Part:BBa_K1065203:Design

Efe+Bba_B0015 in pSpac (BBa_K823026)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 9491
  Illegal suffix found in sequence at 1224
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 9491
  Illegal SpeI site found at 1225
  Illegal PstI site found at 1239
  Illegal NotI site found at 1232
  Illegal NotI site found at 9497
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 9491
  Illegal BglII site found at 319
  Illegal BglII site found at 7085
  Illegal BamHI site found at 2601
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 9491
  Illegal suffix found in sequence at 1225
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 9491
  Illegal XbaI site found at 9506
  Illegal SpeI site found at 1225
  Illegal PstI site found at 1239
  Illegal NgoMIV site found at 17
  Illegal NgoMIV site found at 4148
  Illegal AgeI site found at 1070
  Illegal AgeI site found at 6696
  Illegal AgeI site found at 7658
  Illegal AgeI site found at 8333
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3972
  Illegal BsaI.rc site found at 5411
  Illegal BsaI.rc site found at 7927
  Illegal SapI site found at 2889
  Illegal SapI.rc site found at 6909
  

Design Notes

The coding sequence was taken from Pseudomonas syringae pv. phaseolicola PK2. We firstly optimized the codons for both B. subtilis and E. coli and then synthesized the gene with an RBS sequence upstream of the start codon. We decided to include in the CDS two restriction sites (NgoMIV and AgeI) in order to allow people to use the Freiburg assembly.

GAATTCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGCACC...

...TCGACCGGTTAATACTAGTAGCGGCCGCTGCAG

Source

We used a CDS optimized for both E. coli and B. subtilis that was synthesized by Genescript.