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Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
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Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Published on: April 27, 2017

Re-engineering multicloning sites for function and convenience.

Nathan C Crook1, Elizabeth S Freeman, Hal S Alper

  • 1Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, C0400 Austin, TX 78712, USA.

Nucleic Acids Research
|May 19, 2011
PubMed
Summary
This summary is machine-generated.

Multicloning sites (MCSs) in expression vectors can significantly reduce protein production by causing mRNA structure. Researchers developed a predictive model to design improved MCSs for higher, more consistent protein expression in yeast.

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Principles of Site-Specific Recombinase (SSR) Technology
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Published on: May 29, 2008

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Last Updated: Jun 1, 2026

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
15:13

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Published on: April 27, 2017

Principles of Site-Specific Recombinase (SSR) Technology
07:06

Principles of Site-Specific Recombinase (SSR) Technology

Published on: May 29, 2008

Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • Multicloning sites (MCSs) are standard genetic elements in expression vectors.
  • MCSs are often assumed to be inert, but can influence gene expression.
  • The genetic context of MCSs can impact mRNA secondary structure and translation.

Purpose of the Study:

  • To assess the performance of commonly used MCSs in yeast.
  • To investigate the impact of MCSs on protein expression levels.
  • To design novel MCSs with improved translational control.

Main Methods:

  • Performance-based assessment of MCSs in yeast.
  • Development of a predictive model for structure-based translation inhibition.
  • Design and application of improved MCSs with common yeast promoters (TEF, CYC, GPD).

Main Results:

  • Commonly used MCSs can cause significant reductions in protein expression.
  • Inhibitory effects of MCSs are dependent on the promoter and gene.
  • Designed MCSs minimized inhibitory effects, leading to higher and more consistent protein expression.

Conclusions:

  • MCSs are not benign and significantly impact protein expression.
  • A predictive model enables the design of MCSs for enhanced translational control.
  • Redesigned MCSs are crucial for efficient heterologous protein production and genetic circuit regulation in eukaryotes.