pEF1α-IRES-AcGFP1 is a bicistronic mammalian expression vector that allows the simultaneous, constitutive expression of a protein of interest and the green fluorescent protein AcGFP1. Expression of the bicistronic transcript is driven by the human elongation factor 1 alpha (EF1α) promoter, which continues to be constitutively active even after stable integration of the vector into the host cell genome. Stable expression of the bicistronic transcript allows the monitoring of a variety of cellular processes (such as differentiation in primary or stem cells), without the transgene silencing associated with CMV promoters.
In addition, the vector allows efficient flow cytometric detection of stably or transiently transfected mammalian cells expressing AcGFP1 and a protein of interest, without time-consuming drug and clonal selection.
pEF1α-IRES-AcGFP1 is designed to simultaneously and constitutively express a protein of interest and AcGFP1 in mammalian cells. AcGFP1 is a human-codon-optimized, monomeric green fluorescent protein derived from Aequorea coerulescens (the excitation and emission maxima of native AcGFP1 are 475 nm and 505 nm, respectively). Simultaneous expression of a protein of interest and AcGFP1 is made possible by the presence of an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES; 1, 2) positioned between the multiple cloning site (MCS) and the AcGFP1 gene. The IRES allows a protein of interest and AcGFP1 to be translated from a single bicistronic mRNA. Stable, constitutive expression of the bicistronic transcript is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after vector integration into the host cell genome (3).The vector backbone also 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. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (4). This cassette consists of the SV40 early promoter (PSV40 e), a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. 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
IRES2 (internal ribosome entry site): 1423–2007
AcGFP1 (human-codon-optimized): 2011–2727
SV40 early polyA signal: 2883–2933
f1 origin of replication: 2980–3435 (complementary)
SV40 origin of replication: 3776–3911
PSV40e (SV40 early promoter and enhancer sequences): 3609–3877
Kanr/Neor(kanamycin/neomycin resistance gene): 3960–4754
HSV TK polyA signals: 4990–5008
pUC origin of replication: 5339–5982
Additional Information
pEF1α-IRES2-AcGFP1 can be used to quickly identify cells expressing a gene of interest by screening for AcGFP1 fluorescence. Genes inserted into the MCS must contain a start codon (ATG) and a stop codon. pEF1α-IRES2-AcGFP1 can be introduced into mammalian cells using any standard transfection method. Cells expressing AcGFP1can be detected by flow cytometry or fluorescence microscopy 24 hours after transfection. However, in some cases, up to 48 hours may be required for detection of green-emitting cells. If required, stable transformants can be selected using G418 (4).
Propagation in E. coli
Suitable host strains: DH5α and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM101 or XL1-Blue.
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 AcGFP1
Excitation: 475 nm
Emission: 505 nm
