pTimer-1载体描述 pTimer-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the MCS located upstream of the DsRed1-E5 coding sequence. Shortly after translation, DsRed1-E5 emits green light; its red fluorophore emerges later, hours after translation. Because of its predictable color shift, DsRed1-E5 acts as a timer, useful for monitoring changes in gene activity in living cells. pTimer-1 can be used to study the on-off activity of any cis-acting regulatory element cloned into the MCS upstream of DsRed1-E5. DsRed1-E5 is a mutant of the red fluorescent protein, DsRed1 (1). The cDNA for the wild-type protein, DsRed, was originally isolated by Matz et al., who refer to the protein as drFP583 (2). DsRed1-E5 contains two amino acid substitutions (V105A and S197T), which increase its fluorescence intensity and endow it with a distinct spectral property: As the protein ages, it changes color (3). When first synthesized, DsRed1-E5 is bright green (excitation & emission maxima = 483 nm & 500 nm). As time passes, the green fluorophore undergoes additional changes that cause its fluorescence to shift to longer wavelengths—when fully matured, the protein is bright red (excitation & emission maxima = 558 nm & 583 nm). In mammalian cells transfected with a Tet-inducible DsRed1-E5 expression vector, the green-to-red transition starts about 3 hours after the protein first becomes fluorescent (3). In addition to the amino acid replacements, DsRed1-E5’s coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for high expression in mammalian cells (4).
To further increase translation efficiency in eukaryotic cells, a sequence upstream of DsRed1-E5 has been converted to a Kozak consensus sequence (5). SV40 polyadenylation signals downstream of the DsRed1-E5 gene direct proper processing of the 3' end of the DsRed1-E5 mRNA. The vector backbone 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) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists 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. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.Without addition of a functional promoter, this vector will not express DsRed1-E5
