Integrative vectors for gene deletion and replacement
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Updated: May 10, 2026

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
Published on: December 12, 2017
Marian Sektas1, Magdalena Gregorowicz, Magdalena Kucharska
1Department of Microbiology University of Gdańisk, Gdańsk, Poland. marian.sektas@biol.ug.edu.pl
Abstract:
An improved method for gene deletion or replacement in Escherichia coli was developed. It employs a set of integrative vectors and two helper plasmids, as a temporary source of RecA and Flp activities. The integrative vectors combine several useful features including three different selection markers placed between two parallel oriented Flp recombinase target (FRT) sites. Each marker is flanked by two MCSs, for cloning the chosen homologous fragments of DNA to gene targeting. The vectors contain two properly oriented E. coli Chi sites for recombination enhancement. When required, selection markers can be excised from the chromosome resulting in unmarked strains.
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