Reporter

Part:BBa_J100104

Designed by: Malcolm Campbell   Group: Campbell M Lab   (2012-12-13)

For Testing New Promoters via Golden Gate Assembly v2

J100104 was built by Todd Eckdahl and Malcolm Campbell. This part 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 a reporter gene that turns E. coli blue (J100103) with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. J100104 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. J100103rev represents the reverse complement of J100103 and is designed to be used for Golden Gate Assembly to swap out the blue protein production for a promoter of your choosing. This can be done by simply mixing J100104 with oligos that self-assemble into dsDNA with compatible sticky ends. J100104 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.

[http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate_Assembly_protocol See the full GGA protocol online.]

J100104 insert.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 1611
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 1612
    Illegal PstI site found at 1626
    Illegal NotI site found at 7
    Illegal NotI site found at 1619
  • 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 1612
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 1612
    Illegal PstI site found at 1626
    Illegal AgeI site found at 47
    Illegal AgeI site found at 155
    Illegal AgeI site found at 1484
    Illegal AgeI site found at 1596
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 834
    Illegal BsaI.rc site found at 28


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