■ 基本信息

■ 質(zhì)粒屬性

■ 質(zhì)粒簡介

pDsRed2-C1編碼DsRed2,一種DsRed變體，其設(shè)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(xì)胞DsRed1，含有一系列沉默的堿基對變化,對應(yīng)于哺乳動物細(xì)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(dǎo)致轉(zhuǎn)染細(xì)胞系中紅色熒光的出現(xiàn)更加快速;和R2A，K5E和K9T，其阻止蛋白質(zhì)聚集。（DsRed2可能與DsRed1形成相同的四聚體結(jié)構(gòu)。）在DsRed2組成型表達的哺乳動物細(xì)胞培養(yǎng)物中，轉(zhuǎn)染后24小時內(nèi)可通過熒光顯微鏡檢測發(fā)紅細(xì)胞。在表達DsRed1的細(xì)菌和哺乳動物細(xì)胞系統(tǒng)中經(jīng)常觀察到的蛋白質(zhì)的大不溶性聚集體在表達DsRed2的生物體中顯著降低。更快成熟，更可溶性的紅色熒光蛋白也被宿主細(xì)胞耐受良好;用DsRed2轉(zhuǎn)染的哺乳動物細(xì)胞培養(yǎng)物在測試的這些細(xì)胞系中沒有顯示出降低的存活力的明顯跡象，表達DsRed2的細(xì)胞顯示與非轉(zhuǎn)染對照相同的形態(tài)和生長特征。     質(zhì)粒只保證關(guān)鍵序列正確，不保證表達效果。

pDsRed2-C1 encodes DsRed2, a DsRed variant that has been engineered for faster maturation and lower non-specifc aggregation. Derived from the Discosoma sp. red fuorescent 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 fuorescence 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 fuorescence 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 organisms expressing DsRed2. The faster-maturing, more soluble red fuorescent 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-C1 is positioned between the DsRed2 coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of DsRed2 if they are in the same reading frame as DsRed2 and there are no intervening stop codons. A Kozak consensus translation initiation site upstream of DsRed2 increases the translation efficiency in eukaryotic cells (4). SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector backbone also 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 ), consisting 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli.

pDsRed2-C1 can be used to construct fusions to the C-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-C1 so that it is in frame with the DsRed2 coding sequences, with no intervening in-frame stop codons. The recombinant DsRed2 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pDsRed2-C1 can also be used as a cotransfection marker; the unmodified vector will express DsRed2.

■ 質(zhì)粒圖譜

■ 質(zhì)粒序列

質(zhì)粒序列請下載：ZK434pDsRed2-C1哺乳熒光質(zhì)粒.txt