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pSKI015植物激活标记技术activation tagging载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

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  • 货  号:pSKI015植物激活标记技术activation tagging载体
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pSKI015植物激活标记技术activation tagging载体


Activation tagging激活标记技术是植物基因克隆与基因功能鉴定的一种重要方法,通过构建功能获得突变体库实现。
激活标记技术
具体操作是将多个拷贝的增强子序列构建到T-DNA载体后,将T-DNA插入到植物基因组中,增强子会激活邻近上下游基因的表达,产生激活标记突变;或者将增强子或启动子序列构建到转座子元件(如玉米Ds)后,通过转座酶作用将其随机插入到植物基因组上,增强子或启动子会激活临近基因表达从而获得突变体。


Map图谱

The CaMV 35S enhancers in these vectors correspond to nucleotides -417 to -86 relative to the transcription start.

Recommended Agrobacterium strain is GV3101 pMP90RK. Host and helper plasmid markers are kanamycin, gentamycin and rifampicin resistance.

Plasmid selection in E. coli and in A. tumefaciens is ampicillin resistance.

Sites that leave the pBstKS+ sequences intact and cut only on one side between pBstKS+ and either left or right border can be used for plasmid rescue. Note that there are additional BssS1 sites not shown on the map.

The four enhancer repeats in the construct may be unstable in E. coli and Agrobacterium if stored at +4'C for extended time. Check by PCR with T7 (or M13-20) oligo and this one derived from the RB sequence: 5' acc cgc caa tat atc ctg 3'. This should give 1.4 kb band. Note that these oligos will not work in a transgenic plant, as the RB sequence is not transfered. Using this test, we found that after 1-2 weeks in a fridge the Agrobacterium strain loses on average one copy of a repeat, and after a month in a fridge there is only one copy left. It is a good idea to use a freshly streaked colony from a good -70'C glycerol stock for every infiltration.


  • Vector backbone
    pPCVICEn4HPT

  • Backbone manufacturer
    Hayashi et al, 1992
  • Backbone size w/o insert (bp)10138

  • Vector type
    Plant Expression ; Activation tagging
  • Selectable markers
    Basta

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Growth instructions
    Plasmid grow in standard E. coli at 37 degrees, but the four enhancer repeats may be unstable in E. coli and A. tumefaciens if stored at 4'C for extended time. See comments.
  • Copy number
    High Copy

  • Gene/Insert name
    CaMV 35S enhancers
  • Species
    Cauliflower mosaic virus
  • GenBank ID
    AF187951

  • Cloning methodRestriction Enzyme

  • 5′ cloning siteSee map and paper. (not destroyed)

  • 3′ cloning siteSee map and paper. (not destroyed)

  • 5′ sequencing primerT7


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