Part:BBa_J100091
For Testing New Promoters via Golden Gate Assembly
J100091 was built by Todd Eckdahl 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 [http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate Golden Gate Assembly] 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). Assembly replaces the double terminator in the destination vector with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. Part:BBa_J100091 and Part:BBa_J119044 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 what this part will look like when digested with Bsa I. TT represents the transcriptional terminator Part:BBa_B0014. This part 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 Part:BBa_J100091 with oligos that [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html self-assemble into dsDNA with compatible sticky ends.] Part:BBa_J100091 and its sister part Part:BBa_J119044 are ideal for undergraduates in a teaching lab to [http://gcat.davidson.edu/RFP/ 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 protocol.]
J100091 contains BBa biobrick prefix + BbsI site + 4 base overhang + BsaI site cutting to the left + transcriptional terminator Part:BBa_B0014 + BsaI site that cuts to the right + 4 base overhang + BbsI site + BD18 bicistronic RBS for more reliable translation (Part:BBa_J119024) + mRFP Part:BBa_E1010 + BBa biobrick suffix.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 857 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal SpeI site found at 858
Illegal PstI site found at 872
Illegal NotI site found at 7
Illegal NotI site found at 865 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 858 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16
Illegal SpeI site found at 858
Illegal PstI site found at 872
Illegal AgeI site found at 730
Illegal AgeI site found at 842 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 135
Illegal BsaI.rc site found at 34
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 906 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 907 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 129
Illegal BsaI.rc site found at 28
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