Part:BBa_I50022:Design
From partsregistry.org
Minimal pUC19-derived high copy replication origin
- 10COMPATIBLE WITH RFC[10]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 19
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
Design Notes
BioBrick standard vectors are also designed to be easy to purify. To meet this requirement, we included a pUC19-derived origin (BBa_I50022) in addition to the ccdB selection marker within the BioBrick cloning site of BioBrick standard vectors[1, 2, 3]. The high copy origin encoded by BBa_I50022 means that both base vector DNA and any derived vector DNA are easily purified in large quantities, irrespective of whether the vector replication origin is low copy or not[4, 5]. Cloning a BioBrick part into the BioBrick cloning site removes the high copy origin in the cloning site thereby restoring replication control to the vector origin.
BBa_I50022 was originally designed as BBa_I50020. However, several introduced mutations to the pUC19 origin sequence rendered BBa_I50020 nonfunctional as a replication origin. Hence, these introduced mutations were reverted to make BBa_I50022, a functional high copy replication origin.
Source
pSB1A3 and BBa_I50020 served as the basis of the design of BBa_I50022.
References
- Vieira J and Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 1982 Oct; 19(3) 259-68. pmid:6295879.
- Norrander J, Kempe T, and Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 1983 Dec; 26(1) 101-6. pmid:6323249.
- Yanisch-Perron C, Vieira J, and Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 1985; 33(1) 103-19. pmid:2985470.
- Cabello F, Timmis K, and Cohen SN. Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature 1976 Jan 29; 259(5541) 285-90. pmid:765836.
- Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, Batzer MA, and de Jong PJ. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat Genet 1994 Jan; 6(1) 84-9. doi:10.1038/ng0194-84 pmid:8136839.
