■ 基本信息
別名: | Plasmid #42876 |
復(fù)制子: | p15A ori |
質(zhì)粒分類: | 大腸系列質(zhì)粒；大腸編輯質(zhì)粒；大腸Cas9質(zhì)粒 |
質(zhì)粒大小: | 9326bp |
原核抗性: | Chl |
克隆菌株: | Stbl3 |
培養(yǎng)條件: | 37℃ |
表達(dá)宿主: | 大腸桿菌 |
誘導(dǎo)方式: | 無需誘導(dǎo) |
5'測序引物: | StrepPyoCas9-5UTR-F（CGGTGCCACTTTTTCAAGTTG） |
3'測序引物: | pBRrevBam（GGTGATGTCGGCGATATAGG） |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 大腸桿菌 |
質(zhì)粒用途: | 基因編輯 |
片段類型: |
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片段物種: |
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原核抗性: | 氯霉素 |
真核抗性: |
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熒光標(biāo)記: |
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■ 質(zhì)粒簡介
pCas9質(zhì)粒是CRISPR/Cas9系統(tǒng)類載體，復(fù)制子是p15A ori和ori，質(zhì)粒大小是9326bp，可轉(zhuǎn)化進(jìn)相應(yīng)的宿主細(xì)胞中，通大量養(yǎng)條件是LB，37℃。
pCas9 can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-baseddirected evolution, and combinatorial design. CRISPR-Cas9 nucleases enable efficient genome editing in a wide variety of organisms and cell types . Target site recognition by Cas9 is programmed by a chimeric single guide RNA (sgRNA) that encodes a sequence complementary to a target protospacer5 , but also requires recognition of a short neighboring PAM . SpCas9, the most robust and widely used Cas9 to date, primarily recognizes NGG PAMs and is consequently restricted to sites that contain this motif . It can therefore be challenging to implement genome editing applications that require precision, such as: homology-directed repair (HDR), which is most efficient when DSBs are placed within 10–20 bps of a desired alteration; the introduction of variable-length insertion or deletion (indel) mutations into small size genetic elements such as microRNAs, splice sites, short open reading frames, or transcription factor binding sites by non-homologous end-joining (NHEJ); and allele-specific editing, where PAM recognition might be exploited to differentiate alleles.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請下載：
ZK1672 pCas9大腸敲除質(zhì)粒.txt
