Heat sensitive origin of replication (oriR101 & repA101-ts)
From the plasmid pKD46 maintained inside the E.coli strain BW25113, courtesy of E.coli Genetic Resources at Yale CGSC, The Coli Genetic Stock Center.
OriR101 is an low copy origin of replication that is initiated by the protein repA101. repA101-ts, a mutated form of repA101 would denature at temperatures above 37??C. Hence plasmids using this construct to drive replication can only initiate replication when cultured below 37??C (recommended <30??C). Production of repA101-ts is drived by an unknown constitutive promoter included in the construct.
To make this part compatible with Biobrick standard assembly protocols, overlapping PCR was used to mutate a SpeI cut site originally present inside the construct to render it nonfunctional.
regions not necessary for origin function
point mutation, substitute of C as G to remove SpeI site
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Conversion of the iGEM parts registry to SBOL2.1