pFusionRed-PDHA1 is a mammalian expression vector encoding FusionRed-PDHA1 fusion protein (see reporter description). The vector can be used for fluorescent labeling of PDHA1 in living cells.

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(A) – FoLu cell expressing fusion of human pyruvate dehydrogenase PDHA1 with FusionRed. The fusion protein is localized in the mitochondria.
(B) – HeLa cell expressing fusion of human pyruvate dehydrogenase PDHA1 with FusionRed. The fusion protein is localized in the mitochondria. Scale bar represents 10 μm. Data from Shemiakina et al., 2012

FusionRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human pyruvate dehydrogenase alpha 1 (PDHA1) is fused to the FusionRed N-terminus.

pFusionRed-PDHA1 vector can be used as a source of FusionRed-PDHA1 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 (PCMV 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 (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.


Expression in mammalian cells

pFusionRed-PDHA1 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-PDHA1 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.