pPS-CFP2-C is a mammalian expression vector encoding cyan-to-green fluorescent protein PS-CFP2 (see reporter description). The vector allows generation of fusions to the PS-CFP2 C-terminus and expression of PS-CFP2 fusions or PS-CFP2 alone in eukaryotic (mammalian) cells.

PS-CFP2 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 PS-CFP2 coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PS-CFP2 coding sequence and SV40 polyadenylation signal (SV40 polyA).

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 PS-CFP2 C-terminus when inserted in the same reading frame as PS-CFP2 and no in-frame stop codons are present. PS-CFP2-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express PS-CFP2 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.

pPS-CFP2-C vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of PS-CFP2 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.