pAAVdual-CAG-CcdB

The pAAVdual-CAG-CcdB plasmid serves as a destination vector for efficient ORF cloning into the pAAVdual system using Gateway technology. This system is part of AAVdual platform, which integrates the AAV vector plasmid (pAAVtri) and the Ad helper plasmid (mini-pHelper) into a single vector. The CAG promoter is a synthetic hybrid promoter widely used in molecular biology to drive high levels of gene expression in mammalian cells. It is composed of three key elements: CMV Immediate-Early Enhancer: Derived from the cytomegalovirus (CMV), this enhancer sequence provides strong and ubiquitous transcriptional activation in a variety of cell types. Chick Beta-Actin Promoter: The core promoter region is derived from the chicken beta-actin gene, which is known for its high expression levels in many cell types, particularly in the context of gene expression in vertebrates. Rabbit Beta-Globin Intron: An intronic sequence from the rabbit beta-globin gene is included to enhance transcriptional efficiency and mRNA stability, further boosting expression levels. It is particularly valued for its strength and broad activity across various cell types, making it a popular choice for applications such as gene therapy, transgenic animal models, and research studies that require robust and consistent gene expression. The CcdB gene in the plasmid encodes a toxic protein that disrupts DNA gyrase, a bacterial enzyme essential for DNA replication, leading to cell death. This ensures that only bacterial cells with successful recombination events—those replacing the CcdB gene—survive, facilitating positive selection during cloning. By using the pAAVdual-CAG-CcdB destination plasmid along with the pENTR-GOI vector from the ORFeome collections, researchers can efficiently generate pAAVdual-CAG-GOI plasmids. These plasmids can then be used for AAV packaging within the AAVdual system, enabling the production of corresponding AAV vectors.

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Background

The ORFeome collections provide a comprehensive set of open reading frames (ORFs) from a genome, with each entry vector carrying an ORF flanked by attL1 and attL2 sites. In Gateway cloning, the entry clone undergoes an LR reaction with the destination vector, where the attL and attR sites recombine. This process transfers the gene of interest from the entry vector into the destination vector, replacing the CcdB gene with the ORF, enabling successful cloning for AAV production.NameSKUPriceBuySelect all