pKillerRed-Mem细胞膜定位质粒

基本信息

启动子: CMV promoter

复制子: Ori，F1 ori，SV40 ori

终止子: SV40 poly(A) signal

质粒分类: 哺乳细胞质粒；哺乳报告质粒；荧光定位质粒

质粒大小: 4787bp

质粒标签: C-mem，C-KillerRed

原核抗性: 卡那霉素Kan

筛选标记: 新霉素Neo/G418

克隆菌株: 大肠杆菌DH5α

培养条件: 37℃，有氧，LB

表达宿主: 293T等哺乳细胞

质粒简介

          pKillerRed-mem质粒是一个哺乳细胞红色荧光定位于细胞膜的质粒。pKillerRed-mem is a mammalian expression vector encoding membrane-targeted KillerRed. KillerRed localized on cellular membrane can be used for effective light-induced cell killing.KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al. 1996]. Membrane localization signal (MLS) of neuromodulin is linked to the KillerRed N-terminus. The MLS (Nterminal 20 amino acid residues of neuromodulin) contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets KillerRed to cellular membranes [Skene and Virág 1989]. pKillerRed-mem vector can be used as a source of MLS-KillerRed hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3’-end of the reporter mRNA. SV40 early promoter (PSV40) provides neomycin resistance gene (Neor ) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr ) in E. coli. Kanr /Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.pKillerRed-mem vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of memrane-targeted KillerRed in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman 1985].Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.

质粒图谱

pKillerRed-Mem细胞膜定位质粒使用说明：

     1、收到质粒干粉后请先5000rpm离心1min，再加入20μl无菌水溶解质粒，室温放置1min；

     2、从-80℃冰箱中取出相应的感受态，置于冰盒上解冻，并做好标记；

     3、取2μl质粒加至100μl感受态中，冰浴30min；

     4、42℃热激90s，再冰浴2min；

     5、加入900μl无抗的LB液体培养基，180rpm震荡培养45min；

     6、6000rpm离心5min，仅留100ul上清混匀菌体沉淀；

     7、混匀后的菌液加至对应抗性的LB平板上，倒入适量玻璃珠，涂匀液体；

     8、将平板正向培养1h，再倒置培养12h~16h；

     9、挑取单克隆菌落至对应抗性的LB液体培养基中，震荡培养12h~16h，根据实验需要提取质粒。

pKillerRed-Mem细胞膜定位质粒注意事项：

      1、如果您收到的是甘油菌种，请先四区划线，挑取单克隆培养。

      2、如果第二天转化平板长的过多，请将质粒按比例稀释后再转化。