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URL: http://www.addgene.org/110881
Proper Citation: RRID:Addgene_110881
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: Started with KG#203 (contains pde-4d(+) coding region cDNA in rab-3:: expression vector) and use the QuikChange site directed mutagenesis method to introduce the D448N mutation. The mutation changes codon 448 of pde-4d from GAT(Asp) to AAT (Asn) (HDVDH to HNVDH). Miniprepped 4 clones and chose 2 with the correct size insert to sequence. Sequenced the entire insert of both clones, and kept one clone that has the correct mutation, but no other mutations. After sequencing, this insert was "re-cloned" into a fresh rab-3:: expression vector to ensure that there are no other vector mutations that could affect its performance or expression in transgenic worms. 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 2025 bp coding region of pde-4d (and contains the D448N mutation), and the sequence is taken from the Wormbase WS134 release, gene designation R153.1d (the d isoform appears to be the most common based on analysis of EST cDNAs). 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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Source: Addgene (RRID:SCR_002037)
Description: Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
URL: http://www.addgene.org
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