Note: A plasmid designed for the expression of a human codon-optimized SpCas9 protein along with a chimeric guide RNA.
基本信息
- 载体名称:
- pX330
- 载体抗性:
- Ampicillin
- 载体长度:
- 8504 bp
- 载体类型:
- Mammalian Expression Vectors
- 复制子:
- ori
- 载体来源:
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X,
- 拷贝数:
- High copy number
- 启动子:
- U6
- 培养温度:
- 37℃
产品信息
质粒编号:V011044
质粒名称:pX330
规格:5 μg (冻干粉)
下载资源
我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
确保质粒的关键元件正确，但是我们并不能保证实验效果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
pX330 质粒 (编号: V011044)序列
LOCUS pX330 8504 bp DNA circular SYN 20-JUL-2025
DEFINITION Zhang lab plasmid for expressing a chimeric guide RNA (gRNA)
together with human codon-optimized Cas9. Also known as
pX330-U6-Chimeric_BB-CBh-hSpCas9.
ACCESSION .
VERSION .
KEYWORDS pX330
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8504)
AUTHORS Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X,
Jiang W, Marraffini LA, Zhang F.
TITLE Multiplex genome engineering using CRISPR/Cas systems.
JOURNAL Science 2013;339:819-23.
PUBMED 23287718
REFERENCE 2 (bases 1 to 8504)
AUTHORS Zhang Lab / Addgene #42230
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8504)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8504)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science";
date: "2013"; volume: "339"; pages: "819-23"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT Digest with BbsI to insert annealed oligos encoding the guide
sequence.
NGS sequence provided by Addgene.
FEATURES Location/Qualifiers
source 1..8504
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 51..70
/mol_type="other DNA"
/organism="synthetic DNA construct"
intron 207..434
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 452..454
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 455..520
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 527..547
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=nuclear localization signal of SV40 large T
ant
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 572..4672
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 4673..4720
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 4754..4961
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4970..5110
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5185..5640
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 5922..6026
/label=AmpR promoter
CDS 6027..6884
/label=AmpR
/note="beta-lactamase"
rep_origin 7058..7646
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 7708..7948
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 7975..8050
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 8051..8056
/note="pol III terminator"
/note="RNA polymerase III transcription terminator"
enhancer 8147..8432
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"