BioBrick base vector, as synthesized by Codon Devices
BBa_I51019 is the sequence of the BioBrick base vector as synthesized by Codon Devices, Inc. in Cambridge, MA. The original design of the BioBrick base vector is available as BBa_I51000. BBa_I51001 is the sequence of the BioBrick base vector as reported by Codon Devices, Inc. although the true sequence is something like to BBa_I51019. The exact sequence of the provided DNA is difficult to determine. The corrected BioBrick base vector that was successfully constructed from BBa_I51019 is BBa_I51020.
Synthesis of the BioBrick base vector design (BBa_I51000) was problematic. We encountered two issues while trying to construct the base vector. First, troubleshooting efforts during synthesis compromised the design of our parts: failed attempts to clone the base vector into an ''E. coli'' strain intolerant of expression of the toxic protein CcdB led to an unnecessary redesign of the ''ccdB'' positive selection marker in the BioBrick base vector. As a result, a revised design for the BioBrick base vector (BBa_I51001) was specified instead. Second, faulty part design adversely impacted the synthesis process: our pUC19-based replication origin design (BBa_I50020) was similarly nonfunctional, so the revised base vector (BBa_I51001) could not be propagated as specified. We eventually determined that the provided DNA for the BioBrick base vector was actually a fusion of two slightly different copies of the base vector: one with a nonfunctional version of the pUC19 origin (BBa_I50020) and one with a functional version of the pUC19 origin (BBa_I50022). We corrected the synthesized BioBrick base vector (BBa_I51019) with molecular biology techniques to make the functional BioBrick base vector BBa_I51020 (see Methods). Commercial DNA synthesis processes currently rely on cloning, assembly and propagation of synthesized DNA in ''E. coli''. Difficulties during synthesis stemmed from the inclusion of both a ''ccdB'' positive selection marker that is toxic to most ''E. coli'' strains and a synthetic origin incapable of supporting plasmid replication of the BioBrick base vector. In general, for parts whose function are incompatible with growth and replication of ''E. coli'', the processes of DNA design and DNA synthesis cannot be easily decoupled.
This part contains a stop codon in all 6 reading frames. Useful for ensuring that translation does not proceed through this part.
Topoisomerase I mediated cloning site (forward)
Insertion of this site will enable generation of a TOPO vector. The vector will have a T overhang and permit binding of topoisomerase I for direct cloning of PCR products. See http://openwetware.org/wiki/Topoisomerase_I_mediated_TA_cloning for a complete description of Topoisomerase I mediated cloning.
NheI restriction enzyme site (XbaI, SpeI compatible overhang)
NheI recognition site. Digestion with NheI generates a compatible cohesive end to XbaI and SpeI.
Terminator to be placed upstream of multiple cloning site in new pSB series plasmids.
This part is the standard BioBricks prefix.
Forward primer for sequencing/amplifying BioBrick parts (VF2)
Originally named VF2, primer binds upstream of the standard BBa_MCS on biobrick vectors.
ampicillin resistance cassette, reverse orientation
This contains TetR, a terminator and then AmpR and a terminator
Minimal pUC19-derived high copy replication origin
High copy origin of replication derived from pSB1A3. Designed to omit antibiotic resistance markers and primer binding sites. Lacks mutations that render BBa_I50020 nonfunctional as a replication origin.
We had to leave several restriction sites in because point mutations that eliminated these sites were found to render the origin nonfunctional.
This part is derived from BBa_P1010 but it lacks ccdA-.
This part is a differs from BBa_P1011 in that the mutations introduced in BBa_P1011 to remove restriction sites are not present in this part. It does have the "normal" ccd promoter and ccdB coding region. It also has a double TAA stop codon as per BioBrick conventions.
ccdA- was eliminated since it is an inactive form of ccdA and should not be required for the toxic phenotype of ccdB.
Topoisomerase I mediated cloning site (reverse)
Insertion of this site will enable generation of a TOPO vector. The vector will have a T overhang and permit binding of topoisomerase I for direct cloning of PCR products. See http://openwetware.org/wiki/Topoisomerase_I_mediated_TA_cloning for a complete description of Topoisomerase I mediated cloning. This site belongs 3' of the cloning site. It is the reverse complement of BBa_B0043.
Terminator to be placed downstream of multiple cloning site in new pSB series plasmids. Similar to BBa_B0051.
identical to E. coli MG1655 between tonB and yciA
Reverse of B0052, E.coli transcriptional terminator from the ribosomal RNA rrnC operon.
This part is the standard BioBricks suffix.
Reverse BioBrick primer annealing site (VR binding site)
This is the VR sequence to be used in construction of plasmids which include the VR primer site. It is the reverse complement of BBa_G00101.
High copy origin of replication derived from pSB1A3. Designed to omit antibiotic resistance markers and primer binding sites.
Conversion of the iGEM parts registry to SBOL2.1