The fission yeast Schizosaccharomyces pombe is a powerful model organism for cell biology and molecular biology, as genetic manipulation is easily achieved. Introduction of exogenous genes cloned in episomal plasmids into yeast cells can be done through well-established transformation methods. For expression of genes in S. pombe cells, the multi-copy plasmid pREP1 and its derivatives, including pREP41 and pREP81, have been widely used as vectors. Although recent advancement of technology brought a number of useful genetic elements such as new promoters, selection marker genes and fluorescent protein tags, introduction of those elements into conventional pREP1 requires a large commitment of both time and effort because cloning procedures need to be repeated until the final products are constructed. Here, we introduce materials and methods to construct many pREP1-type plasmids easily and systematically using the Golden Gate shuffling method, which enables one-step ligation of many DNA fragments into a plasmid. These materials and methods support creation of expression plasmids employing a variety of novel genetic elements, which will further facilitate genetic studies using S. pombe.
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