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research article

Minimal shuttle vectors for Saccharomyces cerevisiae

Scutteri, Lorenzo  
•
Barth, Patrick  
•
Rahi, Sahand Jamal  
May 21, 2025
Synthetic Biology

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 centromericshuttle vectors for Saccharomyces cerevisiae, a widely used model organism in synthetic biology. We constructed the plasmids using de novogene synthesis and consisting only of a yeast selection marker (HIS3, LEU2, TRP1, URA3, KanMX, or natMX6), 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: pLS400, pLS410(KanMX); pLS403, pLS413(HIS3); pLS404, pLS414(TRP1); pLS405, pLS415(LEU2); pLS406, pLS416(URA3); pLS408, pLS418(natMX6). This resource substantially simplifies advanced synthetic biology engineering in Saccharomyces cerevisiae.

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Type
research article
DOI
10.1093/synbio/ysaf010
Author(s)
Scutteri, Lorenzo  

École Polytechnique Fédérale de Lausanne

Barth, Patrick  

École Polytechnique Fédérale de Lausanne

Rahi, Sahand Jamal  

École Polytechnique Fédérale de Lausanne

Date Issued

2025-05-21

Publisher

Oxford University Press (OUP)

Published in
Synthetic Biology
Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
UPBARTH  
LPBS  
FunderFunding(s)Grant NumberGrant URL

EPFL

CRSK-3_190526,310030_204938,CRSK-3_221036

Available on Infoscience
May 22, 2025
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/250366
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