Part:BBa_J100091
For Testing New Promoters via Golden Gate Assembly
J100091 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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