載體介紹 pEF1α-IRES-ZsGreen1 is designed to simultaneously and constitutively express a protein of interest and ZsGreen1 in mammalian cells. ZsGreen1 is a human-codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein (ZsGreen) that has been engineered for brighter fluorescence (1–3). The excitation and emission maxima of native ZsGreen1 are 493 nm and 505 nm, respectively. Simultaneous expression of a protein of interest and ZsGreen1 is made possible by the presence of an encephalomyocarditis virus internal ribosome entry site (IRES2; 4, 5) positioned between the multiple cloning site (MCS) and the ZsGreen1 gene. The IRES allows a protein of interest and ZsGreen1 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 (6).
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 (7).
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. 載體特征 PEF1α (human elongation factor 1 alpha promoter): 12–1346
MCS (multiple cloning site): 1348–1422
IRES2 (internal ribosome entry site): 1423–2007
ZsGreen1 (human-codon-optimized): 1999–2703
SV40 early polyA signal: 2859–2909
f1 origin of replication: 2956–3411 (complementary)
SV40 origin of replication: 3752–3887
PSV40e (SV40 early promoter and enhancer sequences): 3585–3853
Kanr/Neor (kanamycin/neomycin resistance gene): 3936–4730
HSV TK polyA signals: 4966–4984
pUC origin of replication: 5315–5958 補(bǔ)充信息 pEF1α-IRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS must contain a start codon (ATG) and a stop codon. pEF1α-IRES2-ZsGreen1 can be introduced into mammalian cells using any standard transfection method. Cells expressing ZsGreen1 can 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. If required, stable transformants can be selected using G418 (7).
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 ZsGreen1
Excitation: 493 nm
Emission: 505 nm
