ArticleNucleic acids research2011
Re-engineering multicloning sites for function and convenience.
Article in Nucleic acids research, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 44 citations in OpenAlex.
- Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.Journal of microscopy · 2025Review
- Concise Overview of Methodologies Employed in the Study of Bacterial DNA Replication.International journal of molecular sciences · 2025Review
- Evolving dual-trait EPSP synthase variants using a synthetic yeast selection system.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Construction and Characterization of Light-Responsive Transcriptional Systems.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Optimization of mammalian expression vector by cis-regulatory element combinations.Molecular genetics and genomics : MGG · 2023Article
- Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor.Nature communications · 2023Article
- Article
- The activity of yeast Apn2 AP endonuclease at uracil-derived AP sites is dependent on the major carbon source.Current genetics · 2021Article
- Design and synthesis of synthetic UP elements for modulation of gene expression inSynthetic and systems biotechnology · 2019Article
- A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production.Biotechnology for biofuels · 2019Article
- Design of RNA hairpin modules that predictably tune translation in yeast.Synthetic biology (Oxford, England) · 2018Article
- Bricks and blueprints: methods and standards for DNA assembly.Nature reviews. Molecular cell biology · 2015Review
- Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris.Microbial cell factories · 2015Article
- Functional optimization of gene clusters by combinatorial design and assembly.Nature biotechnology · 2014Article
- Design of synthetic yeast promoters via tuning of nucleosome architecture.Nature communications · 2014Article
- One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy.Nucleic acids research · 2014Article
- Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.Metabolic engineering · 2013Article
- Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.Microbial cell factories · 2013Article
- Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications.FEMS yeast research · 2013Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Multicloning sites (MCSs) in standard expression vectors are widely used and thought to be benign, non-interacting elements that exist for mere convenience. However, MCSs impose a necessary distance between promoter elements and genes of interest. As a result, the choice of cloning site defines the genetic context and may introduce significant mRNA secondary structure in the 5'-untranslated region leading to strong translation inhibition. Here, we demonstrate the first performance-based assessment of MCSs in yeast, showing that commonly used MCSs can induce dramatic reductions in protein expression, and that this inhibition is highly promoter and gene dependent. In response, we develop and apply a novel predictive model of structure-based translation inhibition to design improved MCSs for significantly higher and more consistent protein expression. In doing so, we were able to minimize the inhibitory effects of MCSs with the yeast TEF, CYC and GPD promoters. These results highlight the non-interchangeable nature of biological parts and represent the first complete, global redesign of a genetic circuit of such widespread importance as a multicloning site. The improved translational control offered by these designed MCSs is paramount to obtaining high titers of heterologous proteins in eukaryotes and to enabling precise control of genetic circuits.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.