Molecular construction of the DEN-2 full-length cDNA plasmids p2 and p2Δ30. A. Diagram of the complete full-length DEN-2 Tonga/74 cDNA plasmid p2 is shown annotated with the restriction enzyme and corresponding cleavage site locations used to assemble the subcloned RT-PCR fragments. Restriction enzyme cleavage sites are numbered relative to nucleotide position in the virus genome. The corresponding genomic regions encoded by each subcloned RT-PCR fragment, X, L, R, A, B, C, and Y, are shown above the plasmid diagram. Relative positions of the SP6 promoter and tetracycline resistance gene (Tetr) are indicated. B. To generate plasmid p2Δ30, 30 nucleotides are removed from the 3'-UTR (Fragment Y). The nucleotide sequence encompassing the Δ30 region is shown for the p2 parent cDNA and the resulting p2Δ30 cDNA. Nucleotide positions in the virus genome are indicated for p2.