Codons corresponding to asparagine residues of RTAs were mutated to aspartate (D) or glutamine (Q) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and confirmed by sequencing. For transfection with 293T cells, calcium phosphate or polyethylenimine was used when cells were of ~50 to 60% confluency. For BAC DNA transfection in SLK cells, Lipofectamine 3000 was used according to the manufacturer's instructions. Cells in each well of a 24-well plate were transfected with approximately 1 μg of BAC DNA, and SLK/iBAC-positive cells were selected with hygromycin B (200 μg/ml) at 48 hours after transfection.

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