Citation: PMID:15489511

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

Comments: Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep). Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 3.8 Kb acy-1 cDNA coding region from KG#61 and cloned into Age I/ Xho I cut KG#59 (rab-3:: expression vector). Transform into XL1-Blue electrocompetent cells. Miniprepped 8 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and made glycerol stock. Features of the expression construct: This expression construct has a promoter (rab-3) that will drive strong and specific expression of acy-1 cDNA in the entire nervous system of juvenile and adult animals. Other features of the expression vector include a "decoy" 5' to the promoter to reduce non-specific expression in the gut (see Fire Lab 1995 Kit documentation for description), the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns (as we are) as opposed to a gene with introns. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG to provide a consensus translation start site. The sequence AT follows the stop codon and immediately precedes the 3' restriction site to provide a consensus post - stop codon sequence. The vector provides the unc-54 3' UTR and control region. 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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