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- 详细信息
- 文献和实验
- 技术资料
- 保存条件:
-20℃
- 保质期:
6个月
- 英文名:
pEXP3-DEST
- 库存:
大量
- 供应商:
钰博生物
- 规格:
1ug/5ug
载体基本信息
| 出品公司: | Ybscience |
|---|---|
| 载体名称: | pEXP3-DEST |
| 质粒类型: | Gateway载体;无细胞表达载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 启动子: | T7 |
| 克隆方法: | Gateway |
| 载体大小: | 4607bp |
| 5' 测序引物及序列: | T7 Forward:TAATACGACTCACTATAGGG |
| 3' 测序引物及序列: | -- |
| 载体标签: | Lumio Tag;His Tag |
| 载体抗性: | 氨苄青霉素 |
| 真核筛选标记: | 无 |
| 备注: | -- |
| 稳定性: | -- |
| 组成型: | -- |
| 病毒/非病毒: | 非病毒 |
载体质粒图谱和多克隆位点信息



载体简介
pEXP3-DEST载体主要特点及功能 The Expressway Lumio Expression and Detection System takes advantage of the Lumio recognition sequence, a small, six amino acid sequence (Cys-Cys-Pro-Gly-Cys-Cys). The Lumio detection reagent binds the recognition sequence with high specificity and affinity, resulting in a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection. In addition, Expressway’s specialized E. coli lysate, derived from a slyD mutant, eliminates nonspecific binding of the Lumio Green Detection Reagent to the endogenous SlyD protein and provides optimal background for detection of recombinant proteins (Figure 1). The Lumio Green Detection Kit is included in the system for real-time detection of protein synthesis as well as easy in-gel protein detection. The Lumio vectors (Figures 2 and 3) also feature: attR sites for efficient recombination with any attL-flanked Gateway entry vector N-terminal Lumio tag (pEXP3-DEST vector) with TEV cleavage site for efficient removal of the Lumio sequence following purification C-terminal Lumio tag (pEXP4-DEST vector) for easy, and immediate in-gel detection T7 promoter, ribosome binding site, and T7 terminator optimally spaced for cell-free protein expression Lumio 检测技术 Expressway Tag-On-Demand Cell-Free E. coliExpression System with Lumio Technology enables convenient, in vitro expression of tagged or untagged protein from a single vector, real-time detection of protein synthesis, and easy in-gel protein detection. With this system, you can: Quickly screen for expression levels of full-length protein Save time and effort by cloning only once to express tagged or native protein Express a C-terminally tagged protein from an Ultimate ORF Clone in an in vitro system The Expressway Tag-On-Demand System includes a specialized E. colilysate derived from a slyD mutant for cell-free synthesis. This lysate eliminates non-specific binding of the Lumio Green Detection Reagent to the endogenous SlyD protein, providing an optimal background for recombinant protein detection. How it works Simply clone your gene of interest containing a TAG (amber) stop codon into the provided pEXP4-DEST (Figure 1). The pEXP4-DEST vector features attR sites for efficient Gateway recombination, C-terminal Lumio and 6xHis tags for simplified identification and purification, and components for cell-free protein synthesis. In the presence of the Expressway Tag-On-Demand Suppressor tRNA, the TAG codon is not recognized and translation continues through the Lumio and 6xHis tags. In the absence of the Suppressor tRNA, the TAG stop codon is recognized and native protein is produced (Figure 2). From the same vector, you can rapidly determine the expression level of the C-terminal-tagged, full-length protein, and then express the native protein free from any adverse affects the tag may have on its structure, activity, or molecular interactions.
载体序列
GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAA TAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCATCATCACCATCACCATGGTGCTGGTG GCTGTTGTCCTGGCTGTTGCGGTGGCGGCGAAAACCTGTATTTTCAGGGAATTATCACAAGTTTG TACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCA TAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGG CGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGAT ATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAA CCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTT ATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATG AAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAAC TGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATT CGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATG TTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGA CAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGC CGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAA TTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATCTAGAGGATCCGGCTTACTAAAAGC CAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATA CCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACA GCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGA ATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTC GCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTA AGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATT GACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTC CCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGG CCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATC AAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTC TGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGC AAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAA AGTGGTTCGATCGAAGCTTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTG CCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG CTGAAAGGAGGAACTATATCCGGATCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC CAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGG TGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT TTAGGGTTCCGATTTAGAGCTTTACGGCACCTCGACCGCAAAAAACTTGATTTGGGTGATGGTTC ACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTA ATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCGCGGTCTATTCTTTTGATTTA TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAATATTTAACGC GAATTTTAACAAAATATTAACGTTTACAATTTCGCCTGATGCGGTATTTTCTCCTTACGCATCTG TGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCA ATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTG CGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTT TCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTT GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGG AGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAG CTGAATGAAGCCATACCAAACGACGAGAGTGACACCACGATGCCTGTAGCAATGCCAACAACGTT GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGG AGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGAT AAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCG CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATA CTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGA TCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGG CTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGT GGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGAT ACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCT TTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG AAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCA
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文献和实验polIII型启动子和终止子序列,RNAi靶序列克隆进入后将在哺乳动物细胞中形成shRNA。目的载体(destinationvector)Gateway®适合的目的载体(DEST)包含attR位点,可以与入门克隆进行重组,构建表达载体。BLOCK-iT™RNAi慢病毒和腺病毒DEST载体接受来自U6入门载体的RNAi框。此外,也有在原核、酵母、昆虫或者哺乳动物系统表达天然蛋白或者融合蛋白的DEST载体可供选择。LRClonase™便于attL和attR位点在LxRGateway®重组反应中进行重组的一种酶
III型启动子和终止子序列,RNAi靶序列克隆进入后将在哺乳动物细胞中形成shRNA。 11、目的载体(destination vector) 适合的目的载体(DEST)包含attR位点,可以与入门克隆进行重组,构建表达载体。BLOCK-iT™ RNAi慢病毒和腺病毒 DEST载体接受来自U6入门载体的RNAi框。此外,也有在原核、酵母、昆虫或者哺乳动物系统表达天然蛋白或者融合蛋白的DEST载体可供选择。 12、LR Clonase 便于attL和 attR位点在LxR
用293FT细胞系和相关的ViraPower™ 试剂制备的高滴度的慢病毒储备液,进行有效的和可控制的导入。一旦制备好了慢病毒储备液,就可以转染细胞以观测RNAi反应(图9)。pLenti6/BLOCK-iT™-DEST 慢病毒 RNAi 载体( pLenti6/BLOCK-iT™-DEST Lentiviral RNAi vector)提供能够长期观察稳定RNAi效应的能力。这个载体包含包装U6RNAi框到病毒所需的全部的元件,因而你能够转染shRNA到正在分裂、难于转染和不分裂的细胞中(图10
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