pKillerOrange-N is a mammalian expression vector encoding photosensitizer KillerOrange (see reporter description). The vector allows generation of fusions to the KillerOrange N-terminus and expression of KillerOrange fusions or KillerOrange alone in eukaryotic (mammalian) cells.

KillerOrange 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 KillerOrange coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between P_{CMV IE} and KillerOrange coding sequence.

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.

A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a fusion to the KillerOrange N-terminus when inserted in the same reading frame as KillerOrange and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. KillerOrange-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express KillerOrange when transfected into eukaryotic (mammalian) cells.

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.

pKillerOrange-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of KillerOrange or its fusions 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.