• Amount - 20 µg • Application: C-terminal mammalian expression vector encoding humanized TagRFP and allowing TagRFP expression and generation of fusions to the TagRFP C-terminus. Generation of pVisionRFP-fusion proteins- A localization signal or a gene of interest should be inserted into MCS of the vector. It will be expressed as a fusion to VisionRFP C-terminus when inserted in the same reading frame as VisionRFP and no intervening stop codons are present. Note: Despite its dimeric structure, VisionRFP is still suitable for generation of fusions with proteins of interest. Expression in mammalian cells- pVisionRFP-C vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of VisionRFP or VisionRFP-tagged fusions in many cell types. If required, stable transformants can be selected using G418. Propagation in E. coli - Suitable host strains: DH5alpha, HB101, and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue. -Selectable marker: plasmid confers resistance to kanamycin (30 µg/ml) to E. coli hosts. - E. coli replication origin: pUC - Copy number: ~500 -Plasmid incompatibility group: pMB1/ColE1
pVisionRFP-C is an eukaryotic (mammalian) expression vector encoding red fluorescent protein VisionRFP from sea anemone Entacmaea quadricolor. The vector allows to generate fusions to the VisionRFP C-terminus and to express VisionRFP fusions or VisionRFP alone in eukaryotic (mammalian) cells. pVisionRFP-C vector carries synthetic VisionRFP gene which codon usage is humanized, i.e. optimized for high expression in mammalian cells. pVisionRFP-C vector contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication and neomycin resistance (Neor) gene for selection in eukaryotic cells. It also contains pUC origin of replication for propagation in E. coli and f1 origin for single-stranded DNA production. Bacterial promoter (P) provides expression of kanamycin resistance gene in E. coli. To increase VisionRFP translation, Kozak consensus translation initiation site is generated upstream of VisionRFP sequence. Multiple cloning site (MCS) is between VisionRFP coding sequence and the SV40 polyadenylation signal (SV40 poly A).
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