Part:BBa_K382003

pORE Expression Series Vector with BioBrick MCS (Kan reistance)

Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has the constitutive promoter pENTCUP2 upstream of multiple cloning site for easy expression of genes in plants. Conforms to Silver BioBrick Assembly Standard 21.

Confirmed functions:

• Kan resistance in bacteria: confirmed by transformation and colony growth of E. coli (DH5alpha) and Agrobacterium tumefaciens
• Kan resistance in plants: confirmed by selective germination of transformed Arabidopsis seeds on kanamycin 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...

• LTP knock-down amiRNA and ihpRNA (BBa_K382065, BBa_K382071)
• Ger3 knock-down ihpRNA (BBa_K382067)
• BetVI knock-down ihpRNA (BBa_K382069)
• LUT2 knock-down amiRNA (BBa_K382061)
• LYC knock-down amiRNA (BBa_K382063)
• Beta Ohase I knock-down amiRNA (BBa_K382062)

Transformed seeds were plated on 50 micrograms/ml kanamycin in Murashige and Skoog agar and we observed approximately 0.1% transgene expression and sprout survival.

Successfully transfected kanamycin resistant Arabidopsis plants, such as the ones shown below, will be tested for BioBrick expression once they have grown large enough for tissue sampling. Shown below are plants transformed with LUT2 BBa_K382061 ("C1") and Beta Ohase I knock-down amiRNA BBa_K382062 ("C2"):

Download the annotated sequence file [http://openwetware.org/images/3/33/V10.gb here]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 5269
  Illegal XbaI site found at 5284
  Illegal SpeI site found at 5298
  Illegal PstI site found at 5312
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5269
  Illegal SpeI site found at 5298
  Illegal PstI site found at 5312
  Illegal NotI site found at 5275
  Illegal NotI site found at 5305
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5269
  Illegal BglII site found at 5182
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 5269
  Illegal suffix found in sequence at 5298
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 5269
  Illegal XbaI site found at 5284
  Illegal SpeI site found at 5298
  Illegal PstI site found at 5312
  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 7160
  Illegal NgoMIV site found at 7795
  Illegal AgeI site found at 341
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 7644
  Illegal SapI.rc site found at 7854