• Amount - 20 µg • Application: C-terminal mammalian expression vector encoding humanized TagGFP and allowing TagGFP expression and generation of fusions to the TagGFP C-terminus. Generation of VisionGFP-tagged fusions A localization signal or a gene of interest should be cloned into MCS of the vector. It will be expressed as a fusion to VisionGFP C-terminus when inserted in the same reading frame as VisionGFP and no in-frame stop codons are present. VisionGFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified pVisionGFP-C vector will express VisionGFP when transfected into eukaryotic (mammalian) cells. Expression in Mammalian Cells pVisionGFP-C can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of VisionGFP or VisionGFP-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
pVisionGFP-C1 vector is a mammalian expression vector encoding green fluorescent protein, VisionGFP. pVisionGFP-C vector is designed to generate fusions to VisionGFP C-terminus for expression, localization and cellular dynamics studies or to express VisionGFP in eukaryotic (mammalian) cells. pVisionGFP-C vector carries synthetic version of the VisionGFP gene which codon usage is humanized, i.e. optimized for high expression in mammalian cells. pVisionGFP-C vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 early promoter provides neomycin resistance gene expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression in E. coli. To increase VisionGFP mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of VisionGFP coding sequence. Multiple cloning site (MCS) is located VisionGFP coding sequence and SV40 polyadenylation signal (SV40 poly A).
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