Description
Quantity: 10 reactions
Unit Size: Each
• Express up to 1% total soluble protein of your gene of interest• Select against gene silencing even over multiple passages• Detect and purify your gene of interest with 6His-TEV and/or V5-His epitope tags• Compatible with seamless assembly for creation of your constructs• Enables you to receive reliable results with exceptional strain viability and purityChlamydomonas pChlamy_4 vectorTransgene expression from the Chlamydomonas nuclear genome offers several advantages over chloroplast expression, such as post-translational modifications and protein-targeting and/or secretion. However, expression from the nuclear genome has typically been less than robust, and the molecular mechanism(s) of poor expression are not completely understood. Possible reasons include poor promoters, genome integration position effects, and transgene silencing. The GeneArt Chlamydomonas Protein Expression Vector provides relative high levels of expressed protein and selection against silencing. Our pChlamy_4 vector is also compatible with seamless cloning, an optional cloning method that results in no extra sequences in your final construct.Features of the vector include:• Endogenous and constitutive promoter, Hsp70A-RbcS2, is composed of activator Hsp70A and two copies of RBC S2 intron sequence, resulting in increased expression of your gene of interest• Hsp70A-Rbc S2 hybrid promoter fused to the bleomycin/Zeocin™-resistance gene allows positive transformants to maintain protein expression levels for multiple passages• Self-cleaving sequence for the 2A peptide from foot-and-mouth-disease-virus (FMDV) mediates proper cleavage between resistance markers and protein-of-interest to yield two discrete proteins• 3’ UTR for proper transcript termination and possible additional benefits like increased translation efficiency, mRNA stability, and polyadenylation signals• Dual protein tags 6His-TEV and V5-His epitopes can be fused to both or either ends of your gene of interest or no tag at allSelection against silencingIn order to circumvent the transgene silencing that often occurs in C. reinhardtii, our new pChlamy_4 vector is designed so that proteins are expressed as transcriptional fusions with the bleomycin/zeocin resistance gene sh-ble. The self-cleaving sequence for the 2A peptide from the foot-and-mouth-disease-virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ∼20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure.
