pECFP-N1哺乳荧光质粒

pECFP-N1哺乳荧光质粒

基本信息

启动子: CMV promoter

复制子: pUC ori,f1 ori

终止子: SV40 poly(A) signal

质粒分类: 哺乳系列质粒；哺乳荧光质粒；哺乳青色质粒

质粒大小: 4733bp

质粒标签: C-ECFP

原核抗性: 卡那霉素Kan（50μg/ml）

筛选标记: 新霉素Neo/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

培养条件: 37℃，5%CO2

诱导方式: 无须诱导，瞬时表达

5'测序引物: CMV-F（CGCAAATGGGCGGTAGGCGTG）

3'测序引物: Sv40-polyA-R（GAAATTTGTGATGCTATTGC）

质粒简介

        pECFP-N1编码Aequorea维多利亚绿色荧光蛋白基因（GFP）的增强的青色荧光变体。ECFP基因含有6个氨基酸取代基。 Tyr-66对Trpsubstitution给出ECFP荧光激发（433nm处的主峰和453nm处的次峰）和与其他青色发射变体类似的发射（475nm处的主峰和501nm的次峰）。其他五个取代（Phe-64至Leu; Ser-65至Thr; Asn-146至Ile; Met-153至Thr;和Val-163至Ala）增强了蛋白质的亮度和溶解度，主要是由于改进的蛋白质折叠性质和生色团形成的效率。除了发色团突变外，ECFP含有> 190个沉默突变，创造出几乎完全是首选人类密码子的开放阅读框架。此外，ECFP侧翼的上游序列已转化为Kozak一致翻译起始位点。这些变化增加了ECFP mRNA的翻译效率，从而增加了哺乳动物和植物细胞中ECFP的表达。     

       pECFP-N1 encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp substitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emission variants (1, 2). The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153 to Thr; and Val-163 to Ala) enhance the brightness and solubility of the protein, primarily due to improved protein folding properties and efficiency of chromophore formation (1, 3, 4). In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (5, 6). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (7). These changes increase the translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian and plant cells.

       The vector contains an SV40 origin of replication and a neomycin resistance (Neor ) gene for selection (using G418) in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. The MCS in pECFP-N1 is between the immediate early promoter of CMV (PCMV IE) and the ECFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of ECFP if they are in the same reading frame as ECFP and there are no intervening stop codons. The inserted gene should include an initiating ATG codon. ECFP fusion proteins retain the fluorescent properties of the native protein in vivo.

       The recombinant ECFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (8). pECFP-N1 can also be used simply to express ECFP in a cell line of interest (e.g., as a transfection marker). ECFP can be used for double-labeling experiments together with EYFP using standard fluorescence microscopy and the appropriate filter sets.

质粒图谱

pECFP-N1哺乳荧光质粒使用说明：

     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。

pECFP-N1哺乳荧光质粒注意事项：

      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。