pLVX-EF1α-DsRed-Monomer-N1慢病毒红色荧光表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pLVX-EF1α-DsRed-Monomer-N1慢病毒红色荧光表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
pLVX-EF1α-DsRed-Monomer-N1慢病毒红色荧光表达载体，EF1a启动子，DsRed Monomer红色荧光报告基因，Puromycin嘌呤霉素细胞筛选标记，Amp抗性。
pLVX-EF1α-DsRed-Monomer-N1 is an HIV-1-based, lentiviral expression vector designed to constitutively express a protein of interest fused to the N-terminus of DsRed-Monomer, a monomeric mutant of the Discosoma sp. red fluorescent protein DsRed (1). The excitation and emission maxima of native DsRed-Monomer are 557 nm and 585 nm, respectively. Stable, constitutive expression of the fusion protein is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after the vector integrates into the host cell genome (2). pLVX-EF1α-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 RNA (3), leading to increased viral titers from packaging 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 (4). Finally, pLVX-EF1α-DsRed-Monomer-N1 also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (5).
In addition to lentiviral elements, pLVX-EF1α-DsRed-Monomer-N1 contains a puromycin resistance gene (Puror ) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr ) for propagation and selection in bacteria. Location of Features 5′ LTR (5′ long terminal repeat): 1–635 PBS (primer binding site): 636–653 Ψ (packaging signal): 685–822 RRE (Rev-response element): 1303–1536 cPPT/CTS (central polypurine tract/central termination sequence): 2028–2151 PEF1α (human elongation factor 1 alpha promoter): 2185–3519 MCS (multiple cloning site): 3560–3599 DsRed-Monomer (human-codon-optimized): 3613–4290 PPGK (phosphoglycerate kinase promoter): 4310–4818 Puror (puromycin resistance gene): 4839–5438 WPRE (woodchuck hepatitis virus posttranscriptional regulatory element): 5452–6043 3′ LTR (3′ long terminal repeat): 6246–6882 pUC origin of replication: 7351–8024 (complementary) Ampr (ampicillin resistance gene; β-lactamase): 8169–9165 (complementary) Additional Information Genes cloned into the MCS must: be in-frame with the DsRed-Monomer coding sequence; contain a start codon (ATG); and lack in-frame stop codons. pLVX-EF1α-DsRed-Monomer-N1 will constitutively express your N-terminal DsRedMonomer fusion when transduced into target cells. Before the vector can be transduced into target cells, however, it must be packaged into viral particles in HEK293T cells, using Lentiviral Packaging System. This packaging system allows the safe production of high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including nondividing and primary cells (6).
Caution!
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.
Selection of Stable Transfectants Selectable marker: plasmid confers resistance to puromycin.
Propagation in E. coli Suitable host strains: DH5α, HB101 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: 557 nm Emission: 585 nm

References
1. Matz, M. V., et al. (1999) Nat. Biotechnol. 17(10):969–973.
2. Wang, R. et al. (2008) Stem Cells Dev. 17(2):279−289.
3. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–2892.
4. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202.
5. Zennou, V. et al. (2000) Cell 101(2):173–185.
6. Wu, X. et al. (2000) Mol. Ther. 2(1):47–55.

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