UBE2D4 CRISPR Activation Plasmid (h): sc-405030-ACT

UBE2D4 encodes a human ubiquitin-conjugating E2 enzyme that cooperates with E3 ligases to attach ubiquitin to substrate proteins, shaping their stability, localization, and signaling output. Through its role in the ubiquitin–proteasome system, UBE2D4 contributes to regulated protein turnover that influences cell-cycle progression, DNA damage responses, and proteostasis under stress. Perturbations in ubiquitination networks are linked to altered signaling fidelity and accumulation of misfolded or aberrant proteins, processes frequently implicated in cancer biology and neurodegeneration. As an E2 component with broad E3 compatibility, UBE2D4 is useful for dissecting pathway-specific ubiquitination events and identifying substrates that control key regulatory nodes.

UBE2D4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous UBE2D4 expression without altering the underlying DNA sequence.

UBE2D4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the UBE2D4 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 UBE2D4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UBE2D4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native UBE2D4 locus and enabling the study of UBE2D4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UBE2D4 pathway restoration in tumor cells with silenced or reduced UBE2D4 expression.

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