■ 基本信息

■ 質粒屬性

■ 質粒簡介

pDsRed2-N1編碼DsRed2，是一種DsRed變體，其設計用于更快的成熟和較低的非特異性聚集。衍生自Discosoma sp。紅色熒光蛋白DsRed2，如其祖細胞DsRed1，含有一系列沉默的堿基對變化，對應于哺乳動物細胞中高表達的人類密碼子使用偏好。除了這些變化外，DsRed2含有六個氨基酸取代：V105A，I161T和S197A，導致轉染細胞系中紅色熒光的出現更加快速;和R2A，K5E和K9T，其阻止蛋白質聚集。（DsRed2可能與DsRed1形成相同的四聚體結構。）在DsRed2組成型表達的哺乳動物細胞培養(yǎng)物中，轉染后24小時內可通過熒光顯微鏡檢測發(fā)紅細胞。在表達DsRed1的細胞和哺乳動物細胞系統(tǒng)中經常觀察到的蛋白質的大不溶性聚集體在表達DsRed2的細胞中顯著降低。更快成熟，更可溶性的紅色熒光蛋白也被宿主細胞耐受良好;用DsRed2轉染的哺乳動物細胞培養(yǎng)物沒有顯示生存力降低的明顯跡象，在測試的那些細胞系中，表達DsRed2的細胞顯示與非轉染對照相同的形態(tài)（例如粘附，光折射）和生長特征。

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).

 質粒只保證關鍵序列正確，不保證表達效果。

■ 質粒圖譜

■ 質粒序列

質粒序列請下載：ZK433pDsRed2-N1哺乳熒光質粒.txt