Minimal shuttle vectors for <i>Saccharomyces cerevisiae</i>

Sophisticated genetic engineering tasks such as protein domain grafting and multi-gene fusions are hampered by the lack of suitable vector backbones. In particular, many restriction sites are in the backbone outside the polylinker region (multiple cloning site; MCS) and thus unavailable for use, and the overall length of a plasmid correlates with poorer ligation efficiency. To address this need, we describe the design and validation of a collection of six minimal integrating or centromeric shuttle vectors for <i>Saccharomyces cerevisiae</i>, a widely used model organism in synthetic biology. We constructed the plasmids using <i>de novo</i> gene synthesis and consisting only of a yeast selection marker (<i>HIS3</i>, <i>LEU2</i>, <i>TRP1</i>, <i>URA3</i>, <i>KanMX</i>, or <i>natMX6</i>), a bacterial selection marker (ampicillin resistance), an origin of replication, and the MCS flanked by M13 forward and reverse sequences. We used truncated variants of these elements where available and eliminated all other sequences typically found in plasmids. The MCS consists of ten unique restriction sites. To our knowledge, at sizes ranging from ~2.6 to 3.5 kb, these are the smallest shuttle vectors described for yeast. Further, we removed common restriction sites in the open reading frames and terminators, freeing up ~30 cut sites in each plasmid. We named our pLS series in accordance with the well-known pRS vectors, which are on average 63% larger: pLS400, pLS410 (<i>KanMX</i>); pLS403, pLS413 (<i>HIS3</i>); pLS404, pLS414 (<i>TRP1</i>); pLS405, pLS415 (<i>LEU2</i>); pLS406, pLS416 (<i>URA3</i>); and pLS408, pLS418 (<i>natMX6</i>). This resource substantially simplifies advanced synthetic biology engineering in <i>S. cerevisiae</i>.