pDsRed-Monomer载体

pDsRed-Monomer is a prokaryotic expression vector that encodes DsRed-Monomer (DsRed. M1), a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1). DsRed-Monomer contains forty-five amino acid substitutions (listed on page 2). The excitation and emission spectra is comparable to that of DsRed-Express (DsRed-Monomer excitation and emission maxima = 557 nm and 592 nm, respectively). The DsRed-Monomer coding sequence has been human codon-optimized for high expression in mammalian cells (2).

In pDsRed-Monomer, the DsRed-Monomer coding sequence is flanked at the 5' and 3' ends by separate and distinct multiple cloning sites (MCS), making it easy to excise the gene for use in other cloning applications. Alternatively, the DsRed-Monomer coding sequence can be amplified by PCR. In E. coli, DsRed-Monomer is expressed from the lac promoter as a fusion with several amino acids, including the first five amino acids of the LacZ protein. Note, however, that if you excise the DsRed-Monomer coding sequence using a restriction site in the 5' MCS, the resulting fragment will encode solely the DsRed-Monomer protein (i.e., without the additional amino acids that are expressed using the lac promoter). A Kozak consensus sequence is located immediately upstream of the DsRed-Monomer gene to enhance translational efficiency in eukaryotic systems (3). The entire DsRed-Monomer expression cassette in pDsRed-Monomer is supported by a pUC19 backbone, which contains a high-copy number origin of replication and an ampicillin resistance gene for propagation and selection in E. coli.

载体应用

pDsRed-Monomer is primarily intended to serve as a source of DsRed-Monomer cDNA. The flanking MCS regions make it possible to excise the DsRed-Monomer coding sequence and insert it into other vector systems of choice. The vector can also be used in bacteria to produce DsRed-Monomer protein.

For Western blotting, the Living Colors® DsRed Polyclonal Antibody (Cat. No. 632496) can be used to recognize the DsRed-Monomer protein. However, to generate optimal results it may be necessary to use a higher concentration of antibody than recommended on the DsRed Polyclonal Antibody Certificate of Analysis.