Part:BBa_J100091
For Testing New Promoters via Golden Gate Assembly
This part works, but it has been superseded by J119137 (pClone Red Part:BBa_J119137) and (pClone Blue Part:BBa_J119313).
J100091 (also called pClone Basic) 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 [http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Golden_Gate 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 J100091 with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. J100091 and its sister part Part:BBa_J119044 incorporate the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org. Related parts are pClone Red and pClone Blue .
Below is a picture of the portion that pops out when digested with [http://www.neb.com/nebecomm/products/productR3535.asp Bsa I.] TT represents the transcriptional terminator Part:BBa_B0014. J100091 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 J100091 with oligos that [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html self-assemble into dsDNA with compatible sticky ends.] 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].
- The only difference between J100091 and Part:BBa_J119044 is the codons used to encode RFP. J100091 uses RFP as encoded in Part:BBa_E1010. Part:BBa_J119044 uses codons optimized for E. coli as designed by GeneArt.
Sequence and Features
J100091 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 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 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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