Figure 4

Schematic showing the experimental design to integrate the marked Pch PRP8 inteins at the CMD1 locus. The top panel shows the composition and organization of the PCR amplified DNA used to transform yeast. The blue hatched area corresponds to the central sequence of the CMD1 gene that was the target for homologous recombination. L1-L5 are linker regions. In the bottom panel are the two proteins produced after splicing: 1) calmodulin with GFP inserted between S79 and N80, and 2) Pch PRP8 intein with genetic markers inserted at the vacant endonuclease site. The junction between L2 and L5 is the splice site. L2 and L5 contain 5 and 4 amino acids from the original Pch Prp8 extein sequences. For a description of the linker sequences see Methods. EF refers to the calcium-binding EF-hand domains. The sizes of the regions are drawn for clarity and are not to scale.