TRAC-1 CRISPR Activation Plasmid (h): sc-409980-ACT

RNF125 encodes TRAC-1, a RING-type E3 ubiquitin ligase that modulates immune signaling by regulating ubiquitination-dependent turnover of key proteins in T cell receptor and cytokine-response pathways. Through control of protein stability and signaling amplitude, TRAC-1 influences lymphocyte activation, antiviral responses, and inflammatory gene programs downstream of pathways such as NF-κB and interferon-associated signaling. Altered RNF125 expression or function has been linked to immune dysregulation and cancer-related immune phenotypes, making it relevant for studying mechanisms of immune surveillance and tumor–immune interactions. In human cell models, RNF125 is frequently examined to understand ubiquitin–proteasome system control of signaling thresholds and lineage-specific transcriptional states.

TRAC-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNF125 expression without altering the underlying DNA sequence.

TRAC-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNF125 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 RNF125 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TRAC-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNF125 locus and enabling the study of TRAC-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TRAC-1 pathway restoration in tumor cells with silenced or reduced RNF125 expression.

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