Measurement

Part:BBa_J119044

Designed by: Todd Eckdahl and A. Malcolm Campbell   Group: Eckdahl_Lab   (2012-01-06)

RFP GGA destination vector BsaI

J119044 was synthesized for GCAT by GeneArt and allows users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see Part:BBa_J119022 as an example or the picture below). Successful GGA assembly replaces the double terminator in J119044 with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. Part:BBa_J119044 and its sister part Part:BBa_J100091 incorporate the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.

Below is a picture of the portion that pops out when digested with Bsa I. TT represents the transcriptional terminator Part:BBa_B0014. J119044 is designed to be used for Golden Gate Assembly to swap out the TT for a promoter of your choosing. This can be done by simply mixing J119044 with oligos that self-assemble into dsDNA with compatible sticky ends. J119044 and its sister part Part:BBa_J100091 are ideal for undergraduates in a teaching lab to discover and characterize new promoters for use in synthetic biology.

See the full GGA protocol online.

J100028.png


Sequence and Features
J119044 contains BBa biobrick prefix + 4 base overhang + BsaI site cutting to the left + transcriptional terminator Part:BBa_B0014 + BsaI site that cuts to the right + 4 base overhang + BD18 bicistronic RBS for more reliable translation (Part:BBa_J119024) + mRFP with E. coli-optimized codons generated by GeneArt + BBa biobrick suffix.

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 906
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 907
    Illegal PstI site found at 921
    Illegal NotI site found at 7
    Illegal NotI site found at 914
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 907
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 907
    Illegal PstI site found at 921
    Illegal AgeI site found at 779
    Illegal AgeI site found at 891
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 129
    Illegal BsaI.rc site found at 28
    Illegal EcoRI site found at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 907
    Illegal PstI site found at 921


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Categories
Parameters
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