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Updated: Apr 23, 2026
Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human
Natalay Kouprina1, Nicholas C O Lee, Artem V Kononenko
1Laboratory of Molecular Pharmacology, National Cancer Institute (NCI, NIH), Bldg 37 Room 4 E28, 37 Covent Drive, Bethesda, MD, 20892-4255, USA, kouprinn@mail.nih.gov.
Abstract:
Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.
