pAcGFP1-Golgi Vector encodes a fusion protein consisting of the N-terminal 81 amino acids of the human beta 1,4-galactosyltransferase (GT; 1) directly upstream of the green fluorescent protein (GFP) from Aequorea coerulescens (AcGFP1). The human beta 1,4-galactosyltransferase sequence contains a membrane-anchoring signal peptide that targets the protein to the transmedial region of the Golgi aparatus (2–4). AcGFP1 (excitation maximum = 475 nm; emission maximum = 505 nm) 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. SV40 polyadenylation signals downstream of the protein coding region direct proper processing of the 3' end of the mRNA. The vector contains an SV40 origin for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells (5). A bacterial promoter (P) upstream of Neor expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
pAcGFP1-Golgi Vector is designed for fluorescent labeling of the Golgi apparatus. pAcGFP1-Golgi can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pAcGFP1-Golgi is not intended as a cloning vector; however, the backbone does contain unique restriction sites upstream and downstream of the AcGFP1-Golgi sequence which permit its excision.
Propagation in E. coli
Suitable host strains: Fusion-Blue competent cells (Cat. No. 636700), DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires a host such as JM109 or XL1-Blue that contains an
F plasmid.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: ≈500
Plasmid incompatibility group: pMB1/ColE1
