Part:BBa_K2448037

pSB3K3 BsaI free

pSB3K3 BsaI free This part is the pSB3K3 backbone vector with a single (A2499T) mutation the non coding region between the p15A origin of replication and the VF2 primer binding site that eliminates the BsaI cloning site.

Usage and Biology

BsaI is a type IIS restriction enzyme used in the Golden Gate assembly [1]. Its presence in the backbone of the pSB3K3 plasmid prevents from using this high-throughtput assembly technique with this vector.

For this reason, we decided to perform a site-directed mutagenesis and disrupt the BsaI recognition site GGTCTC by changing its sequence to GGTCAC. This site is present in the non coding region between the p15A origin of replication and the VF2 primer binding site, so non special considerations had to be taken into account.

Sequence and Features

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 2729
  Illegal NheI site found at 1384
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2735
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal XhoI site found at 177
  Illegal XhoI site found at 1020
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2729
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2729
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2744
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal AgeI site found at 1470
  Illegal AgeI site found at 1793
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.