Difference between revisions of "Part:pSB1A10:Design"
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The process for building pSB1A10,was:
 
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#Build [[Part:BBa_I13534|I13534]] using normal BB construcion.
 
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#Insert [[Part:BBa_I13534|I13534]] into psb1A3 plasmid
 
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#Cap the BB prefix and suffix using [[Part:BBa_I13450|I13450]]
 
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#Re-insert a BB prefix/suffix into the center of [[Part:BBa_I13534|I13534]] using primers to the "insertion region" of [[Part:BBa_I13457|I13457]] and around-the-horn PCR.
 
  
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Some design notes:
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__NOTOC__
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#The inclusion of the 'barcode' was unintentional (it was attached to [[Part:BBa_E1010|E1010]]), it has no (intended) function.
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<partinfo>pSB1A10 short</partinfo>
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#The "spacer artifacts" at position 22 and 5225 are artifacts from around-the-horn PCR for inserting the BB restriction sites into the center of the plasmid.  If you look at [[Part:BBa_I13457|I13457]] you will see the corresponding regions in the section annotated "insertion region".
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#The RNAse E sites are intended to insulate the device being screened from the screening plasmid itself.  See a more detailed explanation [http://openwetware.org/wiki/Endy:Screening_plasmid_1.0 here].
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#The hairpin 5' to [[Part:BBa_E1010|E1010]] is meant to stabilize the mRNA transcript by preventing degredation by RNAse E. <site>keasling</site>
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#*however, there is a C deletion at bp 64 from the sequence used previously<cite>keasling</cite>.  For our current purposes the expression level of E1010 (mRFP1) has been sufficient with this mutated form of the hairpin, so we haven't made an effort to change it.
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==References==
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<partinfo>pSB1A10 SequenceAndFeatures</partinfo>
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<biblio>
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#keasling pmid=11097920
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</biblio>
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===Design Notes===
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===Source===
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===References===

Revision as of 02:17, 5 June 2006


Screening Plasmid v1.0


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 5242
    Illegal NheI site found at 4443
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 5248
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5242
    Illegal BglII site found at 89
    Illegal BamHI site found at 4382
    Illegal XhoI site found at 67
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 5242
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 5242
    Plasmid lacks a suffix.
    Illegal XbaI site found at 5257
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 674
    Illegal AgeI site found at 786
    Illegal AgeI site found at 4217
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2197
    Illegal BsaI.rc site found at 5118
    Illegal SapI site found at 4199


Design Notes

Source

References