Structure of recombinant HCMV BAC DNAs.

To construct the mutant BAC DNAs, a marker cassette containing the RpsL gene, conferring increased sensitivity to streptomycin, and the neomycin resistance marker to provide kanamycin resistance, was inserted into the UL76 ORF with or without a flag epitope fused to N- terminus of the UL77 ORF. Intermediate BAC clones were isolated based on resistance to kanamycin. In a second round of homologous recombination, the entire marker cassette was replaced with the mutated sequence by the counter selection using an oligo as described in the Materials and Methods. Lower case letters indicate mutated bases to insert the TAG stop codon in the UL76 ORF.