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Updated: May 10, 2026

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System
Published on: December 5, 2020
A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation
Abstract:
Repression of the yeast silent mating type loci requires cis-acting sequences located over 1 kb from the regulated promoters. One of these sites, a "silencer," exhibits enhancer-like distance- and orientation-independence. The silencer demonstrates both autonomous replication sequence (ARS) activity and a centromere-like segregation function, suggesting roles for DNA replication and segregation in transcriptional repression. Here we identify three sequences (A, E, and B) involved both in repression and in either ARS or segregation activity. The sequences are functionally redundant: no one is essential for complete transcriptional control, but mutations in any two inactivate the silencer. Surprisingly, elements E and B can each activate transcription from heterologous promoters, and E shows striking homology to several yeast upstream activation sequences. Therefore, sequences individually involved in replication, segregation, and transcriptional activation can, at the silencer, efficiently repress transcription.
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