■ 基本信息

■ 质粒属性

■ 质粒简介

pDsRed2-N1编码DsRed2，是一种DsRed变体，其设计用于更快的成熟和较低的非特异性聚集。衍生自Discosoma sp。红色荧光蛋白DsRed2，如其祖细胞DsRed1，含有一系列沉默的碱基对变化，对应于哺乳动物细胞中高表达的人类密码子使用偏好。除了这些变化外，DsRed2含有六个氨基酸取代：V105A，I161T和S197A，导致转染细胞系中红色荧光的出现更加快速;和R2A，K5E和K9T，其阻止蛋白质聚集。（DsRed2可能与DsRed1形成相同的四聚体结构。）在DsRed2组成型表达的哺乳动物细胞培养物中，转染后24小时内可通过荧光显微镜检测发红细胞。在表达DsRed1的细胞和哺乳动物细胞系统中经常观察到的蛋白质的大不溶性聚集体在表达DsRed2的细胞中显着降低。更快成熟，更可溶性的红色荧光蛋白也被宿主细胞耐受良好;用DsRed2转染的哺乳动物细胞培养物没有显示生存力降低的明显迹象，在测试的那些细胞系中，表达DsRed2的细胞显示与非转染对照相同的形态（例如粘附，光折射）和生长特征。

pDsRed2-N1 encodes DsRed2, a DsRed variant that has been engineered for faster maturation and lower non-specific aggregation. Derived from the Discosoma sp. red fluorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls.

The multiple cloning site (MCS) in pDsRed2-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed2 coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed2. Sequences upstream of DsRed2 have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). SV40 polyadenylation signals  ownstream of the DsRed2 gene direct proper processing of the 3' end of the DsRed2 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette confers kanamycin resistance to E. coli.

pDsRed2-N1 can be used to construct fusions to the N-terminus of DsRed2. If a fusion construct retains the fluorescent properties of the native DsRed2 protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed2-N1 so that it is in frame with the DsRed2 coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed2-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). Unmodified pDsRed2-N1 can also be used to express DsRed2 in a cell line of interest (e.g., for use as a transfection marker).

 质粒只保证关键序列正确，不保证表达效果。

■ 质粒图谱

■ 质粒序列

质粒序列请下载：ZK433pDsRed2-N1哺乳荧光质粒.txt