pIRES2-AcGFP1 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCSand theAequorea coerulescens green fluorescent protein (AcGFP1) coding region.This permits both the gene of interest (cloned into the MCS) and the AcGFP1 gene to be translated from a single bicistronic mRNA. pIRES2-AcGFP1 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing AcGFP1 and the protein of interest.This vector can also be used to express AcGFP1 alone or to obtain stably transfected cell lines without time-consuming drug and clonal selection.AcGFP1 is a green fluorescent protein (GFP) from Aequorea coerulescens. (Excitation maximum = 475 nm; emission maximum = 505 nm). AcGFP1 contains silent mutations that create an open reading frame comprised almost entirely of preferred human codons. These changes increase the translational efficiency of the AcGFP1 mRNA and consequently the expression of AcGFP1 in mammalian and plant cells.
The MCS in pIRES2-AcGFP1 is between the immediate early promoter of cytomegalovirus (PCMVIE) and the IRES sequence. Additional features include SV40 polyadenylation signals downstream of the AcGFP1 gene to direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40T antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418 (3). A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pIRES2-AcGFP1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
