The pBAC/oriV vector permitting its single-copy (SC) maintenance and, alternatively, its conditional, tightly regulated DNA amplification. This new derivative of the pBeloBAC11 vector preserves most of its original specific features, including the plasmid F-derived SC maintenance system based on the oriS–repE–parABC genes (seeKim et al. 1996), but was equipped with a second origin of DNA replication, oriV, from the broad-host-range plasmid RK2 (Stalker et al. 1981). We cloned the NotI-less oriVat the HpaI or XhoI sites, but for reasons not fully anticipated, the TrfA–oriV-directed DNA amplification (see Figs. 2 and 3) was the highest for oriV in the XhoI site. Four derivatives of pBAC/oriV have been constructed: (1) The pBeloBAC11/SceI/oriV (pJW408 = pBAC/oriV/SceI) vector with the I-SceI recognition site at the HpaI site of pBAC/oriV. (2) The pTrueBlue-BAC2/oriV (pJW406) vector, withoriV at the XhoI site. This vector features dark-blue colonies (darker than for original BACs and similarly dark as for pIndigoBAC/oriV; see below), thus allowing more accurate blue/white colony screening. It is based on pTrueBlue-BAC2 (Genomics One 1999 Catalog), which contains four additional cloning sites, as compared with pBeloBAC11, all within the specially constructed lacZαsegment (Slilaty and Lebel 1998). (3) The pTrueBlue-BAC2/oriV/SceI (pJW419) vector with the I-SceI recognition site cloned into the Eco47III site of pTrueBlue-BAC2/oriV. (4) The pIndigoBAC/oriV (pJW550) vector with oriV at the XhoI site. This vector features enhanced, dark-blue-color colony screening and is based on pIndigoBAC-5 (Epicentre 2001 Catalog). Details of the construction of pBAC/oriV and their derivatives are described in Methods. [NotI] Inactivated NotI site.
