Plasmid_Backbone
V0120

Part:BBa_J63010:Design

Designed by: Ira Phillips and Caroline Ajo-Franklin   Group: Silver Lab   (2006-10-18)
Revision as of 14:43, 29 October 2006 by Cajofranklin (Talk | contribs) (→‎Design Notes)


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]. When two parts in this plasmid are fused in the standard assembly method, 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 XbaI site would be methylated by DamI and thus digestion at this site would be blocked.

Source

synthetized DNA

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].