Fig. 2

Overview of strain construction genealogy used in this study and transient PDC1-amdS expression cassette for targeted integration and rapid excision at the ADE2 locus. a The order in which strains were constructed, as well as the modifications made either by (1) targeted integration of PCR product, (2) removal of gene or transient gene cassette or (3) introduction of a gene encoding plasmid. b The transient PDC1 and amdS expression cassettes containing homology to the other respective cassette and to the ADE2 locus were transformed into IMI244 allowing homologous assembly into a full-length cassette and targeted integration at the ADE2 locus when plated on selective media containing acetamide (0.6 g/L) and adenine (20 mg/L) (resulting in IMI275). The resulting integration cassette was flanked by identical tags which have homology to the ADE2 locus such that when plated on selective media containing fluoroacetamide (2.3 g/L) and the absence of adenine, removal of the cassette was induced resulting in reassembly of a functional ADE2 gene (resulting in IMK647)