The three trypanosomatids pathogenic to men， Trypanosoma cruzi， Trypanosoma brucei and Leishmania major， are etiological agents of Chagas disease， African sleeping sickness and cutaneous leishmaniasis， respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle， as there are a high percentage of genes encoding proteins without functional annotation. Also， technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally， such platform would lead to efficient cloning and compatibility with various approaches. Thus， we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.