Part:BBa_J119044
RFP GGA destination vector BsaI
J119044 was synthesized for [http://www.bio.davidson.edu/projects/gcat/GCATSynBio.html GCAT] by [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis.html?CID=fl-genesynthesis 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 [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 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 [http://www.neb.com/nebecomm/products/productR3535.asp 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 [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html 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 [http://gcat.davidson.edu/RFP/ discover and characterize new promoters for use in synthetic biology].
- The only difference between J119044 and Part:BBa_J100091 is the codons used to encode RFP. J119044 uses codons optimized for E. coli as designed by GeneArt. Part:BBa_J100091 uses RFP as encoded in Part:BBa_E1010.
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.
- 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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