pGLuc-Basic2分泌型GLuc荧光报告载体 BioVector NTCC中国质粒载体菌种细胞基因保藏中心

pGLuc-Basic2

BioVector NTCC中国质粒载体菌种细胞基因保藏中心                      

pGluc-Basic2分泌型GLuc荧光报告载体，人源化GLuc分泌型荧光报告蛋白基因，无启动子，N端分泌信号肽可以分泌到细胞外，Neo-G418细胞筛选标记，Amp抗性。

pGLuc-Basic 2 is a cloning vector for expression in mammalian cells, containing a reporter gene but lacking promoter elements. The reporter gene is the secreted luciferase from the copepod Gaussia princeps. Gaussia Luciferase (GLuc) is a 19 kDa protein encoded by a "humanized" sequence, and it contains a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells into the cell culture medium (1,2). The pGLuc-Basic 2 Vector contains a multiple cloning site (MCS) upstream of the GLuc coding sequence. A neomycin resistance gene under the control of an SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.   FeaturesMultiple samples can be obtained from the same transfected cells (i.e., before and after experimental treatments or at multiple time points).90–95% of GLuc activity is found in the cell culture medium, with the remaining 5–10% detectable in cell lysates. This allows flexibility when assaying GLuc along with other co-transfected reporters.The activity of GLuc is high and the GLuc assay is sensitive enough to detect very small amounts of GLuc enzyme activity.GLuc is very stable in the cell culture medium so the GLuc activity detected reflects the amount of GLuc secreted by the transfected cells over a period of several days. GLuc can also be stored at 4°C for several days without any loss in activity.GLuc does not use the same substrate as Cypridina Luciferase. Therefore, it is possible to assay both GLuc and CLuc independently in cell culture medium from cells expressing both reporters (3,4).The pGLuc-Basic 2 Vector can be transfected into cells using any standard transfection protocol and stable cell lines can be established using Neomycin selection.Recommended Sequencing Primers for pGLuc-Basic 2 Vector (not available from NEB)Upstream of MCS: (23-mer)5´-GGGGTTCCGCGCACATTTCCCCG-3´(4917–4939)pBasic Reverse Primer (25-mer)5´-TCAGAAGCCATAGAGCCCACCGCAT-3´(785–761)GLuc 3´ End Forward Primer (20-mer)5´-GCCAGCAAGATCCAGGGCCA-3´(580–599)GLuc 5´ End Reverse Primer (24-mer)5´-TCAGGGCAAACAGAACTTTGACTC-3´(103–80)ApplicationsThe pGLuc-Basic 2 Vector can be used to test promoters by cloning promoter element of interest into the MCS upstream of the GLuc reporter gene. For constitutive expression of GLuc, vectors containing promoters are available (See Companion Products Sold Separately).GLuc can be used as a stand alone reporter or in conjunction with other compatible reporters such as Cypridina Luciferase (CLuc) (3). GLuc and CLuc are ideally suited for co-expression as both are secreted and highly active enzymes providing ease of use and sensitivity (3,4).HighlightsPolylinker MCS: 12–68GLuc coding: 76–633Start codon: 76–78Stop codon: 631–633Signal peptide: 76–126Synthetic poly-A site: 642–690Neo promoter (SV40): 1276–1611Neomycin resistance gene: 1663–2457Bacterial replication ori (pMB1): 3791–3203Amp resistance: 4822–3962All pGLuc 2 vectors and plasmids have improved polyadenylation-transcription termination of the luciferase transcript. The polyadenylation signal is a synthetic polyadenylation sequence based on the β-globin gene (5)