Part:BBa_K1223012:Design

pKD46 functional unit

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1207
  Illegal EcoRI site found at 2716
  Illegal SpeI site found at 4505
  Illegal PstI site found at 2211
  Illegal PstI site found at 2458
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1207
  Illegal EcoRI site found at 2716
  Illegal SpeI site found at 4505
  Illegal PstI site found at 2211
  Illegal PstI site found at 2458
  Illegal NotI site found at 4824
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1207
  Illegal EcoRI site found at 2716
  Illegal BamHI site found at 1146
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1207
  Illegal EcoRI site found at 2716
  Illegal SpeI site found at 4505
  Illegal PstI site found at 2211
  Illegal PstI site found at 2458
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1207
  Illegal EcoRI site found at 2716
  Illegal SpeI site found at 4505
  Illegal PstI site found at 2211
  Illegal PstI site found at 2458
  Illegal AgeI site found at 981
  Illegal AgeI site found at 2638
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 963
  

Design Notes

just need to assemble with selection gene of choice to get any plasmid from the pKD family - pKD46 carries the bla gene (ampicillin resistance), pKD78 carries the cat gene(chloramphenicol resistance), pKD119 carries the tet gene (tetracycline resistance)or create new plasmid with kanamicin resistance.

temperature sensitive origin of replication. grow the bacteria at 30C to keep and amplify the plasmid or grow at 42C to eliminate it.

Source

genbank.

Kirill A. Datsenko and Barry L. Wanner One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products PNAS 2000 97 (12) 6640-6645; published ahead of print May 30, 2000, doi:10.1073/pnas.120163297