UBE2B CRISPR Activation Plasmid (h): sc-404361-ACT

UBE2B (ubiquitin-conjugating enzyme E2 B) is a human E2 ubiquitin–conjugating enzyme that partners with E3 ligases to catalyze ubiquitin transfer to protein substrates, shaping proteostasis through the ubiquitin–proteasome system. It contributes to regulated protein turnover and signaling control, with established roles in DNA damage responses and chromatin-associated processes including ubiquitination events that influence repair pathway choice. Through these activities, UBE2B impacts cell-cycle progression, genome stability, and stress signaling networks. Dysregulated ubiquitination and DNA repair capacity are broadly relevant to oncogenesis and other disorders linked to impaired proteostasis and genome maintenance, making UBE2B a useful node for mechanistic studies.

UBE2B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous UBE2B expression without altering the underlying DNA sequence.

UBE2B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the UBE2B locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the UBE2B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UBE2B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native UBE2B locus and enabling the study of UBE2B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UBE2B pathway restoration in tumor cells with silenced or reduced UBE2B expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.