Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of Shuttle Vector DNA
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Updated: Aug 18, 2026
09:02
Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
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Cold Spring Harbor Protocols
|April 3, 2020
Abstract:
In two-step bacterial artificial chromosome (BAC) engineering, a single plasmid is introduced into the BAC-carrying cell lines. The shuttle vector pLD53.SCAB (or pLD53.SCAEB) carries the recA gene and the R6Kγ origin, which requires the π protein to replicate. PIR2 cells, expressing π, are typically used for the amplification of the vector and maintain about 15 copies/cell of the donor vector, which is relatively stable in this host.
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