pLenti-UBA3-sgRNA

Cat. No.: P751467

Specifiche

Product Name

pLenti-UBA3-sgRNA

Product Line

Prodotti biologici

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at -80°C

Spedito in

Dry ice packs + Cold packs

Stabilità e conservazione

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

Application Protocols

Item-specific tested applications and recommended conditions are Not specified for this item; refer to CoA/Spec Sheet.

General workflow for pLenti-sgRNA use (literature/general)

1. Plasmid prep and verification

• Transform into a suitable E. coli strain; select on appropriate antibiotic plates (marker Not specified for this item; refer to CoA/Spec Sheet).
• Miniprep and Sanger-sequence the sgRNA region to verify spacer integrity.

2. Lentiviral production (if viral delivery desired)

• Co-transfect HEK293T cells with transfer vector + packaging plasmids under BSL-2 conditions.
• Harvest supernatants at 48–72 h; filter (0.45 µm); concentrate if needed (ultracentrifugation or PEG).

3. Target cell transduction and selection

• Transduce with defined MOI; include polybrene if tolerated. Begin selection per vector cassette (Not specified for this item; refer to CoA/Spec Sheet). Maintain appropriate controls (non-targeting sgRNA, Cas9-only).

4. Edit validation

• Assess indels via ICE/TIDE or amplicon NGS; confirm UBA3 pathway impact by monitoring cullin neddylation status.

These steps are general recommendations and not product-specific validated protocols.

Biological Roles

Item-specific details

• Specific guide sequence(s), species, and intended isoform targeting: Not specified for this item; refer to CoA/Spec Sheet.

Literature/general on UBA3 biology

• UBA3 encodes the catalytic subunit of the NEDD8-activating enzyme (NAE; E1 for neddylation), which activates NEDD8 in an ATP-dependent manner, forming a UBA3~NEDD8 thioester prior to transfer to E2s (e.g., UBC12). Neddylation regulates cullin-RING ligases (CRLs), broadly influencing protein turnover, cell cycle progression, and stress responses.
• Genetic perturbation of UBA3 typically diminishes global neddylation, reduces CRL activity, and stabilizes CRL substrates. This can be leveraged to dissect proteostasis, DNA damage response, and signaling networks that rely on ubiquitin-like modifications.
• Cellular context: UBA3 is essential in many cell types; complete knockout may be lethal. Conditional strategies (e.g., inducible Cas9, mosaic editing, or hypomorphic edits) are often used for viability studies (literature/general).
• Readouts: Monitor NEDD8-conjugated cullins by immunoblot; assess CRL substrate stabilization, cell cycle markers, and transcriptional changes. Complement with rescue using sgRNA-resistant UBA3 constructs to confirm specificity.

Note: All biological roles described are general literature context and not item-specific claims.

Buffer Applications

This product is a nucleic acid/viral vector reagent rather than a classical buffer component. Standard buffer formulation guidance (e.g., buffer pKa, concentration for buffering ranges) is not directly applicable.

Practical handling buffers (literature/general)

• Plasmid storage: TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) or nuclease-free water are typical. EDTA helps chelate Mg2+ to inhibit nucleases.
• Viral stock storage: Sterile PBS or HBSS with 0.1% BSA and/or 5–10% glycerol is commonly used to stabilize lentiviral particles at −80°C. For this item, exact formulation is Not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

This product is a biological nucleic acid reagent rather than a solvent or commodity chemical; conventional green chemistry solvent substitution does not directly apply.

Sustainability considerations for genetic tools (literature/general)

• Minimizing single-use plastics: Use reusable racks and low-binding tubes judiciously; consolidate aliquots to reduce waste while balancing freeze–thaw constraints.
• Energy efficiency: Store working aliquots at −20°C when feasible for plasmids (short-term) and reserve −80°C for long-term/viral stocks. Maintain freezer inventory to limit door-open time.
• Safer auxiliaries: Prefer non-phenolic DNA purification kits and avoid chloroform/phenol when possible. Choose enzymatic cleanup methods that reduce hazardous waste.
• Shipping impact: Coordinate batch orders to reduce shipments requiring dry ice. Consider on-site amplification of plasmids from glycerol stocks to minimize repeated cold-chain shipments.
• Deactivation: Use validated biocidal procedures (e.g., bleach, autoclave) to ensure safe disposal and prevent environmental release of recombinant materials.

Pharmaceutical Uses

This product is labeled For research use only and is not intended for human or veterinary use, diagnostic procedures, or as a therapeutic.

Literature/general context

• Research-grade lentiviral vectors and plasmids are used in discovery and preclinical laboratories for target validation, pathway interrogation, and cell line engineering. They are distinct from GMP-grade materials required for any clinical manufacturing.
• If translation is anticipated, analogous design principles (vector architecture, promoter selection, safety features) inform later GMP builds, but this research reagent is not qualified for pharmaceutical manufacturing. No pharmacopeial monographs apply.

Item-specific regulatory/grade details

• Any claims regarding GMP status, sterility testing, endotoxin limits, residual host cell DNA, or release criteria are Not specified for this item; refer to CoA/Spec Sheet.

Physical Properties

Item-specific facts (from Product Data)

• Appearance, concentration, solvent/buffer: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight/formula: Not specified for this item; refer to CoA/Spec Sheet.

Literature/general properties for similar reagents

• Physical form: pLenti plasmids are typically supplied as aqueous DNA solutions (e.g., in TE buffer) or as glycerol stocks of E. coli harboring the plasmid; lentiviral particles (if provided) are typically in buffered saline with cryoprotectant. For this specific item, the supplied form is Not specified for this item; refer to CoA/Spec Sheet.
• Nucleic acid characteristics: dsDNA plasmid lengths often range 8–14 kb for lentiviral backbones; A260/A280 ~1.8–2.0 (literature). These values are general guidance and not specifications.
• Stability: DNA plasmids are stable at −20 to −80°C; repeated freeze–thaw can shear DNA. Lentiviral particles (if applicable) are labile at room temperature and require −80°C storage; avoid >2–3 freeze–thaw cycles (literature).

Not provided/specification reminder

• Any exact BP/MP, density, pKa, logP, or refractive index values are not applicable to DNA constructs and are Not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

Item-specific grade/purity

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

General guidance for this reagent class (literature/general)

• Plasmid DNA quality: For transfection, endofree/low-endotoxin preparations reduce cytotoxicity and improve expression. Common benchmarks include A260/A280 of 1.8–2.0 and supercoiled fraction >80%, but these are not item specifications.
• Viral titer metrics: If supplied as lentiviral particles, quality is often reported as genomic copies/mL or TU/mL, with sterility and mycoplasma testing. No titers are specified for this item.
• Sequence verification: sgRNA-bearing plasmids should be confirmed by Sanger sequencing across the U6 promoter, spacer insertion site, and scaffold to ensure fidelity. For production use, full backbone verification by restriction analysis or NGS is recommended.
• Stabilizers/antibiotic markers: Presence of antibiotic resistance genes (e.g., ampicillin) for bacterial propagation and selection cassettes (e.g., puromycin) for mammalian selection are common in pLenti backbones; exact features for this item are Not specified for this item; refer to CoA/Spec Sheet.
• Documentation: For cGMP-incompatible research tools, certificate of analysis (CoA) typically lists concentration, purity, endotoxin (if applicable), and sterility; consult the item’s CoA/Spec Sheet.

Reaction and Applications

Item-specific applications

• Manufacturer applications: Not specified for this item; refer to CoA/Spec Sheet.

General applications for pLenti-sgRNA constructs (literature/general)

• CRISPR/Cas9 knockout or knockdown: pLenti-UBA3-sgRNA would be used to express an sgRNA targeting UBA3. In cells stably expressing Cas9 (or via co-delivery), this guides Cas9 to generate indels at the UBA3 locus, enabling functional studies in the neddylation pathway.
• Stable gene editing via lentiviral integration: Lentiviral packaging (with helper plasmids) allows transduction of difficult-to-transfect cells, including primary cells, followed by antibiotic selection to enrich edited populations.
• Pooled screening: If compatible with barcoding/selection, UBA3-directed sgRNA can be used as a positive control or pathway-specific perturbation in pooled CRISPR screens.
• Rescue and mechanistic studies: Combine UBA3 perturbation with complementation constructs (e.g., cDNA resistant to sgRNA) to validate on-target effects.

Practical notes (literature/general)

• Validate spacer: Sequence the sgRNA insert; confirm on-target cutting with T7E1, ICE/TIDE, or amplicon NGS.
• Off-target assessment: Use in silico design tools to minimize off-targets; orthogonal validation recommended.
• Lentiviral work: Produce virus with second- or third-generation packaging systems under BSL-2; titrate MOI empirically; select edited cells appropriately.

Reaction Conditions

Classical chemical reaction conditions (solvent, temperature, catalysts, yields) do not apply to this biological vector.

Molecular biology conditions (literature/general)

• Transfection: Deliver plasmid DNA to producer cells (e.g., HEK293T) with PEI or lipid reagents; optimize DNA:reagent ratios per vendor protocols. Culture at 37°C, 5% CO2; harvest viral supernatant at 48–72 h.
• Transduction: Apply filtered viral supernatant with polybrene (e.g., 4–8 µg/mL) to target cells; adjust MOI to control multiplicity of integration. Spinoculation can enhance efficiency for refractory cells.
• Antibiotic selection: Begin 48–72 h post-transduction if the vector encodes a selectable marker; titrate antibiotic to cell line kill-curve. Exact selection cassette for this item is Not specified for this item; refer to CoA/Spec Sheet.
• Editing assessment: Analyze indel frequency 3–7 days post-delivery via PCR amplicon sequencing or ICE/TIDE; extend culture for clonal isolation if needed.

Note: The above are general literature practices and not item specifications.

Safety and Handling

Item-specific hazard statements

• Signal word, H-statements, GHS class, pictograms: Not specified for this item; refer to CoA/Spec Sheet and the SDS.

General biosafety guidance (literature/general)

• Classification: Lentiviral vectors are generally handled under BSL-2 conditions when producing or concentrating viral particles. Plasmid DNA itself is typically handled at BSL-1. Follow your institution’s IBC approvals.
• PPE: Lab coat, gloves, and eye protection. Use a certified biosafety cabinet for aerosol-generating steps (e.g., virus production, transductions).
• Incompatibilities: Avoid DNases and strong oxidants for plasmid DNA. For viral preparations, avoid repeated freeze–thaw and exposure to elevated temperatures.
• First aid (overview): If exposure occurs, wash skin with water and soap; flush eyes with water for 15 minutes; move to fresh air if inhaled aerosols suspected. Seek medical attention as per institutional policy. Defer to SDS for authoritative guidance.
• Waste and decontamination: Treat lentiviral materials and contaminated disposables as biohazardous; decontaminate with 0.5–1% sodium hypochlorite or 70% ethanol and autoclave as appropriate (literature/general). Quench bleach after appropriate contact time.
• Special risks: Replication-incompetent lentiviral vectors can integrate into host genomes; implement engineering controls and procedural safeguards during packaging/transduction.

Solvent Selection

This product is a genetic construct (plasmid DNA and/or lentiviral preparation), not a small-molecule solute. Classical solvent selection parameters (polarity, dielectric constant, miscibility) are not typically applicable.

Practical handling notes (literature/general)

• Plasmid DNA is commonly supplied or stored in aqueous buffers (e.g., TE: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0) or nuclease-free water. Avoid organic solvents that denature nucleic acids.
• For viral stocks, maintain in sterile, buffered saline solutions with cryoprotectants (e.g., 5–10% glycerol), aliquot to avoid freeze–thaw, and handle on ice.
• If concentrating or exchanging buffers, use centrifugal ultrafiltration (e.g., 30–100 kDa MWCO) with nuclease-free, low-binding plastics.

Storage and Reconstitution

Item-specific storage and shipping (from Product Data)

• Storage conditions: Store at −80°C.
• Shipped in: Dry ice packs + Cold packs.

General guidance (literature/general)

• Upon receipt: Inspect packaging temperature. Keep on dry ice during unpacking. Immediately transfer to −80°C storage.
• Aliquoting: To minimize freeze–thaw cycles, prepare small, single-use aliquots in nuclease-free tubes. For viral stocks, avoid more than 2–3 freeze–thaw cycles.
• Thawing: Thaw on ice. Mix by gentle flicking; avoid vigorous vortexing that can shear DNA or denature viral particles.
• Reconstitution: If provided lyophilized (not specified for this item), reconstitute in nuclease-free water or TE buffer to the desired concentration using low-EDTA formulations for transfection compatibility. For this specific item, any reconstitution instructions are Not specified for this item; refer to CoA/Spec Sheet.
• Short-term storage: Keep working aliquots at 4°C for only a few hours (viral) or ≤1–2 days (plasmid DNA) to limit degradation.
• Long-term stability: Maintain at −80°C. Avoid frost-free freezers. Document aliquot IDs, concentrations, and freeze–thaw counts.
• Research use: For research use only; follow institutional biosafety and SDS guidance.

Structure and Identity

Item-specific facts (from Product Data)

• Product name: pLenti-UBA3-sgRNA (SKU: P751467)
• CAS/CID/InChIKey/SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula and molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

General description (literature/general)

• Reagent class: Lentiviral CRISPR/Cas system sgRNA expression construct designed to target the human UBA3 locus (UBA3 encodes the NAE1/UBA3 subunit of the NEDD8-activating enzyme E1 complex).
• Typical vector architecture (literature): pLenti sgRNA vectors commonly include a U6 or H1 promoter driving a single guide RNA scaffold; lentiviral LTRs (5′ and self-inactivating 3′ LTR), ψ packaging signal, RRE, cPPT, and selection markers (e.g., puromycin) or reporters. Presence/absence of these elements for this specific item is Not specified for this item; refer to CoA/Spec Sheet.
• Targeting content: While the product name indicates UBA3-directed sgRNA, the exact spacer sequence(s), strand orientation, and any PAM context are Not specified for this item; refer to CoA/Spec Sheet.

2D structural features described in words (literature/general)

• The construct comprises modular DNA cassettes: promoter → sgRNA spacer + scaffold, flanked by lentiviral replication-incompetent elements enabling packaging and genomic integration in dividing and non-dividing mammalian cells when used with appropriate helper plasmids. No small-molecule structural identifiers apply.

Synthetic Utility

Not typically applicable. This product is a genetic construct used for genome engineering rather than a small-molecule building block or reagent in organic synthesis.

Related molecular biology utility (literature/general)

• Enables targeted gene perturbation of UBA3 via CRISPR, facilitating pathway mapping in the neddylation system.
• Can be combined with donor templates for HDR-mediated edits (e.g., tagging endogenous loci) when co-delivering Cas9 and repair donors.
• Useful in generating isogenic cell panels differing at UBA3 to probe structure–function relationships.

For chemical synthesis applications such as cross-coupling, oxidations, or catalysis, please refer to appropriate chemical reagents rather than this genetic tool.

Target Specificity

Item-specific targeting information

• sgRNA spacer sequence(s), PAM, exon/intron context, species specificity, and any validated off-target assessment: Not specified for this item; refer to CoA/Spec Sheet.

General context (literature)

• UBA3-targeting sgRNAs should be designed to unique exonic regions conserved in the intended species to ensure efficient on-target editing. Use current genome builds and off-target prediction tools to confirm specificity. Empirical validation (amplicon NGS, rescue experiments) is recommended.

Domande frequenti

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-UBA3-sgRNA); cross-reactivity in untested species may exist but is not guaranteed. Contact technical support for specific species inquiries.