pLV3-His-Tagged Vectors: Product Specifications and Usage Guide

pLV3-His-tagged vectors are a series of constitutive lentiviral expression vectors offered by the Miaoling Plasmid Platform. They are commonly used in gene overexpression and protein purification research. Driven by strong CMV or CAG promoters, these vectors support C-terminal fusion of target proteins to His tags of different lengths, including 6×His and 8×His. Depending on the construct, they also contain a puromycin resistance marker (Puro) for selection and a fluorescent reporter, such as CopGFP, EGFP, or mCherry, for visual tracking of transduced cells.

The vector backbones are high-copy-number plasmids with ampicillin resistance (Amp) for bacterial selection. They are suitable for propagation in Stbl3 competent cells under the recommended culture conditions, typically at 37°C.

Miaoling Biotechnology is a high-tech enterprise with a 4,000 m² standardized laboratory facility and a P2 laboratory for viral-vector work. Supported by proprietary plasmid-extraction technologies and an ISO 9001 quality management system, Miaoling constructs, sequence-verifies, and quality-controls these vectors. In-stock vectors undergo multiple quality-control checks.

Key Specifications of Miaoling’s pLV3-His-Tagged Vector Series

Empty Vectors (Base Backbones)

pLV3-CMV-MCS-6×His-Puro (Catalog No. P47113)

• Vector type: Constitutive lentiviral expression vector
• Promoter: CMV
• Restriction sites: EcoRI and BamHI
• Bacterial selection marker: Ampicillin resistance (Amp)
• Recommended bacterial strain: Stbl3 competent cells
• Tag: C-terminal 6×His
• Background vector: Reported as derived from P1268 with the addition of a C-terminal 6×His tag

pLV3-CMV-MCS-8×His-CopGFP-Puro (Catalog No. P59125)

• Vector type: Constitutive lentiviral expression vector
• Promoter: CMV
• Restriction sites: XbaI and EcoRI
• Bacterial selection marker: Ampicillin resistance (Amp)
• Recommended bacterial strain: Stbl3 competent cells
• Tag: C-terminal 8×His
• Fluorescent reporter: CopGFP (green fluorescence)
• Background vector: Reported as derived from P0268 with the addition of an 8×His tag at the C-terminus

Representative In-Stock Vectors Carrying Target Genes

pLV3-CMV-VHL(human)-8×His-CopGFP-Puro (Catalog No. P71340)

• Target gene length: 639 bp
• Plasmid length: 8,870 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Fluorescent reporter: CopGFP (green fluorescence)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: P59125, pLV3-CMV-MCS-8×His-CopGFP-Puro

pLV3-CMV-Maged1(mouse)-6×His-Puro (Catalog No. P71563)

• Target gene length: 2,325 bp
• Plasmid length: 9,724 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: P47113, pLV3-CMV-MCS-6×His-Puro

pLV3-CMV-Pdcd1(mouse)-6×His-Puro (Catalog No. P72791)

• Target gene length: 864 bp
• Plasmid length: 8,263 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: P47113, pLV3-CMV-MCS-6×His-Puro

pLV3-CMV-Rac1(mouse)-8×His-CopGFP-Puro (Catalog No. P72870)

• Target gene length: 633 bp
• Plasmid length: 8,864 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Fluorescent reporter: CopGFP (green fluorescence)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: P59125, pLV3-CMV-MCS-8×His-CopGFP-Puro

pLV3-CMV-Axl(mouse)-8×His-CopGFP-Puro (Catalog No. P72961)

• Target gene length: 2,664 bp
• Plasmid length: 10,895 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Fluorescent reporter: CopGFP (green fluorescence)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: P59125, pLV3-CMV-MCS-8×His-CopGFP-Puro

pLV3-CMV-MTOR(human)-6×His-mCherry-Puro (Catalog No. P73215)

• Target gene length: 7,647 bp
• Plasmid length: 15,819 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Fluorescent reporter: mCherry (red fluorescence)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F

pLV3-CMV-PBX1(human)-EGFP-6×His-Puro (Catalog No. P73228)

• Target gene length: 1,290 bp
• Plasmid length: 9,436 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Fluorescent reporter: EGFP (green fluorescence)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: pLV3-CMV-MCS-6×His-Puro

pLV3-CMV-SLC22A3(human-Toxic-opt)-6×His-Puro (Catalog No. P73530)

• Target gene length: 1,671 bp
• Plasmid length: 9,067 bp
• Copy number: High
• Promoter: CMV
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: CMV-F
• Background vector: P47113, pLV3-CMV-MCS-6×His-Puro

pLV3-CAG-MIDN(human)-4-6×His-Puro (Catalog No. P74384)

• Target gene length: 1,404 bp
• Plasmid length: 10,181 bp
• Copy number: High
• Promoter: CAG
• Selection markers: Ampicillin resistance (Amp) and puromycin resistance (Puro)
• Recommended bacterial strain: Stbl3 competent cells
• Bacterial culture temperature: 37°C
• Forward sequencing primer: pCAGGS-F
• Background vector: Reported as P47113, pLV3-CAG-MCS-4-6×His-Puro

How to Use pLV3-His-Tagged Vectors in Different Experiments

Plasmid Propagation and Purification

Transform the pLV3-His-tagged plasmid into Stbl3 competent cells and propagate the bacteria in LB medium containing ampicillin at 37°C. Purify the plasmid using an endotoxin-free plasmid preparation kit. As a general quality check, an A260/A280 ratio of approximately 1.8–2.0 is commonly expected for relatively pure DNA.

Miaoling’s proprietary magnetic-bead-based extraction process is designed to achieve an endotoxin level of less than 0.1 EU/μg DNA and a supercoiled DNA fraction of at least 85%, supporting applications that require high-quality plasmid DNA, including experiments involving hard-to-transfect cells. Confirm the specifications for the particular product and preparation scale before use.

Lentiviral Packaging

Co-transfect the pLV3 lentiviral transfer vector with appropriate packaging plasmids, such as psPAX2 and pMD2.G, into 293T cells using a validated lentiviral packaging protocol. Collect the viral supernatant approximately 48–72 hours after transfection, then concentrate and aliquot the viral preparation as appropriate. Store viral stocks at −80°C.

The exact packaging system, collection schedule, and handling conditions should follow the validated protocol for the vector and target application.

Cell Transduction

Optimize the multiplicity of infection (MOI) for the target cell type. Add an appropriate amount of viral preparation and, when compatible with the cells, polybrene at a starting concentration of 5–10 μg/mL. After approximately 6–12 hours, replace the medium with fresh complete medium.

These values are starting references rather than universal conditions. Cell sensitivity and viral transduction efficiency can vary substantially, so preliminary optimization is recommended.

Stable Cell-Line Selection

Approximately 48 hours after transduction, begin puromycin selection using a concentration appropriate for the target cell line. A range of 1–10 μg/mL is provided as a general starting reference, but the effective concentration must be established experimentally, ideally using a puromycin kill curve. Selection commonly continues for 7–14 days, depending on cell growth and survival.

Protein Expression and Purification

Lyse the cells and use Ni-NTA agarose resin to purify His-tagged fusion proteins by immobilized metal affinity chromatography. After washing and elution, assess the purity and integrity of the recovered protein before downstream functional assays.

Successful purification depends on factors such as protein expression level, tag accessibility, protein solubility, and buffer composition. Confirm that the His tag is correctly positioned and remains accessible in the expressed protein.

Fluorescence Detection

Vectors carrying CopGFP, EGFP, or mCherry reporters can be monitored using an appropriate fluorescence microscope or other compatible detection system. Fluorescence can help assess reporter expression and provide an indication of transduction efficiency.

However, reporter fluorescence alone does not necessarily confirm expression, localization, or function of the target protein. Use an additional assay, such as Western blotting or another target-specific method, when independent confirmation is required.

Frequently Asked Questions

Q1: Which cell types are suitable for pLV3-His-tagged vectors?

These vectors may be used in a range of mammalian cell types, including 293T, HeLa, HepG2, SH-SY5Y, and some primary cells. Transfection and transduction efficiencies vary by cell type, however, and primary cells can be particularly sensitive to experimental conditions. Pilot experiments are recommended to optimize delivery and selection.

Q2: Should I choose a 6×His tag or an 8×His tag?

The 6×His tag is a widely used standard for affinity purification and detection. An 8×His tag provides additional histidine residues, which may affect binding under some conditions, but it does not guarantee higher purification affinity or better recovery for every protein. Consider the target protein, tag accessibility, and purification conditions when choosing a construct.

Q3: Do all pLV3-His-tagged vectors include a fluorescent reporter?

No. Some constructs include CopGFP, EGFP, or mCherry, while others contain a puromycin resistance marker without a fluorescent reporter. For example, the P59125 backbone includes CopGFP, whereas the P47113 backbone is listed as a Puro-selection vector without a fluorescent reporter. Check the individual product specification and vector map to confirm the features of your selected construct.

Custom Vector Construction Services

If the available empty vectors do not meet your experimental requirements, Miaoling offers custom vector construction services. Its R&D team can customize features such as tags, promoters, and selection markers to suit specific experimental designs. Sequencing and restriction-digest verification reports can be provided as applicable, helping researchers develop tailored solutions for protein overexpression and purification.