pFusionRed-Cx43 is a mammalian expression vector encoding FusionRed-Cx43 fusion protein (see reporter description). The vector can be used for fluorescent labeling of connexin 43 in living cells.

Pig kidney fibroblast cells expressing fusion of rat connexin 43 with FusionRed.

Multiple small gap junction plaques fuse to the larger plaque as it moves along the cell-cell border. Movie from Shemiakina et al., 2012.

FusionRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Rat connexin 43 is fused to the FusionRed N-terminus.

pFusionRed-Cx43 vector can be used as a source of FusionRed-Cx43 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.

Expression in mammalian cells

pFusionRed-Cx43 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-Cx43 fusion 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.