Chromosomal building in E. coli. Recombination with circular targeting vectors. Chromosomal building is one of several techniques that allow seamless mutagenesis. It is based on a targeting vector that carries: i) an antibiotic resistance cassette e.g. ampicillin (amp); ii) the sequence to be introduced into the EBV-BAC (represented by the grey shading and star) flanked by EBV-specific sequences (designated as A and B on the EBV BAC and A' and B' on the targeting vector) that will determine its site of insertion; iii) the gene that encodes the lacZ enzyme; and iv) a temperature-sensitive origin of replication that is operative only at 30°C. The targeting vector is introduced into E.coli cells that carry the EBV BAC. Recombination between both prokaryotic episomes is performed by a recA recombinase present on the targeting vector, whose expression is driven by an arabinose-inducible promoter. Homologous recombination can be initiated anywhere within the regions of homology (indicated by an arrow). The antibiotic resistance cassettes present on the targeting vector (amp) and on the EBV BAC (cam) allow the selection of co-integrates, which are a fusion vector comprising the targeting vector and the EBV BAC. Propagation at 42°C (non-permissive temperature) forces the loss of free targeting vectors. A second round of recombination resolves the co-integrates and reconstitutes both the EBV BAC and the targeting vector. Depending on which flanking region initiates resolution of the co-integrate, a recombinant EBV BAC containing either the foreign sequence (a) or the wild type (b) will be generated. Reconstituted targeting vectors are eliminated by culture at non-permissive temperature. Candidate clones are assessed for their sensitivity to ampicillin and expression of the lacZ gene. LacZ-negative and ampicillin-sensitive clones, indicative of reconstituted EBV BACs, are then submitted to restriction enzyme analysis, colony PCR or any other appropriate technique. goi: gene of interest.