Minimal integrating shuttle vectors for Saccharomyces cerevisiae depleted of restriction sites outside the polylinker region

Abstract

Many plasmids harbor unnecessary elements that complicate or hinder cloning tasks such as inserting one gene into another for protein domain grafting. In particular, restriction sites may be present 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 these concerns, there has been a growing interest in minimal plasmids. Here, we describe the design and validation of a collection of six minimal integrating shuttle vectors for genetic manipulation in Saccharomyces cerevisiae. We constructed the plasmids using de novo gene synthesis and consisting only of a yeast selection marker (HIS3, TRP1, LEU2, URA3, natMX6, or KanMX), a bacterial selection marker (Ampicillin resistance), an origin of replication (ORI), and the MCS flanked by M13 forward and reverse sequences. We use 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 approximately 2.6 kb to 3.5 kb, these are the smallest shuttle vectors described for yeast. Further, we removed common restriction sites in the open reading frames (ORFs) and terminators, freeing up approximately 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: pLS403 (HIS3), pLS404 (TRP1), pLS405 (LEU2), pLS406 (URA3), pLS408 (natMX6), and pLS410 (KanMX). These minimal vector backbones open up new opportunities for efficient molecular biology and genetic manipulation in Saccharomyces cerevisiae.