pAAVone-AAVs-CAG-CcdB

The pAAVone-AAVs-CAG-CcdB plasmids serves as destination vectors for efficient ORF cloning into the AAVpme system using Gateway technology. This system is part of AAVone platform, which integrates the AAV vector plasmid (pAAVtri) and the Ad helper plasmid (mini-pHelper) and AAV heper plasmid (pRCap) into a single plasmid. 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 pAAVone-AAVs-CAG-CcdB destination plasmids along with the pENTR-GOI vectors from the ORFeome collections, researchers can efficiently generate pAAVone-AAVs-CAG-GOI plasmids. These plasmids can then be used for AAV packaging with the AAVone 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.