Bodo saltans SSU-GFP-Neo cassette v1

This plasmid is designed to integrate within the small subunit ribosomal RNA gene region of B. saltans. It carries 1 kb of the B. saltans small subunit ribosomal RNA gene. To construct this cassette, we first sequenced the complete ribosomal operon of B. saltans, which has a total length of ̴9 kb. The structure of the ribosomal operon of B. saltans is similar in the closely related kinetoplastida Leishmania, where the ribosomal RNA genes are arranged in head- to-tail repeats (Figure 4b and Yan et al.,Molecular and biochemical parasitology103, no. 2 (1999): 197-210). For the cassette construction, we used only a 1 kb region of the 18S ribosomal RNA gene, which is represented in the schematic as Homologous Recombination Region 1 (HR1) and Homologous Recombination Region 2 (HR2) each, of which is 500 bp . The GFP and Neo genes are located in the middle of the construct and their expression is controlled by the presence of the B. saltans tubulin intergenic region (Bs TUB IGR) at the 3’ and 5’ ends of each gene. As the transcription in B. saltans is polycistronic, the presence of the tubulin intergenic region is necessary for trans-splicing and polyadenylation.