pLenti-CDK5RAP3-sgRNA

Cat. No.: P744760

Specifications

Product Name

pLenti-CDK5RAP3-sgRNA

Product Line

Produits biologiques

Stockage et expédition

Conditions de stockage de stockage

Store at -80°C

Expédié en

Dry ice packs + Cold packs

Stabilité et stockage

Stored at -20 ℃, valid for 3 months;Stored at -80 ℃, valid for at least one year.

Application Protocols

Item-specific (from Product Data)

• Manufacturer-tested applications, recommended conditions, and positive controls: Not specified for this item; refer to CoA/Spec Sheet.

General workflow outlines (literature; adapt to your system)

• Transient CRISPR editing (Cas9-positive cells):
  1. Thaw plasmid on ice; briefly spin. 2) Dilute to working concentration in sterile nuclease-free water. 3) Mix with lipid transfection reagent at recommended ratios; incubate 10–20 min. 4) Add to cells at 50–70% confluence. 5) Replace medium after 6–24 h. 6) Analyze editing after 48–96 h using TIDE/ICE or amplicon NGS.
• Lentiviral delivery:
  1. Co-transfect producer cells with transfer, packaging, and envelope plasmids. 2) Harvest at 48/72 h; filter. 3) Optionally concentrate virus. 4) Transduce target cells with polybrene if suitable. 5) Select and expand. 6) Validate genotype/phenotype.
• Clonal isolation and validation: Single-cell clone; expand; confirm on-target edits; screen for biallelic frameshifts for knockouts.

Note: Exact spacer sequence, antibiotic selection, and MOI recommendations are not provided for this item and must be determined empirically.

Biological Roles

General background on the target (literature)

• Gene: CDK5RAP3 (CDK5 regulatory subunit-associated protein 3), also known as C53/LZAP.
• Cellular roles: Reported to interact with components of the cyclin-dependent kinase 5 (CDK5) pathway and to participate in cellular stress responses, protein quality control, and aspects of cell cycle regulation. It has been implicated in modulating translational control and signaling hubs such as NF-κB and p53 pathways in various experimental contexts.
• Localization: Predominantly cytoplasmic with nuclear presence reported depending on cell type and conditions (literature).
• Complexes: Associations with chaperone-like and ribosome-associated factors have been described, consistent with roles in proteostasis and co-translational surveillance (literature).

Relevance to this product

• The pLenti-CDK5RAP3-sgRNA construct enables targeted perturbation of CDK5RAP3 to interrogate these cellular roles experimentally. Specific spacer sequence, on-target score, and predicted off-targets are not provided for this item; users should verify and, if needed, redesign or complement with additional sgRNAs.

Caveats

• Functional outcomes can be cell-type and context dependent. Always include appropriate genetic and phenotypic controls and validate edits at the genomic and protein levels.

Note: No medical or clinical claims are made; this information is provided for basic research planning.

Buffer Applications

Not a classical buffering reagent.

• This product is a plasmid DNA construct; it does not serve as a buffer component. For handling and storage, commonly used buffers include nuclease-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0). EDTA chelates divalent cations to inhibit nucleases; Tris stabilizes pH in the physiological range (literature). If electroporating, avoid high-salt buffers; if transfecting, follow reagent-specific compatibility.
• Any item-specific buffer composition or concentration is not specified for this item; refer to the CoA/Spec Sheet.

Green Alternatives

Applicability note

• Green solvent alternatives are not relevant to this plasmid DNA product. Environmental considerations for this workflow focus on minimizing hazardous reagents used alongside (stains, solvents) and adopting greener molecular biology practices.

Greener practice suggestions (general)

• Nucleic acid stains: Prefer SYBR Safe or GelRed/GelGreen over ethidium bromide for gel electrophoresis.
• Decontamination: Use DNA-away or 70% ethanol instead of halogenated solvents; avoid excessive bleach where compatible with materials.
• Packaging reduction: Aliquot once and store to minimize plastic waste; select recyclable ice packs/dry ice reuse programs where available.
• Energy: Consolidate freezer storage; use high-efficiency −80°C freezers set to −70°C where validated to reduce energy consumption.
• Endotoxin-free water systems: Centralized preparation reduces single-use plastics from small bottles.

Note: These are general sustainability practices; they do not alter specifications of this item.

Pharmaceutical Uses

Not applicable to this catalog item.

• This is a research-use-only plasmid DNA construct for laboratory studies. It is not manufactured under GMP, and no pharmacopeial status, excipient role, or clinical/formulation use is stated or implied. Do not use for diagnostic, therapeutic, or human/animal administration.

Physical Properties

Item-specific (from Product Data)

• Appearance, concentration, buffer, OD260/280, endotoxin: Not specified for this item; refer to CoA/Spec Sheet.

General/literature values for plasmid DNA constructs

• Physical state: Aqueous DNA solution or lyophilized DNA, depending on formulation (not specified here).
• Solubility: Plasmid DNA is water-soluble; readily dissolves in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5–8.0) or nuclease-free water.
• pKa/logP: Not applicable for macromolecular polyanion; DNA does not have a classical logP.
• UV characteristics: DNA exhibits a peak absorbance at 260 nm (A260) with A260/A280 ~1.8–2.0 for pure plasmid DNA (literature). Extinction coefficient depends on plasmid length/base composition.
• Stability: Stable to multiple years at −80°C; weeks to months at −20°C; days at 4°C if free of nucleases (literature). Repeated freeze–thaw cycles can shear or reduce transfection performance.
• Viscosity: Concentrated plasmid solutions can be viscous; avoid vigorous pipetting to prevent shearing (literature).
• DNase sensitivity: Highly susceptible to nuclease contamination; maintain sterile, nuclease-free conditions (literature).

Note: Any specific mass, base pair length, antibiotic resistance marker, and concentration are not provided for this item and should be verified on the CoA/Spec Sheet.

Quality and Grades

Item-specific (from Product Data)

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.

Context and expectations (general)

• Plasmid quality metrics typically include: supercoiled percentage, A260/A280 and A260/A230 ratios, residual genomic DNA/RNA, endotoxin level (EU/µg DNA), and sterility. For transfection- or transduction-grade DNA, low endotoxin content is often required to ensure high transfection efficiency and cell viability.
• Sequence identity: Confirmation by Sanger/NGS of the sgRNA spacer, scaffold, and junctions is standard. Restriction digest patterns and full plasmid map verification are recommended.
• Application-driven grades (literature):
  • Cloning grade: Suitable for bacterial propagation and subcloning; endotoxin not necessarily minimized.
  • Transfection grade/EndoFree: Produced with endotoxin-reduction steps for mammalian cell work.
  • GMP-like or ancillary material: For manufacturing environments; requires formal quality systems (not implied for this catalog item unless explicitly stated).

Documentation

• For this specific SKU, consult the CoA/Spec Sheet for: backbone name, antibiotic resistance marker, plasmid length (bp), sgRNA spacer sequence, recommended antibiotic for bacterial propagation, concentration, and any endotoxin/sterility testing. In the absence of item-specific declarations, assume research-use, non-GMP material.

Reaction and Applications

Item-specific (from Product Data)

• Intended use: pLenti-CDK5RAP3-sgRNA — a lentiviral plasmid expressing an sgRNA targeting the CDK5RAP3 gene for research use.

General applications (literature/practice)

• CRISPR workflows: Use as the sgRNA component in CRISPR/Cas9 gene knockout, repression (CRISPRi), or activation (CRISPRa) systems depending on paired effector (SpCas9 nuclease, dCas9-KRAB, dCas9-VP64, etc.).
• Lentiviral delivery: The pLenti backbone supports packaging into pseudotyped lentiviral particles when co-transfected with packaging (gag/pol, rev) and envelope (e.g., VSV-G) plasmids in producer cells. This enables stable delivery and selection in target mammalian cells.
• Functional genomics: Gene function interrogation, pathway mapping, and construction of isogenic knockouts/perturbations centered on CDK5RAP3.
• Stable cell line generation: Following viral transduction and selection, isolate clonal lines for downstream assays (omics, imaging, biochemical analyses).

Practical tips (literature)

• Verify target: Confirm CDK5RAP3 transcript variants in your cell model; ensure the sgRNA spacer sequence (not provided here) matches the intended locus and PAM.
• Controls: Include non-targeting sgRNA controls and multiple independent sgRNAs to deconvolute off-target effects.
• Validation: Assess editing by TIDE/ICE analysis, targeted amplicon NGS, or Surveyor assays; confirm functional knockdown/knockout at mRNA/protein levels.
• Packaging safety: Follow BSL-2 practices for lentiviral production; use third-generation packaging systems to enhance biosafety.

Reaction Conditions

General guidance for common workflows (literature; adjust empirically)

• Bacterial propagation: Transform into chemically competent or electrocompetent E. coli (e.g., DH5α, Stbl3 for LTR-containing plasmids). Plate on the appropriate antibiotic (not specified for this item). Grow 30–37°C; avoid recombination-prone strains for lentiviral LTRs. Miniprep/Midiprep using endotoxin-minimizing kits for mammalian use.
• Mammalian transfection (Cas9 already present): Transfect 0.5–2 µg plasmid per well (6-well format) using a lipid reagent per manufacturer’s protocol. Assess sgRNA expression at 24–48 h; evaluate editing 48–96 h post-transfection. Optimize DNA:reagent ratios.
• Lentiviral packaging: Co-transfect producer cells (e.g., HEK293T) with this transfer plasmid plus packaging and envelope plasmids. Typical ratios 4:3:1 (transfer:gag/pol:VSV-G) by mass are common starting points. Harvest viral supernatant at 48 and 72 h; filter 0.45 µm. Titrate virus; transduce target cells at MOI 0.3–5 depending on application. Perform selection 48–72 h post-transduction if a selectable marker is present.
• Selection and cloning: Apply antibiotic per backbone (not specified) to enrich; clone by limiting dilution or single-cell sorting. Validate edits by PCR/NGS and protein assays.

Important

• Exact antibiotic marker, promoter strengths, and sgRNA spacer sequence are not provided for this item; consult the CoA/Spec Sheet and adjust conditions accordingly.

Safety and Handling

Item-specific (from Product Data)

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.
• Storage: Store at −80°C. Shipped on dry ice packs + cold packs.

General guidance for plasmid DNA reagents (literature/practice)

• Hazard profile: Plasmid DNA solutions are typically not classified as hazardous under GHS. Nonetheless, handle using good laboratory practice and consult the SDS for authoritative guidance.
• Biosafety: This product is a plasmid DNA construct (non-replicating in mammalian cells without packaging). It is not a lentiviral particle preparation. Standard BSL-1 molecular biology practices are generally adequate for plasmid handling; cloning or production of lentiviral particles would require BSL-2 containment per institutional biosafety policies.
• PPE: Lab coat, gloves (nitrile), and safety glasses. Work in a clean area to avoid nuclease contamination.
• Incompatibilities: Avoid strong acids/bases that can hydrolyze DNA, oxidants, and nucleases. Do not mix with DNase-containing reagents. Avoid metal contamination that may promote nuclease activity.
• First aid (overview): In case of skin/eye contact with buffer, rinse with water. If inhaled as aerosol, move to fresh air. If ingested, rinse mouth. Seek medical attention as needed; defer to SDS.
• Special risks: None known for plasmid DNA; principal risks are biological (downstream viral packaging) and ergonomic/chemical (e.g., ethidium bromide, solvents) used alongside in workflows.
• Waste: Dispose of DNA and related consumables per institutional biosafety and chemical hygiene policies.

Solvent Selection

Not typically applicable.

• This item is a plasmid DNA construct, not a small-molecule solute. It is normally handled in aqueous, nuclease-free buffers (e.g., TE or water). Organic solvent selection is not relevant. For experimental planning, refer instead to Reaction & Applications and Application Protocols for aqueous delivery (transfection, electroporation) considerations.

Storage and Reconstitution

Item-specific (from Product Data)

• Storage: Store at −80°C.
• Shipping: Dry ice packs + Cold packs.
• Appearance, buffer composition, concentration: Not specified for this item; refer to CoA/Spec Sheet.

General handling guidance (literature/practice)

• Upon receipt: Keep frozen. Minimize time at ambient temperature. Record lot and any accompanying sequence map/CoA.
• Thawing: Thaw on ice. Mix gently by slow pipetting; avoid vortexing to minimize shearing.
• Aliquoting: Prepare small, single-use aliquots (e.g., 5–20 µL) in sterile, nuclease-free tubes to avoid repeated freeze–thaw cycles.
• Short-term storage: 4°C for up to 24–72 h in sterile nuclease-free buffer if processing; otherwise keep at −20°C to −80°C. For long-term integrity and low endotoxin workflows, maintain at −80°C.
• Reconstitution: If lyophilized (not specified), reconstitute in nuclease-free water or TE buffer to the desired concentration. Allow to fully dissolve on ice. Spin briefly to collect contents.
• Working buffers: Use endotoxin-free water/TE for mammalian applications. Filter-sterilize if needed (0.22 µm) using DNA-grade low-binding filters.
• Freeze–thaw: Avoid repeated cycles; each thaw can reduce supercoiling fraction and performance.
• Documentation: Retain plasmid map, sequence, and antibiotic resistance information from the CoA/Spec Sheet for correct propagation and selection.

Structure and Identity

Item-specific (from Product Data)

• Product name: pLenti-CDK5RAP3-sgRNA (SKU: P744760)
• Type: Lentiviral plasmid backbone carrying an sgRNA expression cassette targeting CDK5RAP3
• Storage: Store at -80°C
• Shipped: Dry ice packs + Cold packs
• Research use: For research use only
• CAS, SMILES, InChIKey, Molecular formula, Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

General description (literature)

• Construct class: pLenti-family plasmid, typically containing 5′LTR, packaging signal (Ψ), Rev-response element (RRE), central polypurine tract (cPPT), and 3′LTR modifications for self-inactivation (in many modern backbones). The sgRNA cassette is usually driven by a U6 or H1 promoter; optional selection markers or reporters (e.g., puroR, GFP) may be present depending on exact clone.
• Target: CDK5RAP3 (CDK5 regulatory subunit-associated protein 3). The sgRNA spacer sequence is designed to direct Cas9 to a CDK5RAP3 genomic locus. Specific spacer/protospacer sequence and PAM context are not provided for this item.
• 2D “structure” in words: Circular double-stranded DNA plasmid consisting of lentiviral long terminal repeats flanking a cloning backbone with bacterial origin of replication and antibiotic resistance marker for propagation in E. coli, plus an RNA Pol III promoter–sgRNA scaffold unit targeting CDK5RAP3.
• Stereochemistry/functional groups: Not applicable to plasmid DNA; macromolecular polyanion composed of nucleotide polymers.

Synthetic Utility

Interpretation for molecular biology workflows

• Vector engineering: Serves as a modular backbone for cloning alternative sgRNA spacers, swapping promoters, or adding selection/reporting cassettes. Standard restriction/ligation, Gibson assembly, or Golden Gate cloning can be used (literature).
• Library construction: Can be adapted into pooled CRISPR libraries targeting CDK5RAP3 neighborhoods or interacting genes; barcoding compatible backbones facilitate NGS deconvolution (literature).
• Retrosynthetic planning (analogy): Functional elements include the sgRNA expression cassette (Pol III promoter + scaffold), selection marker, bacterial origin, and LTR/packaging elements. Each can be re-engineered via PCR, site-directed mutagenesis, or recombineering to yield derivatives suited to CRISPRi/a or base/prime editing (with appropriate editors supplied in trans).
• Downstream utility: Enables generation of isogenic knockouts for mechanistic biochemistry, proteomics, and cell-based assays centered on CDK5RAP3 pathways.

Item-specific notes

• Exact backbone features (antibiotic resistance, promoters, reporters) and spacer sequence are not specified for this item; consult the CoA/Spec Sheet for cloning sites and propagation conditions.

Target Specificity

Item-specific (from Product Data)

• Target: CDK5RAP3 (CDK5 regulatory subunit-associated protein 3) — inferred from product name. Specific spacer sequence, strand orientation, PAM, and transcript isoform targeting are not specified for this item; refer to CoA/Spec Sheet if available.

Scope limitations

• No additional details on species specificity, off-target analysis, validated cell lines, or performance metrics are provided in the product data. Users should perform in silico off-target prediction and empirical validation in their system.

Foire aux questions

How should this product be stored?

Store at ?80 °C. Ultra-low temperature storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships frozen with dry ice and cold packs. Unpack on arrival and transfer it to the storage condition stated above; handle dry ice with insulated gloves in a ventilated area.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.

Which species reactivity has this antibody been validated against?

Validated reactivity is listed on the PDP and on each lot's datasheet. We validate against the most commonly tested species for the target (pLenti-CDK5RAP3-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.