pCDNA3.1(-)是来自pCDNA3的5.4kb载体，设计用于哺乳动物宿主中的高水平稳定和瞬时表达。大多数哺乳动物细胞可以进行高水平的稳定和非复制性瞬时表达。人类巨细胞病毒立即早期（CMV）启动子，用于在广泛哺乳动物细胞中的高水平表达。前向（+）和反向（-）方向上的多个克隆位点，以促进克隆用于选择稳定细胞系的新霉素抗性基因。在潜伏感染SV40或表达SV40大T抗原（例如COS-1，COS-7）的细胞系中的异常复制。
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.