Cdc25B CRISPR Activation Plasmid (h): sc-401457-ACT

CDC25B encodes Cdc25B, a dual-specificity phosphatase that promotes cell-cycle progression by dephosphorylating inhibitory sites on CDK1 and related cyclin-dependent kinases, facilitating G2/M transition. Its activity is integrated with checkpoint signaling and stress response pathways, including regulation by ATM/ATR–CHK1/CHK2 and modulation by 14-3-3 proteins that influence subcellular localization and timing of mitotic entry. Through coordination of DNA damage checkpoints and mitotic control, CDC25B expression and dysregulation are frequently examined in contexts of genomic instability and aberrant proliferation. As a node in kinase–phosphatase circuitry, Cdc25B is widely studied for its contributions to cell-cycle control, replication stress adaptation, and pathway crosstalk relevant to cancer biology.

Cdc25B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CDC25B expression without altering the underlying DNA sequence.

Cdc25B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CDC25B 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 CDC25B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Cdc25B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CDC25B locus and enabling the study of Cdc25B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Cdc25B pathway restoration in tumor cells with silenced or reduced CDC25B expression.

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