3xFLAG-dCas9/pMXs-neo 载体 (V012352)

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。

所有的产品都严格仅供科学研究使用,不能应用于临床试验,包括人体摄入、注射或外用的药理学用途。

确保质粒的关键元件正确,但是我们并不能保证实验结果。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。

载体名称:
3xFLAG-dCas9/pMXs-neo
载体抗性:
Ampicillin
载体长度:
10444 bp
载体类型:
CRISPR Plasmids
复制子:
ori
载体来源:
Fujita T, Fujii H.
筛选标记:
Neomycin/G418(Geneticin)
拷贝数:
High copy number

3xFLAG-dCas9/pMXs-neo 载体图谱

3xFLAG-dCas9/pMXs-neo10444 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000long terminal repeat from Moloney murine leukemiavirusMMLV Psigag (truncated)pol regionATG3xFLAGdCas9SV40 NLSSV40 promoterNeoR/KanRLTRoriAmpRAmpR promoter

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。

3xFLAG-dCas9/pMXs-neo 载体序列

LOCUS       3xFLAG-dCas9_pMX       10444 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Retroviral vector with a G418 resistance marker, encoding 
            FLAG(R)-tagged dCas9 for engineered ChIP (enChIP) purification of 
            specific genomic regions.
ACCESSION   .
VERSION     .
KEYWORDS    3xFLAG-dCas9 pMXs-neo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10444)
  AUTHORS   Fujita T, Fujii H.
  TITLE     Identification of proteins associated with an IFN-gamma-responsive 
            promoter by a retroviral expression system for enChIP using CRISPR.
  JOURNAL   PLoS ONE 2014;9:e103084.
  PUBMED    25051498
REFERENCE   2  (bases 1 to 10444)
  AUTHORS   Fujii Lab / Addgene #51260
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10444)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS ONE"; 
            date: "2014"; volume: "9"; pages: "e103084"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             22..615
                     /note="long terminal repeat from Moloney murine leukemia
                     virus"
     misc_feature    678..1035
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus
                     (MMLV)"
     CDS             1094..1510
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
     misc_feature    1520..1894
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     CDS             1948..1950
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             1951..2016
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
     CDS             2020..6123
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             6136..6156
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     promoter        6341..6670
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             7027..7818
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     LTR             7889..8482
                     /label=LTR
                     /note="long terminal repeat from Moloney murine leukemia
                     virus"
     rep_origin      complement(8719..9307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9474..10331)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(10332..10436)
                     /label=AmpR promoter