pSKI015
(Plasmid #11570)

• Depositing Lab: Detlef Weigel
• Publication: Weigel et al Plant Physiol. 2000 Apr . 122(4):1003-13.

Backbone

• 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

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• 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

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

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: See map and paper. (not destroyed)
• 3′ cloning site: See map and paper. (not destroyed)
• 5′ sequencing primer: T7

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Max Planck Institute

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.