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Updated: Jun 4, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
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High-Throughput Cloning of Open Reading Frames (ORFs): Assembling Large Sets of Expression Constructs.

Andrew J Link1, Joshua Labaer

  • 1Vanderbilt University School of Medicine, Nashville, TN 37232-8575, USA.

CSH Protocols
|March 2, 2011
PubMed
Summary

This study introduces a flexible recombinational cloning system for protein expression. It enables rapid, efficient transfer of protein-coding sequences into various vectors for functional proteomics research.

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Area of Science:

  • Molecular Biology
  • Proteomics
  • Biotechnology

Background:

  • Studying protein function requires expressing proteins from cloned sequences.
  • A validated set of protein-coding clones is crucial for functional proteomics.
  • Different protein studies necessitate diverse expression vectors, demanding a flexible cloning system.

Purpose of the Study:

  • To describe a protocol for transferring protein-coding sequences into expression vectors.
  • To highlight the utility of recombinational cloning for functional proteomics.
  • To demonstrate the efficiency and flexibility of the Gateway system for protein expression.

Main Methods:

  • Utilizing recombinational cloning (Gateway system) for DNA fragment transfer.
  • Creating a master clone containing the protein-coding sequence.

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Functional Cloning Using a Xenopus Oocyte Expression System
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Related Experiment Videos

Last Updated: Jun 4, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
08:31

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit
06:28

Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit

Published on: September 2, 2025

Functional Cloning Using a Xenopus Oocyte Expression System
09:40

Functional Cloning Using a Xenopus Oocyte Expression System

Published on: January 30, 2016

  • Transferring the sequence from the master clone into various destination vectors (bacterial, mammalian, viral).

    • Main Results:

    • Recombinational cloning allows single-step, in-frame, and mutation-free transfer of DNA fragments.
    • The Gateway system facilitates high-throughput automation and is highly efficient.
    • Identical protein-coding sequences can be rapidly moved into multiple expression vectors.

    Conclusions:

    • A flexible vector system employing recombinational cloning is essential for functional proteomics.
    • The Gateway system provides an efficient method for creating protein expression constructs.
    • This approach streamlines the process of protein functional analysis across different systems.