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Gene manipulation and epitope tagging are essential tools for understanding the molecular function of specific genes. The opportunistic human pathogen  Candida albicans  is a diploid fungus that utilizes a non-canonical genetic code. Since selection markers available in this organism are scarce, several tools based on recyclable markers have been developed for gene disruption, such as the  Clox  system. This system relies on the Cre recombinase, which recycles selection markers flanked by  loxP  sites with high efficiency, facilitating single marker or multi-marker recycling. However, PCR-based modules for epitope tagging, such the pFA-modules, mainly use limited non-recyclable auxotrophic markers. To solve this problem, we have used a Gibson assembly strategy to construct a set of new plasmids where the auxotrophic markers of the pFA vectors were swapped with five recyclable marker modules of the  Clox  system, enhancing the versatility of the pFA plasmids. This new toolkit, named pFA- Clox , is composed of 36 new vectors for gene disruption and epitope tagging (GFP, 3xGFP, mCherry, 3xHA, 5xmyc and TAP). These plasmids contain the dominant  NAT1  marker, as well as  URA3 ,  HIS1  and  ARG4  cassettes, thereby permitting functional analysis of laboratory strains as well as clinical isolates of  C .  albicans . In summary, we have adapted the  Clox  system to the pFA-backbone vectors. Thus, the set of primers used for the amplification of previously published pFA modules can also be utilized in this new pFA- Clox  system. Therefore, this new toolkit harbors the advantages of both systems, allowing accelerated gene modification strategies that could reduce time and costs in strain construction for  C .  albicans .

A new toolkit for gene tagging in Candida albicans containing recyclable markers