pmKate2-f-mem is a mammalian expression vector intended for far-red fluorescent labeling of plasma membrane in living cells. The vector encodes far-red fluorescent protein mKate2 (see reporter description) targeted to plasma membrane by 20 amino acid farnesylation signal from c-Ha-Ras [Aronheim et al., 1994; Hancock et al., 1991]. The farnesylation signal is fused to the mKate2 C-terminus.

mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2-f-mem coding sequence [Kozak, 1987].

pmKate2-f-mem vector can be used as a source of mKate2-f-mem hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.

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.

pmKate2-f-mem vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-f-mem in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].

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.

Transiently transfected HeLa cells expressing mKate2 targeted to plasma membrane by 20-amino acid farnesylation signal from c-Ha-Ras.