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V011361
p3xFLAG-CMV-14
5 μg

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。

p3xFLAG-CMV-14 is a mammalian expression vector designed for the constitutive expression of a target protein in mammalian cells.
p3xFLAG-CMV-14 features the strong cytomegalovirus (CMV) promoter to drive high levels of gene expression. The multiple cloning site (MCS) contains three FLAG tags, allowing for easy detection and immunoprecipitation of the expressed protein. Additionally, the vector includes the SV40 origin of replication, enabling efficient replication in mammalian cells. The ampicillin resistance gene confers resistance to ampicillin, facilitating selection of transformed cells.
p3xFLAG-CMV-14 is commonly used in protein overexpression studies, subcellular localization analysis, and protein-protein interaction assays in mammalian cell culture systems.

载体名称:
p3xFLAG-CMV-14
载体抗性:
Ampicillin
载体长度:
6310 bp
载体类型:
Mammalian Expression Vectors
复制子:
ori
载体来源:
Sigma-Aldrich
筛选标记:
Neomycin/G418(Geneticin)
拷贝数:
High copy number
启动子:
CMV
克隆方法:
Enzyme digestion and ligation
5'引物:
M13 fwd
3'引物:
M13 rev
感受态:
DH10B
培养温度:
37℃

p3xFLAG-CMV-14 载体图谱

p3xFLAG-CMV-146310 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300M13 fwdCMV enhancerCMV promoterMCS3xFLAGhGH poly(A) signalSV40 promoterNeoR/KanRSV40 poly(A) signalT7 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。

p3xFLAG-CMV-14 载体序列

LOCUS       p3xFLAG-CMV-14.        6310 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector with a neomycin resistance marker for expression of 
            C-terminally 3xFLAG-tagged proteins in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    p3xFLAG-CMV-14
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6310)
  AUTHORS   Sigma-Aldrich
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6310)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6310
                     /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"
     misc_feature    924..990
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             993..1058
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
     polyA_signal    1069..1691
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     promoter        1733..2030
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2084..2875
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    3530..3664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3670..3688)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3702..3718)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3726..3742)
                     /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(3750..3780)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3795..3816)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4104..4692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4866..5723)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5724..5828)
                     /label=AmpR promoter
     rep_origin      5855..6310
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"