Part:BBa_K382002

pORE Expression Series Vector with BioBrick MCS, Basta resistance

Vector for transformation of plants. Cloning in E. Coli, transfer to agrobacteria, integrates in the plant genome. Kanamycin resistance in bacteria, confers Basta resistance in plants. Contains the pENTCUP2 promoter upstream of MCS for constitutive expression of gene of interest.

Confirmed functions:

• Kan resistance in bacteria: confirmed by transformation and colony growth of E. coli (DH5alpha) and Agrobacterium tumefaciens
• Basta resistance in plants: confirmed by selective germination of transformed Arabidopsis seeds on basta agar plates (See photos [http://2010.igem.org/Team:Harvard/results]).
  
  

Confirmation pending: Function of the ENTCUP2 promoter will be tested by checking for expression for several BioBrick parts in adult Arabidopsis plants. These parts include...

• miraculin (BBa_K382021)
• brazzein (BBa_K382052)
• GFP knock-down amiRNA (BBa_K382054)

Transformed seeds were plated on 5 micrograms/ml basta in Murashige and Skoog agar and we observed approximately 0.1% transgene expression and sprout survival.

Successfully transfected basta resistant Arabidopsis plants, such as the one shown below (brazzein BBa_K382052), will be tested for BioBrick expression once they have grown large enough for tissue sampling:

Download the annotated sequence file [http://openwetware.org/images/d/d2/V9.gb here]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 5326
  Illegal XbaI site found at 5299
  Illegal SpeI site found at 5378
  Illegal PstI site found at 5356
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5326
  Illegal NheI site found at 4766
  Illegal SpeI site found at 5378
  Illegal PstI site found at 5356
  Illegal NotI site found at 5341
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5326
  Illegal BglII site found at 5212
  Illegal BglII site found at 7771
  Illegal BamHI site found at 5312
  Illegal XhoI site found at 4790
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 5326
  Illegal XbaI site found at 5299
  Illegal SpeI site found at 5378
  Illegal PstI site found at 5356
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 5326
  Illegal XbaI site found at 5299
  Illegal SpeI site found at 5378
  Illegal PstI site found at 5356
  Illegal NgoMIV site found at 625
  Illegal NgoMIV site found at 749
  Illegal NgoMIV site found at 2181
  Illegal NgoMIV site found at 2772
  Illegal NgoMIV site found at 7220
  Illegal AgeI site found at 341
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 7241