pAcGFP1-Hyg-C1載體描述 pAcGFP1-Hyg-C1 encodes a green fluorescent protein (GFP) from Aequorea coerulescens. (Excitation maximum = 475 nm; emission maximum = 505 nm). The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1). The MCS in pAcGFP1-Hyg-C1 is located downstream of the AcGFP1 coding region, allowing the construction of a C-terminal fusion protein with AcGFP1 when genes are cloned in the same reading frame as AcGFP1 and there are no intervening stop codons. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbonealso contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A hygromycin resistance cassette (Hygr), consisting of the SV40 early promoter, the hygromycin resistance gene, and SV40 polyadenylation signals, allows stably transfected eukaryotic cells to be selected using hygromycin. A bacterial promoter upstream of the ampicillin gene expresses ampicillin resistance in E. coli. The pAcGFP1-Hyg-C1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Fusions to the C terminus of AcGFP1 retain the fluorescent properties of the native protein, allowing the localization of the fusion protein in vivo. The target gene should be cloned into pAcGFP1-Hyg-C1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using hygromycin. pAcGFP1-Hyg-C1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as a transfection marker).
Propagation in E. coli
Suitable host strains: DH5α and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Reference
1. Haas, J., et al. (1996) Curr. Biol. 6:315–324.
