Cdc50A CRISPR Activation Plasmid (h): sc-403231-ACT

TMEM30A encodes the human Cdc50A subunit of P4-ATPase phospholipid flippase complexes that maintain plasma membrane phospholipid asymmetry by translocating aminophospholipids across bilayers. By supporting vesicular trafficking, endocytosis, and membrane protein recycling, Cdc50A influences cell polarity, apoptotic signaling linked to phosphatidylserine exposure, and lipid-dependent signaling microdomains. Disruption of TMEM30A/Cdc50A-dependent lipid homeostasis has been associated with altered receptor turnover, inflammatory responses, and cellular stress phenotypes relevant to neurobiology, immunity, and cancer-associated membrane remodeling. These functions make Cdc50A a useful entry point for studying membrane asymmetry, trafficking pathways, and lipid-regulated signaling networks in human cells.

Cdc50A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TMEM30A expression without altering the underlying DNA sequence.

Cdc50A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TMEM30A 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 TMEM30A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Cdc50A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TMEM30A locus and enabling the study of Cdc50A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Cdc50A pathway restoration in tumor cells with silenced or reduced TMEM30A expression.

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