pLVX-DsRed-Monomer-N1 Vector Information

(PR0X3715; published October 2010)

pL VX-DsRed-Monomer-N1 Vector Information PT3992-5

Catalog No. 632152

pL VX-DsRed-Monomer-N1 Vector Map and Multiple Cloning Site (MCS).

Description

pLVX-DsRed-Monomer-N1 is an HIV-1-based, lentiviral expression vector that allows you to express your gene of interest fused to DsRed-Monomer, a monomeric mutant of the Discosoma sp. red fluorescent protein. Genes cloned into the multiple cloning site (MCS), located upstream of the DsRed-Monomer coding sequence, are expressed as N-terminal fusions of the DsRed-Monomer protein. Expression of the fusion protein is driven by the constitutively active human cytomegalovirus immediate early promoter (P CMV IE ) located just upstream of the MCS. Lentiviral particles derived from the vector allow the expression of DsRed-Monomer fusion proteins in virtually any cell type, including primary cells. The unmodified vector expresses DsRed-Monomer, and may be used to produce marker virus to optimize infection protocols.

pLVX-DsRed-Monomer-N1 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral and transgene RNA (1), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (2). Finally, pL VX-DsRed-Monomer-N1 also contains a central polypurine tract (cPPT) element that increases nuclear importation of the viral genome during target cell infection, resulting in improved vector integration and more efficient transduction (3).

Puro

r

2816

CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG ACG GTA CCG CGG GCC CGG GAT CCA

EcoRI

CCG GTC GCC 2870Start of DsRed-Monomer

BstBI

BamHI

SmaI

XmaI

ApaI

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V e c t o r I n f o r m a t i o n

Clontech Laboratories, Inc. https://www.wendangku.net/doc/f011437467.html,

Protocol No. PT3992-5 2 Version No. PR0X3715

In addition to lentiviral elements, pLVX-DsRed-Monomer-N1 contains a puromycin resistance gene (Puro r ) under the control of the murine phosphoglycerate kinase (PGK) promoter (P PGK ) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Amp r ) for propagation and selection in https://www.wendangku.net/doc/f011437467.html,e

pL VX-DsRed-Monomer-N1 constitutively expresses your gene of interest from P CMV IE when transduced into target cells. Before the vector can be transduced into cells, however, it must be transfected into 293T packaging cells with our Lenti-X? HTX Packaging System (Cat. Nos. 631247 and 631249). T his packaging system allows you to safely produce high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including non-dividing and primary cells (4).Location of Features ? 5' LTR: 1–635

? PBS (primer binding site): 636–653? Ψ (packaging signal): 685–822

? RRE (Rev-response element): 1303–1536? cPPT(central polypurine tract): 2028–2151

? P CMV IE (human cytomegalovirus immediate early promoter): 2185–2788? MCS (multiple coning site): 2816–2868

? DsRed-Monomer (Discosoma sp. red fluorescent protein monomer): 2882–3559? P PGK (phosphoglycerate kinase promoter): 3579–4087? Puro r (puromycin resistance gene ): 4108–4707

? WPRE (woodchuck posttranscriptional regulatory element): 4721–5312? 3' LTR: 5516–6152

? pUC origin of replication: 6622–7292 (complementary)

? Amp r (ampicillin resistance gene; β-lactamase): 7437–8433 (complementary)Selection of Stable T ransfectants

? Selectable marker: plasmid confers resistance to puromycin.

Propagation in E. coli

? Suitable host strains: DH5α, DH10B and other general purpose strains.

? Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.? E. coli replication origin: pUC ? Copy number: high

Excitation and emission maxima of DsRed-Monomer ? Excitation maximum = 557 nm ? Emission maximum = 592 nm

Notes:

The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. T his vector has not been completely sequence.

The viral supernatants produced by this lentiviral vector could contain potentially hazardous recombinant virus. Due caution must be exercised in the production and handling of recombinant lentivirus. Appropriate NIH, regional, and institutional guidelines apply.References

1. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–289

2.

2. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202.

3. Zennou, V. et al. (2000) Cell 101(2):173–185.

4. Wu, X. et al. (2000) Mol. T her. 2(1):47–5

5.

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Clontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc.

This product and its use are the subject of U.S. Pat. No. 6,682,907

The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot disclose information, sell or otherwise transfer this product, its components or materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for anycommercial purposes. If the buyer is not willing to accept the limitations of this limited use statement, Clontech is willing to accept return of the product with a full refund. For information on purchasing a license to the DNA-Flap technology for purposes other than research, contact the T ransfer of T echnology Office, Institut Pasteur, 28 rue du Docteur Roux, 75 724 Paris Cedex 15 (www.pasteur.fr). The DsRed Monomer and the Fruit Fluorescent Proteins are covered by one or more of the following U.S. Patents: 7,157,566; 7,393,923; 7,005,511 and 7,250,298.

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Clontech has a license to sell products containing the WPRE, under the terms described below. Any use of WPRE outside of Clontech’s Product or the Product’s intended use, requires a license as detailed below. Before using the Product containing WPRE, please read the following license agreement. If you do not agree to be bound by its terms, contact Clontech within 10 days for authorization to return the unused Product containing WPRE and to receive a full credit.Patents: T he WPRE technology is covered by one or more of the following US patents and corresponding patent claims outside the US: 6,136,597 ; 6,284,469 ; 6,312,912 ; 6,287,814 , issued to T he Salk Institute for Biological Studies; Individual License Agreement: Clontech grants you a non-exclusive license to use the enclosed Product containing WPRE in its entirety for its intended use. The Product containing WPRE is being transferred to you in furtherance of, and reliance on, such license. Any use of WPRE outside of Clontech’s Product or the Product’s intended use, requires a license from the Salk Institute for Biological Studies.Termination of License: This license agreement is effective until terminated. You may terminate it at any time by destroying all Products containing WPRE in your control. It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement. Y ou shall, upon termination of the license agreement, destroy all Products containing WPRE in your control, and so notify Clontech in writing. T his License shall be governed in its interpretation and enforcement by the laws of the State of California.Contact for WPRE Licensing:The Salk Institute for Biological Studies , 10010 North Torrey Pines Road, La Jolla, CA 92037, Attn.: Office of T echnology Management , Phone: 858.453.4100 ext. 1275 , Fax: 858.546.8093

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Protocol No. PT3992-5 https://www.wendangku.net/doc/f011437467.html, Clontech Laboratories, Inc. Version No. PR0X3715 3