Plasmid_Backbone
V0120

Part:BBa_J63010:Design

Designed by: Ira Phillips and Caroline Ajo-Franklin   Group: Silver Lab   (2006-10-18)
Revision as of 14:51, 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 - biofusion assembly], which remains compatible with standard biobricks and uses the standard biobricks assembly methods. However, unlike the standard biobricks assembly, when two parts in this plasmid are fused, 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 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].