Takara pTimer Vector \20 ug

Takara pTimer is a prokaryotic expression vector that encodes DsRed1-E5, a fluorescent protein that changes color as it ages. 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.

DsRed1-E5 is a mutant of the red fluorescent protein, DsRed1. The wild-type protein, DsRed, was originally referred to as drFP583. 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. 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. In addition to the amino acid replacements, DsRed1-E5s coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for high expression in mammalian cells.

pTimer is primarily intended to serve as a convenient source of the DsRed1-E5 cDNA. The DsRed1-E5 coding sequence is flanked by separate and distinct multiple cloning sites at the 5 and 3 ends so that the gene can easily be excised for use in other expression systems. Alternatively, the DsRed1-E5 coding sequence can be amplified by PCR.

The DsRed1-E5 gene was inserted in frame with the lacZ initiation codon from pUC19 so that DsRed1-E5 is expressed from the lac promoter (P_lac) in E. coli host cells. A Kozak consensus sequence is located immediately upstream of DsRed1-E5 to enhance translational efficiency should you wish to express the gene in eukaryotic systems. The entire DsRed1-E5 expression cassette in pTimer is supported by a pUC backbone, which contains a high-copy number origin of replication and an ampicillin resistance gene for propagation and selection in E. coli.

When expressed in multicellular organisms, DsRed1-E5s invariable green-to-red shift can be used as a timer to track the on-off phases of gene expression during embryogenesis and cell differentiation. - TAKBGN (Additional S&H or Hazmat Fees May Apply)