CNT1 CRISPR Activation Plasmid (h): sc-404790-ACT

SLC28A1 encodes concentrative nucleoside transporter 1 (CNT1), a high-affinity, sodium-dependent plasma membrane transporter that mediates cellular uptake of pyrimidine nucleosides such as uridine and cytidine. By controlling nucleoside salvage, CNT1 supports nucleotide pool homeostasis required for DNA/RNA synthesis, cell-cycle progression, and responses to metabolic stress. CNT1 activity intersects with purine/pyrimidine metabolism and can influence sensitivity to nucleoside analog–based experimental perturbations through altered intracellular substrate availability. Dysregulated SLC28A1 expression has been reported across multiple disease contexts, including cancer-associated metabolic reprogramming, making it relevant for studies of transporter biology, proliferation, and drug-transport mechanisms.

CNT1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC28A1 expression without altering the underlying DNA sequence.

CNT1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC28A1 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 SLC28A1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CNT1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC28A1 locus and enabling the study of CNT1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CNT1 pathway restoration in tumor cells with silenced or reduced SLC28A1 expression.

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