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.
  
  

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

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.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 906
  
• 12
  INCOMPATIBLE 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
  
• 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 907
  
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 129
  Illegal BsaI.rc site found at 28