出品公司: Clontech
载体名称: pNFkB-DD-AmCyan1
质粒类型: 哺乳动物细胞表达载体;荧光报告载体
高拷贝/低拷贝: 高拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: PTA
载体大小: 4734 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: --
载体抗性: 卡那霉素
筛选标记: 新霉素(Neomycin)
克隆菌株: DH5α, HB101
宿主细胞(系): 常规细胞系,293、CV-1、CHO等
备注: pNFkB-DD-AmCyan1载体含有NFkB增强子元件,用来检测NFkB在细胞内的活性;
DD-AmCyan1与AmCyan1相比,N端融合了ProteoTuner destabilization domain (DD)去稳定结构域,
产品目录号: 631083
稳定性: 瞬表达 或 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒



pNFkB-DD-AmCyan1 载体特征



pNFkB-DD-AmCyan1 is a reporter vector that allows you to monitor NFkB activation in mammalian cells. The vector contains an NFkB enhancer element (composed of four tandem copies of the NFkB consensus binding sequence; 1) upstream of a minimal TA promoter (PTA), which consists of the TATA box from the herpes simplex virus thymidine kinase (HSV-TK) promoter. The vector encodes the reporter protein DD-AmCyan1, a ligand-dependent, destabilized cyan fluorescent protein that minimizes background fluorescence from leaky promoters.
AmCyan1 is a human codon-optimized variant of the wild-type Anemonia majano cyan fluorescent protein (AmCyan) that exhibits enhanced emission characteristics (excitation and emission maxima: 458 and 489, respectively; 2, 3). DD-AmCyan1 is a modified version of AmCyan1 that is tagged on its N-terminus with the ProteoTuner destabilization domain (DD; 4). The presence of this destabilization domain causes rapid, proteasomal degradation of the fluorescent fusion protein; however, when the membrane permeant ligand Shield1 is added to the medium, it binds to the destabilization domain and protects the fusion protein from degradation.
In the absence of Shield1, the destabilization domain causes the degradation of any DD-AmCyan1 reporter protein produced prior to promoter activation, thus reducing background fluorescence. In order to analyze NFκB activation, an inducer of choice is added to the medium along with the Shield1 stabilizing ligand, which effectively stabilizes the reporter protein, allowing it to accumulate. As a result, only the reporter molecules expressed during promoter induction will contribute to the fluorescence signal, providing a considerably higher signal-to-noise ratio than that obtained with non-destabilized or constitutively destabilized reporter systems. The high signal-to-noise ratio also allows the monitoring of NFκB activation during discrete windows of time when Shield1 is added to the cell medium for discrete periods of time.

The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large 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 (5). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
The pNFkB-DD-AmCyan1 Reporter vector, available as part of the NFkB DD Cyan1 Reporter System (Cat. No. 631083), can be used to monitor NFkB activation in live cells as well as in vivo. pNFkB-DD-AmCyan1 Reporter can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418.

Propagation in E. coli
 Recommended host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production
requires a host containing an F plasmid such as JM109 or XL1-Blue.
 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
 E. coli replication origin: pUC
 Copy number: high
 Plasmid incompatibility group: pMB1/ColE1

Excitation and emission maxima of AmCyan1
 Excitation maximum = 458 nm
 Emission maximum = 489 nm









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