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pTet-Off-Advanced 载体 (V011072)

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。

In the Tet-Off expression system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the strong transactivating domain(sequence: PTDALDDFDLDML) of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter. In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive. The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change(E19G, A56P, D148E, H179R) in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

载体名称:
pTet-Off-Advanced
载体抗性:
Ampicillin
载体长度:
7140 bp
载体类型:
Mammalian Expression Vectors
复制子:
ori
载体来源:
Clontech
筛选标记:
Neomycin/G418(Geneticin)
拷贝数:
High copy number
启动子:
SV40

pTet-Off-Advanced 载体图谱

pTet-Off-Advanced7140 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900CMV enhancerCMV promotertTA-AdvancedSV40 poly(A) signaloriAmpRAmpR promoterSV40 poly(A) signalSV40 NLSsmall t intronNeoR/KanRSV40 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。

pTet-Off-Advanced 载体序列

LOCUS       pTet-Off-Advance        7140 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector for expressing the tTA-Advanced transactivator in the 
            Tet-Off(R) Advanced system.
ACCESSION   .
VERSION     .
KEYWORDS    pTet-Off-Advanced
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7140)
  AUTHORS   Clontech
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7140)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        89..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             775..1518
                     /label=tTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
     polyA_signal    1531..1665
                     /label=SV40 poly(A) signal
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2385..2973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3147..4004)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4005..4109)
                     /label=AmpR promoter
     polyA_signal    complement(4222..4356)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4781..4801)
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     intron          complement(4931..4996)
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5419..6210)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        complement(6571..6900)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"