pKillerOrange-dMito is a mammalian expression vector encoding mitochondria-targeted KillerOrange (see reporter description). KillerOrange codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Duplicated mitochondrial targeting sequence (MTS) is fused to the KillerOrange N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995].

pKillerOrange-dMito vector can be used as a source of dMTS-KillerOrange hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, dMTS-KillerOrange coding sequence can be amplified by PCR.

Note: The plasmid DNA was isolated from dam⁺-methylated E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam^- host and make fresh DNA.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV IE}) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

SV40 early promoter (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Expression in mammalian cells

pKillerOrange-dMito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mitochondria-targeted KillerOrange in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].

Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.