Part:BBa_J63010:Design

Protein fusion vector (Silver lab standard)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3246
  Illegal XbaI site found at 3261
  Illegal SpeI site found at 1
  Illegal PstI site found at 15
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3246
  Illegal SpeI site found at 1
  Illegal PstI site found at 15
  Illegal NotI site found at 8
  Illegal NotI site found at 3252
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3246
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Illegal EcoRI site found at 3246
  Illegal XbaI site found at 3261
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3246
  Illegal XbaI site found at 3261
  Illegal SpeI site found at 1
  Illegal PstI site found at 15
  Illegal NgoMIV site found at 2825
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 1393
  Illegal SapI site found at 310
  

Design Notes

This plasmid was designed to permit in-frame assembly of protein fusions. Although it contains the standard biobrick ends (EcoRI, XbaI, SpeI, PstI), a part immediately follows the XbaI restriction site and immediately precedes a SpeI site. These changes form a [http://hdl.handle.net/1721.1/32535 revised biobrick assembly strategy - biofusion assembly], which remains compatible with standard biobricks and uses the standard biobricks assembly methods. However, unlike the standard biobricks assembly, when two parts from the biofusion plasmid are fused together, six nucleotides (ACTAGA) separate the two parts, thus keeping the coding sequences in-frame.

• Like the biobrick standard, parts cannot contain EcoRI, XbaI, SpeI or PstI restriction sites.
• An additional design criteria is that a part should not begin with the nucleotides TC. If a part did, the [http://www.neb.com/nebecomm/products/productR0145.asp XbaI site would be methylated by DamI] and thus digestion at this site would be blocked.

Source

Synthetized DNA from Blue Heron.

References

[http://hdl.handle.net/1721.1/32535 1. Ira Phillips and Pam Silver. "A New Biobrick Assembly Strategy Designed for Facile Protein Engineering." DSpace at MIT].