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pLenti6/V5 Directional TOPO® Cloning Kit

pLenti6/V5 Directional TOPO® Cloning Kit

(Invitrogen™)

Description

The pLenti6⁄V5 Directional TOPO® Cloning Kit contains the TOPO®-adapted ViraPower™ lentiviral expression vector, pLenti6⁄V5-D-TOPO® for quick PCR-based cloning and high-level expression of a target gene in dividing and non-dividing mammalian cells. The vector has the CMV promoter for driving high-level, constitutive expression of the target gene and the blasticidin selection marker for stable selection in mammalian cells.

Advantages
• High efficiency and rapid cloning
• Constitutive gene expression in dividing and non-dividing mammalian cells in vitro or in vivo
• Produces replication-incompetent virus for enhanced biosafety of the system

Key Features
• Directional TOPO® Cloning site for rapid and efficient directional cloning of blunt-end PCR products
• Rouse Sarcoma Virus (RSV) enhancer⁄promoter for Tat-independent production of viral mRNA in the producer cell line
• Modified HIV-1 5’ and 3’ Long Terminal Repeats (LTR) for viral packaging and reverse transcription
• HIV-1 psi (ψ) packaging sequence for viral packaging
• HIV Rev response element (RRE) for Rev-dependent nuclear export of unspliced viral mRNA
• (CMV) immediate early promoter for high-level constitutive expression of the gene of interest in mammalian cells
• C-terminal V5 epitope for detection of the recombinant protein
• Blasticidin (bsd) resistance gene for selection in E. coli and mammalian cells
• Ampicillin resistance gene for selection in E. coli
• pUC origin for high-copy replication and maintenance of the plasmid in E. coli

Kit includes
pLenti6⁄V5-D- TOPO® Reagents
One Shot® Stbl3™ Chemically Competent E. coli

For research use only. Not intended for any therapeutic or diagnostic use.

Catalog Number

K4955-10

Size: 20 reactions

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Figure 1

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Specifications

General Specifications

Promoter:	CMV
Protein Tag or Fusion:	V5 Epitope Tag
Constitutive or Inducible System:	Constitutive
Delivery Method:	Lentiviral
Cloning Method:	Directional TOPO®
Key Function:	Viral Expression
Selection Agent (Eukaryotic):	Blasticidin
Vector Type:	Directional TOPO Vectors, pLenti
Product Size:	20 reactions
Regulatory Statement:	For Research Use Only. Not for use in diagnostic procedures.

Contents and Storage

pLenti⁄V5-D-TOPO® reagents (Box 1) Store at -20°C
• pLenti6⁄V5-D-TOPO® Reagents (15-20 ng⁄µl linearized plasmid DNA in: 50% glycerol, 50 mM Tris-HCl, pH 7.4 (at 25°C),1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 100 µg⁄ml BSA, 30 µM bromophenol blue)
• dNTP Mix (12.5 mM each dATP, dCTP, dGTP, dTTP in water, pH8 at a total volume of 10 µl)
• Salt Solution (1.2 M NaCl and 0.06 M MgCl2, volume of 50 µl)
• Water (1 ml)
• CMV Forward Sequencing Primer (0.1 µg⁄µl in TE Buffer, pH 8, pH8 at a total volume of 20 µl)
• Control PCR Primers (0.1 µg⁄µl each in TE Buffer, pH 8, pH8 at a total volume of 10 µl)
• Control PCR Template (0.1 µg⁄µl in TE Buffer, pH 8, pH8 at a total volume of 10 µl)
• pLenti6⁄V5-GW⁄lacZ (lyophilized in TE Buffer, pH 8, 10 µg)

One Shot® Stbl3™ Chemically Competent E. coli (Box 2) Store at -80°C
• S.O.C. Medium (2% Trypton, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose. 6ml) May be stored at +4°C or room temperature
• Stbl3™ Cells (21 x 50 µl)
• pUC19 Control DNA (10 pg⁄µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8. 50 µl)