Part:BBa_K2448036:Design

pSB1C3 BsmBI free

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2049
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2055
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2049
  Illegal XhoI site found at 1033
  Illegal XhoI site found at 1925
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2049
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2049
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2064
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

Design Notes

To disrupt the BsmBI recognition site CGTCTC by site-directed mutagenesis we choose to change its sequence to CGTGTC. This site is present in the chloramphenicol resistance gene so care was taken that the mutation should not affect the amino acid composition of the protein. The chosen mutation is a synonymous one GTC(Val) to GTG(Val).

                       >BsmBI
                        |
pSB1C3:        1390 - TTC GTC TCA - 1382
                      F   V   S

BBa_K2448036:  1390 - TTC GTG TCA - 1382
                      F   V   S

Source

site directed mutagenesis on the pSB1C3 bearing the BBa_J04450 reporter.

References

[1] Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One (2008) 3, e3647.