pHcRed1-C1 is a mammalian expression vector designed to express a protein of interest fused to the C-terminus of the far-red fluorescent protein HcRed1. pHcRed1-C1 can be used to monitor gene expression and protein localization in vivo or as a cotransfection marker; the unmodified vector will express HcRed1 in mammalian cells.

HcRed1 was generated by mutagenesis of a non-fluorescent chromoprotein from the reef coral Heteractis crispa. The coding sequence for HcRed1 is human codon-optimized for higher expression in mammalian cells. Genes cloned into the multiple cloning site (MCS) downstream of the HcRed1 coding sequence are expressed as fusions to the C-terminus of HcRed1. The MCS in pHcRed1-C1 is positioned between the HcRed1 coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of HcRed1 if they are in the same reading frame as HcRed1 and there are no intervening stop codons. A Kozak consensus sequence upstream of HcRed1 increases the translation efficiency in eukaryotic cells. SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neor)-consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene-allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. pHcRed1-C1 can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418.

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