Schematic of the modification of pBRf to arrive at a self-excisable construct (pBR). A. The TK sequences present in plasmid #5 were inserted into plasmid pEPMCS-in-Belo containing flanks A and B homologous to sequences within in the mini-F vector present in pBRf as well as an I-Sce I site and aphAI, which confers resistance to kanamycin, resulting in plasmid #10. B. The construct was then integrated into the mini-F sequence of pBRf in inverse orientation via en passant mutagenesis. After transfection of the generated construct in eukaryotic cells, vector sequences are lost through two antiparallel recombination events, which are indicated by the cross-over lines.