载体简介
pEF1α-DsRed-Monomer-N1 is designed to 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 DsRed-Monomer coding sequence has been human-codon-optimized for high expression in mammalian cells (2). The excitation and emission maxima of native DsRed-Monomer are 557 nm and 585 nm, respectively. Expression of fusion proteins that retain the fluorescence properties of unmodified DsRed-Monomer can be monitored by flow cytometry and localized by fluorescence microscopy.
The multiple cloning site (MCS) in pEF1α-DsRed-Monomer-N1 is positioned between the EF1 promoter (PEF1α) and the DsRed-Monomer coding sequence. Expression of the fusion protein is driven by the EF1α promoter, which remains constitutively active even after stable integration of the vector into the host cell genome (3). A Kozak consensus sequence, located immediately upstream of the DsRed-Monomer gene, enhances the translational efficiency of DsRed-Monomer in eukaryotic systems (4), and SV40 polyadenylation signals direct proper processing of the 3' end of the DsRed-Monomer mRNA.
The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycinresistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418 (5). This cassette consists of the SV40 early promoter (PSV40 e), the Tn5 neomycin
namycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette drives expression of the kanamycin resistance gene in E. coli.
Location of Features
PEF1α (human elongation factor 1 alpha promoter): 12–1346
MCS (multiple cloning site): 1348–1422
Kozak consensus sequence: 1429–1439
DsRed-Monomer (human-codon-optimized): 1436–2110
SV40 polyA signal: 2267–2301
f1 origin of replication: 2364–2819 (complementary)
PSV40 e (SV40 early promoter and enhancer sequences): 2993–3261
SV40 origin of replication: 3160–3295
Kanr/Neor (kanamycin/neomycin resistance gene): 3344–4138
HSV TK polyA signals: 4374–4392
pUC origin of replication: 4723–5366
Additional Information
The gene of interest must be cloned into pEF1α-DsRed-Monomer-N1 so that it is in-frame with the DsRed-Monomer coding sequence. The gene must contain a start codon (ATG), and lack in-frame stop codons. Cells expressing DsRed-Monomer fusions can be detected by flow cytometry or fluorescence microscopy 12–16 hr after transfection. If required, stable transfectants can be selected using G418 (5). pEF1α-DsRed-Monomer-N1 can also be used as a cotransfection marker, as the unmodified vector will express DsRed-Monomer in mammalian cells.
Propagation in E. coli
Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid, such as the JM109 or XL1-Blue strains.
Selectable marker: plasmid confers resistance to kanamycin (50 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
