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平臺編號：bio-109457
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質(zhì)粒信息： 宿主：BL21
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規(guī)格：2ml甘油菌 20ul質(zhì)粒
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用途：酵母細胞質(zhì)粒；酵母雜交質(zhì)粒
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服務費用：
加載中…… - 訂購
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注意事項：僅用于科學研究或者工業(yè)應用等非醫(yī)療目的不可用于人類或動物的臨床診斷或治療，非藥用，非食用（產(chǎn)品信息以出庫為準）
pGADT7-Rec2
| 培養(yǎng)條件 | LB,37℃，氨芐 |
| 啟動子 | ADH1 |
| 復制子 | 2U |
| 質(zhì)粒分類 | 酵母系列，酵母單雜交載體 |
| 質(zhì)粒大小 | 7.6kb |
| 原核抗性 | Amp |
| 篩選標記 | LEU2 |
| 克隆菌株 | DH5a |
| 表達宿主 | 酵母細胞 |
| 5'測序引物 | T7:TAATACGACTCACTATAGGG |
| 3'測序引物 | 根據(jù)序列設計引物 |
| 載體宿主 | 酵母菌 |
| 載體用途 | 雜交 |
| 原核抗性 | Amp |
| 真核抗性 | LEU2 |
| 質(zhì)粒簡介 | pGADT7-Rec2 is a cloning vector that can be used in yeast to express a protein of interest as a GAL4 activation domain (GAL4 AD) fusion. Transcription starts with the constitutive ADH1 promoter (PADH1) and ends with the ADH1 termination signal (TADH1). The GAL4 AD sequence includes the SV40 nuclear localization signal (SV40 NLS; 1) so that fusions translocate to the yeast nucleus. GAL4 AD fusions also contain a hemagglutinin (HA) epitope tag. The T7 promoter in pGADT7-Rec2 can be used for in vitro transcription and translation of the HA-tagged fusion protein. It also provides a binding site for sequencing with the T7 Promoter Sequencing Primer. pGADT7-Rec2 is a shuttle vector; it can be maintained in both yeast and bacteria. It contains an autonomous replication sequence (ARS4) and a LEU2 nutritional marker for replication and selection in yeast (2, 3). A centromeric sequence (CEN6) is included to ensure proper segregation of the plasmid during cell division (2, 3). For propagation and selection in E. coli, the vector contains a pUC origin of replication (pUC ori) and an ampicillin resistance gene (Ampr). pGADT7-Rec2 is derived from pGADT7-Rec, a cloning vector used in the Matchmaker (Two-Hybrid) Library Construction & Screening Kit (Cat. No. 630445). It was constructed by replacing the 2μ ori in pGADT7-Rec with the ARS4 and CEN6 elements. The ARS and CEN elements ensure stable, low-copy propagation of the vector. Unlike pGADT7-Rec, which is able to replicate multiple times during the yeast cell cycle, pGADT7-Rec2, with its ARS and CEN elements, can replicate only once during the cell cycle, so its copy number is restricted. Low-copy plasmids such as pGADT7-Rec2 are preferred for one-hybrid screening because they generate fewer false positives. In the original Matchmaker One-Hybrid System, copy number was restricted not by using low-copy, autonomously replicating plasmids, but by integrating the reporter construct into the yeast genome. With the development of pGADT7-Rec2 (and pHIS2, a low-copy reporter vector), integration is no longer necessary because each plasmid, with its CEN and ARS elements, now behaves like a minichromosome, both mitotically and meiotically. |
