Difference between revisions of "Part:BBa K4727003:Design"

 
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===Design Notes===
 
===Design Notes===
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Starting from the commercialy available pTZ19R (ThermoFisher scientific) a 45bp long region containing 3 prohibited restriction sites (EcoRI, XbaI and PstI) was deleted. Through PCR mutagenesis the BioBrick RFC[10] prefic and suffix were added.
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The design of this part started from the commercially available pTZ19R (ThermoFisher scientific). While this construct exhibited several favorable attributes aligned with our objectives, certain notable issues necessitated resolution. Primarily, there existed an obstacle due to the presence of a multiple cloning site embedded within the LacZ gene. This site encompassed three restricted cut sites, a circumstance that could be readily rectified through targeted deletion, achieved using a primer pair that excludes this region. Another significant concern was the absence of the BioBrick RFC[10] prefix and suffix. This challenge was similarly addressed straightforwardly, utilizing PCR mutagenesis to seamlessly incorporate the two required sequences.
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===Source===
 
===Source===

Latest revision as of 08:18, 23 September 2023


M13 standard phagemid


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 623
    Illegal suffix found at 645
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 623
    Illegal SpeI site found at 646
    Illegal PstI site found at 660
    Illegal NotI site found at 629
    Illegal NotI site found at 653
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 623
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 623
    Illegal suffix found at 646
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 623
    Plasmid lacks a suffix.
    Illegal XbaI site found at 638
    Illegal SpeI site found at 646
    Illegal PstI site found at 660
    Illegal NgoMIV site found at 127
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2026
    Illegal SapI site found at 943


Design Notes

The design of this part started from the commercially available pTZ19R (ThermoFisher scientific). While this construct exhibited several favorable attributes aligned with our objectives, certain notable issues necessitated resolution. Primarily, there existed an obstacle due to the presence of a multiple cloning site embedded within the LacZ gene. This site encompassed three restricted cut sites, a circumstance that could be readily rectified through targeted deletion, achieved using a primer pair that excludes this region. Another significant concern was the absence of the BioBrick RFC[10] prefix and suffix. This challenge was similarly addressed straightforwardly, utilizing PCR mutagenesis to seamlessly incorporate the two required sequences.

Source

Deirived from pTZ19R by ThermoFischer scientific, a pUC19 plasmid containig the phage f1 intergenic region anche the T7 promoter

References