The study introduces a new gene disruption cassette, loxP–kanMX–loxP, designed for repeated use in budding yeast. This cassette combines the advantages of the heterologous kan' marker gene and the Cre–loxP recombination system. The cassette integrates efficiently via homologous recombination at the correct genomic locus (70% efficiency). Upon expression of the Cre recombinase, the kanMX module is excised, leaving a single loxP site. This system allows for the functional analysis of gene families by enabling repeated use of the kan' marker gene. The authors demonstrate the efficiency of the cassette in gene disruption experiments and show that the kan' marker can be efficiently rescued using the Cre recombinase. Additionally, they confirm that a second gene can be disrupted in a loxP-carrying yeast strain without affecting the first disruption. The system is particularly useful for yeast strains with incomplete deletions of standard markers or for industrially used strains lacking standard auxotrophic markers.The study introduces a new gene disruption cassette, loxP–kanMX–loxP, designed for repeated use in budding yeast. This cassette combines the advantages of the heterologous kan' marker gene and the Cre–loxP recombination system. The cassette integrates efficiently via homologous recombination at the correct genomic locus (70% efficiency). Upon expression of the Cre recombinase, the kanMX module is excised, leaving a single loxP site. This system allows for the functional analysis of gene families by enabling repeated use of the kan' marker gene. The authors demonstrate the efficiency of the cassette in gene disruption experiments and show that the kan' marker can be efficiently rescued using the Cre recombinase. Additionally, they confirm that a second gene can be disrupted in a loxP-carrying yeast strain without affecting the first disruption. The system is particularly useful for yeast strains with incomplete deletions of standard markers or for industrially used strains lacking standard auxotrophic markers.