FIG. 3.

Construction of the M83 revertant rΔM83, using the EGFP-puro cassette. (A) DNA sequences of the ΔM83 genome were recombined in NIH 3T3 cells with homologous sequences (indicated by identical shading and cross-hatching) in the rescue plasmid cassette pM83Res13.6 (B). Selected restriction sites used for subcloning or for reference are indicated with abbreviations as in Fig. 2. Bam, BamHI. (C) Recombination between both homologous regions yields an rΔM83 intermediate virus in which the EGFP-puro cassette and full-length M83 ORF have been inserted into the ΔM83 genome as indicated. Intramolecular recombination between M84 direct-repeat sequences in the rΔM83 intermediate would be expected to delete intervening marker gene sequences (bracketed in panel C), resulting in an EGFP⁻ LacZ⁻ virus with a wild-type (wt) HindIII C region (D).