pAcGFP1-Actin载体描述 pAcGFP1-Actin encodes a green fluorescent protein (GFP) from Aequorea coerulescens (Excitation maximum = 475 nm; emission maximum = 505 nm) and the gene encoding human cytoplasmic β-actin (1). SV40 polyadenylation signals downstream of the AcGFP1-Actin fusion direct proper processing of the 3' end of the AcGFP1 mRNA. AcGFP1 contains silent mutations that create an open reading frame comprised almost entirely of optimized human codons. These changes increase the translational efficiency of the AcGFP1 mRNA and consequently the expression of AcGFP1 in mammalian and plant cells. The vector backbone also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-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. A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pAcGFP1-Actin backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
The pAcGFP1-Actin Vector expresses the AcGFP1-Actin fusion protein in mammalian cells. The protein is incorporated into growing actin filaments and allows for visualization of actin-containing subcellular structures in living and fixed cells (2, 3). This vector is not intended to be used as a cloning vector; however, unique restriction sites at the 5' end of AcGFP1, and between AcGFP1 and the β-actin open reading frame, allow excision or insertion of DNA. pAcGFP1-Actin can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (4). 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 JM109 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC; copy number: ≈500
Plasmid incompatibility group: pMB1/ColE1
