■ 基本信息
啟動子: | CMV |
復(fù)制子: | pUC |
終止子: | SV40 poly(A) signal |
質(zhì)粒分類: | 哺乳系列質(zhì)粒；哺乳熒光質(zhì)粒；哺乳青色質(zhì)粒 |
質(zhì)粒大小: | 4733bp |
質(zhì)粒標(biāo)簽: | C-ECFP |
原核抗性: | Kan |
真核抗性: | G418 |
克隆菌株: | DH5a |
培養(yǎng)條件: | 37℃，5%CO2 |
表達(dá)宿主: | 293T等哺乳細(xì)胞 |
誘導(dǎo)方式: | 無須誘導(dǎo)，瞬時表達(dá) |
5'測序引物: | CMV-F（CGCAAATGGGCGGTAGGCGTG） |
3'測序引物: | Sv40-polyA-R（GAAATTTGTGATGCTATTGC） |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細(xì)胞 |
質(zhì)粒用途: | 蛋白表達(dá) |
片段類型: | ORF
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片段物種: |
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原核抗性: | Kan |
真核抗性: | G418
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熒光標(biāo)記: |
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■ 質(zhì)粒簡介
pECFP-N1編碼Aequorea維多利亞綠色熒光蛋白基因（GFP）的增強(qiáng)的青色熒光變體。ECFP基因含有6個氨基酸取代基。 Tyr-66對Trpsubstitution給出ECFP熒光激發(fā)（433nm處的主峰和453nm處的次峰）和與其他青色發(fā)射變體類似的發(fā)射（475nm處的主峰和501nm的次峰）。其他五個取代（Phe-64至Leu; Ser-65至Thr; Asn-146至Ile; Met-153至Thr;和Val-163至Ala）增強(qiáng)了蛋白質(zhì)的亮度和溶解度，主要是由于改進(jìn)的蛋白質(zhì)折疊性質(zhì)和生色團(tuán)形成的效率。除了發(fā)色團(tuán)突變外，ECFP含有> 190個沉默突變，創(chuàng)造出幾乎完全是首選人類密碼子的開放閱讀框架。此外，ECFP側(cè)翼的上游序列已轉(zhuǎn)化為Kozak一致翻譯起始位點(diǎn)。這些變化增加了ECFP mRNA的翻譯效率，從而增加了哺乳動物和植物細(xì)胞中ECFP的表達(dá)。
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.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請下載：
ZK459pECFP-N1哺乳熒光質(zhì)粒.txt
