Part:BBa_I50042:Design

pSC101 origin of replication

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The original design for BBa_I50042 was BBa_I50040. Synthesis of BBa_I50040 (designed pSC101 origin) was straightforward but testing revealed that BBa_I50040 was nonfunctional as a replication origin presumably due to introduced mutations.

In constructing BBa_I50042, the replication origin boundaries were chosen quite liberally (i.e. they might include more sequence than is strictly necessary) in part because BBa_I50040 was shown to be non-functional.

BBa_I50042 is very similar to the replication origin used in Bujard's pZS4Int-1 vector and in Michael Elowitz's repressilator. However, a mutation was introduced to eliminate a SpeI site for compatibility with the BioBrick assembly standard. BBa_I50042 is also the origin of replication in pSB4* series plasmids.

Source

We constructed BBa_I50042 by PCR using pSB4A3-P1010 as a template and amplification primers I50042-f (5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTGTCAGACCAAGTTTACGAG-3') and I50042-r (5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTACATTGTCGATCTGTTC-3').

References

<biblio>

1. Cohen-J-Bacteriol-1977 pmid=334752
2. Lutz-Nucleic-Acids-Res-1997 pmid=9092630
3. Elowitz-Nature-2000 pmid=10659856

</biblio>