■ 基本信息

■ 质粒属性

■ 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

■ 质粒序列

质粒序列请下载：ZK459pECFP-N1哺乳荧光质粒.txt