Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of Shuttle Vector DNA.

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 R6Kgamma origin, which requires the pi protein to replicate. PIR2 cells, expressing pi, 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.