pAcGFP1-Mito encodes a fusion of a mitochondrial targeting sequence derived from the precursor of subunit VIII of human cytochrome C oxidase (1, 2) and the green fluorescent protein (GFP) from Aequorea coerulescens (AcGFP1; excitation maximum = 475 nm; emission maximum = 505 nm). The mitochondrial targeting sequence is fused to the N-terminus of AcGFP1. 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 contains an SV40 origin for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells (3). 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-Mito is designed for fluorescent labeling of mitochondria. The fluorescence from pAcGFP1-Mito expression can be observed within the mitochondrial matrix inside the inner membrane. pAcGFP1-Mito can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (3). pAcGFP1- Mito is not intended as a cloning vector; however, the backbone does contain unique restriction sites upstream and downstream of the AcGFP1-Mito sequence which permit excision of the AcGFP1-Mito sequence.
Propagation in E. coli
Suitable host strains: 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) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ≈500
Plasmid incompatibility group: pMB1/ColE1
