Cdc50A CRISPR/Cas9 KO Plasmid (m): sc-427588
- Target species: mouse
- 20 µg of transfection-ready, purified plasmid DNA; Suitable for up to 20 transfections
- Cdc50A CRISPR/Cas9 Knockout (KO) Plasmid (m) is a pool of plasmids, each encoding Cas9 nuclease and a target-specific 20 nt guide RNA (gRNA) designed for maximum knockout efficiency using sequences derived from the GeCKO v2 library
- gRNA sequences direct Cas9 to induce site-specific double-strand breaks (DSBs) in the Cdc50A genomic locus, resulting in gene knockout through non-homologous end joining (NHEJ)
- Cdc50A HDR Plasmid (m) (sc-427588-HDR) is recommended for co-transfection with Cdc50A CRISPR/Cas9 KO Plasmid (m) to enable selection of successfully edited cells through HDR-mediated integration of a puromycin resistance cassette and RFP reporter gene
- Cdc50A HDR Plasmid (m) is a pool of plasmids, each containing a homology-directed repair (HDR) template corresponding to the gRNA target sites in the Cdc50A CRISPR/Cas9 KO Plasmid (m)
- Each HDR plasmid contains two ~800 bp homology arms flanking the puromycin resistance and RFP cassettes, designed to bind genomic DNA sequences surrounding the Cas9-induced double-strand break site and facilitate precise HDR-mediated integration
- The puromycin resistance and RFP genes are flanked by LoxP sites, enabling removal of selection markers via Cre recombinase (Cre Vector: sc-418923) after establishing stable knockout cell lines
Ordering Information
| Product Name | Catalog # | UNIT | Price | Qty | FAVORITES | |
Cdc50A CRISPR/Cas9 KO Plasmid (m) | sc-427588 | 20 µg | $397.00 | |||
Cdc50A HDR Plasmid (m) | sc-427588-HDR | 20 µg | $445.00 |
Overview
Tmem30a encodes Cdc50A, an essential CDC50 family subunit that complexes with P4-ATPase lipid flippases to maintain plasma membrane phospholipid asymmetry by translocating aminophospholipids across bilayers. By regulating phosphatidylserine distribution, Cdc50A influences vesicle trafficking, endocytosis, membrane protein recycling, and the formation of functional membrane microdomains that shape receptor signaling. Disruption of Cdc50A-dependent lipid homeostasis can perturb cell polarity, apoptosis-associated membrane remodeling, and immune recognition cues linked to phosphatidylserine exposure. These processes make Tmem30a/Cdc50A a relevant node for studying membrane dynamics in neurobiology, hematopoiesis, and inflammatory disease mechanisms in mouse models.
Cdc50A CRISPR/Cas9 KO Plasmid (m) is a pool of plasmids designed for targeted disruption of the Tmem30a gene in mouse cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the Tmem30a locus, alongside the Streptococcus pyogenes Cas9 nuclease, and encodes GFP to enable fluorescent identification and enrichment of successfully transfected cells. This multi-guide strategy increases the likelihood of inducing frameshifts or deletions that produce a functional knockout, offering a more robust alternative to single-guide approaches. DSBs induced at multiple sites are resolved through non-homologous end joining (NHEJ) or, when used with the included HDR donor template, homology-directed repair (HDR) at a defined target site within the locus.
When used in conjunction with the RFP-expressing HDR donor, GFP and RFP fluorescence can be used together to distinguish transfected from edited cell populations, streamlining flow cytometry-based sorting and clone selection workflows.
Homology-Directed Repair (HDR) Donor — Puromycin Cassette with RFP Reporter
For applications requiring confirmed, selectable knockout clones, Cdc50A HDR Plasmid (m) includes an HDR donor construct containing a puromycin resistance cassette (PuroR) and a red fluorescent protein (RFP) reporter, flanked by homology arms specific to a defined Tmem30a target site.
When co-transfected with Cdc50A CRISPR/Cas9 KO Plasmid (m):
- The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the Tmem30a open reading frame.
- RFP fluorescence provides an immediate visual indicator of successful integration, enabling fluorescence-based identification or sorting of edited cells prior to or alongside puromycin selection.
- Successfully edited cells are confirmed through puromycin resistance, substantially reducing clone screening burden.
- This selection strategy is ideal for generating stable, clonal KO cell lines for downstream functional studies, drug screening, or model development.
Cre-lox Cassette Removal System
The HDR donor construct features loxP sites flanking the PuroR-RFP selection cassette to allow clean marker removal following clone confirmation. Transient expression of Cre recombinase via the included Cre Vector: sc-418923 excises the cassette, leaving a minimal residual loxP site within the Tmem30a locus and eliminating potential confounding effects on downstream assays.
This two-step approach:
- Minimizes disruption to local chromatin architecture and neighboring regulatory elements
- Restores a near-native genomic context at the edited locus
- Enables reuse of the puromycin selection strategy in the same cell line for additional edits
Key Features
- gRNA targeting Tmem30a exon(s) critical for Cdc50A function
- Co-expression of SpCas9 and sgRNA from a single plasmid for simplified delivery
- HDR donor with puromycin resistance for positive clone selection
- loxP-flanked PuroR cassette with Cre recombinase vector for seamless marker removal
- Supplied ready to use for delivery by transfection
For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.