Sgk223 CRISPR Activation Plasmid (h): sc-406338-ACT

PRAG1 encodes the human Sgk223 protein, an atypical serine/threonine kinase–like scaffolding factor implicated in receptor-proximal signaling and cytoskeletal regulation. Sgk223 interacts with SH3 domain–containing adaptors and tyrosine kinase networks to influence cell adhesion, migration, and growth-related signaling cascades, including pathways downstream of receptor tyrosine kinases. Dysregulated PRAG1/Sgk223 activity and altered expression patterns have been reported in contexts relevant to oncogenic signaling and aberrant cell motility, supporting its use as a molecular node for studying signaling plasticity and invasion-associated phenotypes. PRAG1 is therefore of interest for mechanistic research linking membrane-proximal signaling to transcriptional programs and cellular behavior.

Sgk223 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PRAG1 expression without altering the underlying DNA sequence.

Sgk223 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PRAG1 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 PRAG1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Sgk223 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PRAG1 locus and enabling the study of Sgk223-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Sgk223 pathway restoration in tumor cells with silenced or reduced PRAG1 expression.

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