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URL: http://www.addgene.org/110880

Proper Citation: RRID:Addgene_110880

Insert Name: rab-3 promoter

Organism: Caenorhabditis elegans

Bacterial Resistance: Ampicillin

Defining Citation: PMID:16624912

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

Comments: Used StrataScript Reverse Transcriptase and a primer engineered with a restriction site to make the full length pde-4d cDNA (2025 bp). We then nicked the duplexed RNA with RNAse H and synthesize the 2nd strand of cDNA using E. coli DNA Polymerase I, which can do nick translation. The DNA Pol I uses the nicked RNA as primers to synthesize the second strand (it chews up the RNA in front of it and replaces with DNA). We then used Accuprime Pfx and primers engineered with restriction sites to amplify and clone the cDNA into Nhe I/ Age I cut KG#59 (rab-3:: expression vector; 4.9 Kb). Transformed into XL1-Blue electrocompetent cells. Qiagen miniprepped 2 clones to found those with correct insert, then submitted the 2 clones for sequence. Choose one clone that had correct sequence and made glycerol stock. Features of the expression construct: This expression construct has a promoter (rab-3) that will drive expression of the cDNA throughout the nervous system. This construct includes only the coding region of pde-4d, and the sequence is taken from the Wormbase WS134 release, gene designation R153.1d. This 'd' isoform seems to be the most common splice variant. 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. Three introns (all located in untranslated regions of the vector) are present 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). The vector includes the unc-54 3' untranslated end containing a poly A addition signal to stop transcription and provide a 3' UTR for the transcript. The vector also 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.

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