Plasmid backbones/Version 5/Features

1. A complete BioBrick® cloning site for easy cloning and assembly of BioBrick® parts.
2. A low or medium copy replication origin to reduce consumption of cellular resources during device or system operation.
3. Forward and reverse terminators both upstream and downstream of the BioBrick® cloning site to insulate the vector from read-through transcription originating in the cloned BioBrick® device or system and to insulate the cloned BioBrick® system from the vector.
4. Stop codons in all reading frames to prevent inadvertent translation into or out of the BioBrick cloning site.
5. Primer binding sites for the standard BioBrick® verification primers VF2 (BBa_G00100) and VR (BBa_G00101). These primers are located for convenient sequencing and [http://openwetware.org/wiki/Colony_PCR screening by colony PCR] of cloned BioBrick® devices and systems.

Plasmid backbones are distributed by the Registry with a default insert. There are just a handful of default plasmid inserts used in the Registry. Many the available plasmid backbones include the ccdB positive selection marker (both BBa_I52001 and BBa_I52002) as the default plasmid insert within the BioBrick® cloning site. The ccdB gene ensures that when assembling two BioBrick® parts together, the uncut plasmid is not transformed. However, inclusion of the ccdB gene means that these vectors must be propagated in a ccdB tolerant strain, such as E. coli strain DB3.1 (BBa_V1005).

Plasmid backbones with the default plasmid insert of BBa_I52002 also include a high copy replication origin in the default insert. Thus, these plasmid backbones are easily [http://openwetware.org/wiki/Miniprep/Qiagen_kit purified in large quantities]. Cloning or assembly of a BioBrick® part into the BioBrick® cloning site of the plasmid backbone eliminates the default insert returning the plasmid to the control of the replication origin in the plasmid backbone.