FIG 1.

Insertion of a fluorescent marker for viral early gene expression into the mini-F region of pBRF. The PK1L-mRFP1-aphAI fragment was amplified from transfer vector pEP-MVA-dVI-PK1L-mRFP using the primers mRFPfw and mRFPrv. The cassette was integrated downstream of the existing late gene expression marker (eGFP) by en passant mutagenesis, resulting in pBRFeR. In a second en passant mutagenesis, the K1L promoter was exchanged for a previously published synthetic early promoter (Psyn7.5), resulting in pBRFseR. Dashed lines indicate recombination events between homologous sequences.