Featured Parts:CloningFacilitationTools

This page describes a set of plasmid variants to facilitate cloning. Basically, these elements are BioBrick parts that insert between the EcoRI and XbaI sites, or between SpeI and PstI site rather than usual BioBrick parts that insert between SpeI and PstI. All the elements described here can be used with standard EcoRI-XbaI-part-SpeI-PstI polylinker plasmids (pSB1A2, pSB1AK3, pSB2K3, pSB3C6, etc.) With respect to nomenclature, modifications outside of the XbaI/SpeI sites change the name of the vector. So, if you insert a cassette between the EcoRI and XbaI sites of plasmid pSB1AK3-b0015, the result would still be a *-b0015 plasmid, but a new name must replace the "*". At present, each basic plasmid part is receiving a standard BioBrick part name, but this nomenclature may change later.

Inserting a Ptet promoter between EcoRI and XbaI

Part J23006 is a pSB1A2-derived plasmid for constructing basic BioBrick parts under the control of an external promoter. Insertion of parts such as a protein generator or a RNA key between the XbaI and SpeI (or) PstI sites places the part under the control of the part r0040 Tet promoter. The resulting plasmid retains the normal sequence context of a r0040.newpart composite with one point mutation changing the ACTAGA linker to TCTAGA.

Inserting an RBS-RFP-Terminator cassette between SpeI and PstI

Plasmid J61002 is a pSB1A2-derived plasmid for constructing basic BioBrick promoter parts. When new promoters or libraries of promoters are placed within the XbaI and SpeI sites, they control the expression of RFP while retaining their ability to be used in BioBrick standard assembly.

Inserting a Ptet-RBS cassette between EcoRI and SpeI

Plasmids Part:BBa_J61003, J23018 and J23019 allow the construction of open reading frame basic parts while allowing their constitutive expression on a colE1 (J61003, J23018) or p15A origin of replication (J23018).

Inserting a large Gentamicin resistance cassette between EcoRI and XbaI

Part J61035 facilitates cloning with of small BioBrick parts such as ribosome binding sites. By placing a selectable marker (gentamicin resistance) upstream of a small biobrick part, the large EcoRI/SpeI fragment can be transferred into the EcoRI/XbaI region of another biobrick part, and the product of cloning can be selected on gentamicin plates. Additionally, the large size of the cassette decreases the parent vector background when displacing this cassette with an EcoRI/SpeI fragment of another BioBrick part.