3xFLAG-dCas9/pCMV-7.1 载体 (V012353)

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/pCMV-7.1
载体抗性:
Ampicillin
载体长度:
8848 bp
载体类型:
CRISPR Plasmids
复制子:
ori
载体来源:
Fujita T, Fujii H.
拷贝数:
High copy number
启动子:
SV40

3xFLAG-dCas9/pCMV-7.1 载体载体图谱

3xFLAG-dCas9/pCMV-7.18848 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800M13 fwdCMV enhancerCMV promoterATG3xFLAGdCas9SV40 NLShGH poly(A) signalSV40 promoterT7 promoterM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterf1 ori

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/pCMV-7.1 载体载体序列

LOCUS       3xFLAG-dCas9_pCM        8848 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expression in mammalian cells of FLAG(R)-tagged 
            catalytically inactive dCas9, for engineered ChIP (enChIP) 
            purification of specific genomic regions.
ACCESSION   .
VERSION     .
KEYWORDS    3xFLAG-dCas9 pCMV-7.1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8848)
  AUTHORS   Fujita T, Fujii H.
  TITLE     Efficient isolation of specific genomic regions and identification 
            of associated proteins by engineered DNA-binding molecule-mediated 
            chromatin immunoprecipitation (enChIP) using CRISPR.
  JOURNAL   Biochem. Biophys. Res. Commun. 2013;439:132-6.
  PUBMED    23942116
REFERENCE   2  (bases 1 to 8848)
  AUTHORS   Fujii Lab / Addgene #47948
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8848)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biochem. 
            Biophys. Res. Commun."; date: "2013"; volume: "439"; pages: "132-6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             928..930
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             931..996
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
     CDS             1000..5103
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5116..5136
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    5189..5811
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     promoter        5840..6169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(6208..6226)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6240..6256)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6264..6280)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6288..6318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6333..6354)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6642..7230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7404..8261)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8262..8366)
                     /label=AmpR promoter
     rep_origin      8393..8848
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"