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. 2013 Nov 20;3:3279. doi: 10.1038/srep03279

Figure 2. Schematic workflow of the knockout of OCH1 using a flipper cassette.

Figure 2

The regions 5′ OCH1 and 3′ OCH1 represent sequences upstream and downstream of the OCH1 ORF, respectively. The 34 bp flipper recombinase target (FRT) sequences flank the AOX1 promoter (P AOX1), the FLP recombinase ORF, the CYC1 transcription terminator (CYC TT), a constitutive eukaryotic and a prokaryotic promotor (P ILV and P EM72, respectively), a ble ORF mediating Zeocin™ resistance and an AOD transcription terminator (AOD TT). A double homologous recombination event replaced the OCH1 ORF in the genome with the flipper cassette. Growth of recombinant cells on methanol induced the production of the FLP recombinase which recognized the two FRT sites and excised the inner sequence, leaving only one FRT site in the genome. Single fragments are not drawn to scale.