■ 基本信息
別名: | pCDNA3.1- |
啟動(dòng)子: | CMV |
復制子: | pUC |
終止子: | BGH poly（A）signal |
質(zhì)粒分類(lèi): | 哺乳系列質(zhì)粒；哺乳表達質(zhì)粒；pCDNA系列質(zhì)粒 |
質(zhì)粒大小: | 5427bp |
原核抗性: | Amp |
真核抗性: | G418 |
克隆菌株: | DH5a |
培養條件: | 37℃，5%CO2 |
表達宿主: | 293T等哺乳細胞 |
誘導方式: | 無(wú)需誘導 |
5'測序引物: | pCDNA3.1-F（CTAGAGAACCCACTGCTTAC） |
3'測序引物
| pCDNA3.1-R（TAGAAGGCACAGTCGAGG）
|
備注: | 需要添加起始密碼子ATG |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細胞 |
質(zhì)粒用途: | 蛋白表達 |
片段類(lèi)型: | ORF
|
片段物種: | 空載體
|
原核抗性: | Amp |
真核抗性: | G418
|
熒光標記: |
|
■ 質(zhì)粒簡(jiǎn)介
pCDNA3.1(-)是來(lái)自pCDNA3的5.4kb載體，設計用于哺乳動(dòng)物宿主中的高水平穩定和瞬時(shí)表達。大多數哺乳動(dòng)物細胞可以進(jìn)行高水平的穩定和非復制性瞬時(shí)表達。人類(lèi)巨細胞病毒立即早期（CMV）啟動(dòng)子，用于在廣泛哺乳動(dòng)物細胞中的高水平表達。前向（+）和反向（-）方向上的多個(gè)克隆位點(diǎn)，以促進(jìn)克隆用于選擇穩定細胞系的新霉素抗性基因。在潛伏感染SV40或表達SV40大T抗原（例如COS-1，COS-7）的細胞系中的異常復制。 質(zhì)粒只保證關(guān)鍵序列正確，不保證表達效果。
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.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請下載：
ZK346pCDNA3.1(-)哺乳表達質(zhì)粒.txt
