pEF1α-AcGFP1-N1 is designed to express a protein of interest fused to the N-terminus of AcGFP1, a human-codonoptimized, monomeric green fluorescent protein derived from Aequorea coerulescens. The excitation and emission maxima of the native AcGFP1 protein are 475 nm and 505 nm, respectively. Expression of fusion proteins that retain the fluorescence properties of the unmodified AcGFP1 protein can be monitored by flow cytometry and localized by fluorescence microscopy.
The multiple cloning site (MCS) in pEF1α-AcGFP1-N1 is positioned between the EF1 promoter (PEF1α) and the
AcGFP1 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 (1). A Kozak consensus sequence, located immediately upstream of the AcGFP1 gene, enhances the translational efficiency of AcGFP1 in eukaryotic systems (2), and SV40 polyadenylation signals direct proper processing of the 3' end of the AcGFP1 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 neomycin resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418 (3). This cassette consists of the SV40 early promoter (PSV40 e), the Tn5 neomycin/kanamycin 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
AcGFP1(human-codon-optimized): 1430–2146
SV40 polyA signal: 2302–2336
f1 origin of replication: 2399–2854 (complementary)
PSV40 e (SV40 early promoter and enhancer sequences): 3028–3296
SV40 origin of replication: 3195–3333
Kanr/Neor (kanamycin/neomycin resistance gene): 3379–4173
HSV TK polyA signals: 4409–4427
pUC origin of replication: 4758–5401 Additional Information The gene of interest must be cloned into pEF1α-AcGFP1-N1 so that it is in-frame with the AcGFP1 coding sequence. Thegene must contain a start codon (ATG), and lack in-frame stop codons.
The pEF1α-AcGFP1-N1 vector can be transfected into mammalian cells using any standard transfection method. pEF1α-AcGFP1-N1 can be used as a cotransfection marker, as the unmodified vector will express AcGFP1 in mammalian cells.If required, stable transfectants can be selected using G418 (3). 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 AcGFP1
Excitation: 475 nm
Emission: 505 nm
