pCDNA3.1(-)质粒

基本信息
启动子: CMV
复制子: pUC ori，F1 ori
终止子: BGH poly（A）signal
质粒分类: 哺乳细胞真核载体；蛋白过表达质粒
质粒大小: 5427bp
原核抗性: 氨苄青霉素(Ampicillin) 筛选标记: 新霉素(Neomycin)/遗传霉素(Geneticin/G418)
克隆菌株: DH5α等大肠杆菌
培养条件: 37℃，有氧，LB
表达宿主: 293T等哺乳细胞
5'测序引物: pcDNA3.1-F:CTAGAGAACCCACTGCTTAC
3'测序引物: pcDNA3.1-R (BGH-R):TAGAAGGCACAGTCGAGG
备注: 需要添加起始密码子ATG
质粒简介
pCDNA 3.1(-) is 5.4 kb vector derived from pcDN3 and designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells Multiple cloning sites in the forward (+) and reverse (C) orientations to facilitate cloning Neomycin resistance gene for selection of stable cell lines Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7) The control plasmid, pcDNA3.1/CAT, is included for use as a positive control for transfection and expression in the cell line of choice. Use the following outline to clone and express your gene of interest in pcDNA 3.1. 1. Design the multiple cloning 2. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on LB plates containing 100 g/ml ampicillin 3. Analyze your transformants for the presence of insert by restriction digestion. 4. Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation. 5. Transfect your construct into the mammalian cell line of interest using your own method of choice. Generate a stable cell line, if desired. 6. Test for expression of your recombinant gene by western blot analysis or functional assay.