Help:Protocol/DNA Synthesis


Constructing a BioBrick part via direct synthesis=

If you are constructing a BioBrick part via direct synthesis ... especially a coding sequence (in which the sequence is flexible), you may want to consider eliminating the following restriction sites that might be useful in the future to others. This list of sites is prioritized.

These sites MUST be absent

The exception to this rule is for the BioBricks prefixes, BioBricks suffixes and the multiple cloning site of BioBricks vectors.

Strongly prefer that these sites be absent

These restriction enzymes are those that generate compatible cohesive ends to the BioBrick sites and therefore can be useful for various projects.

These restriction sites are used in other assembly standards

  • BamHI, BglII, XhoI (Berkeley standard)
  • NgoMIV, AgeI (Freiburg standard)

Would prefer that these sites be absent

Offset cutters

These are offset cutters that can generate arbitrary overhangs. In addition, some are used in the BioBricks++ assembly scheme.

Nicking enzymes

  • Nt.BstNBI, BbvCI, Nt.AlwI (best effort to at least remove sites near each other)

Homing endonucleases

These enzymes have very long recognition sites and are unlikely to be in your part.

  • I-CeuI, I-SceI, PI-PspI, PI-SceI, I-PpoI

Others

Would be convenient if these sites were removed

These are common, efficient cutters that people might want to use.

  • HindIII, BamHI, XhoI, NcoI, SacI, NdeI

Here are other low priority cut sites to remove

  • KasI, MssI, NgoMIV, PacI, PmeI, SalI, SfiI, SgfI, SmiI, SrfI, SwaI, XmaI, ZraI

Sites to include

  • Having GATC sites in your part can sometimes be useful because it allows plasmid purified DNA to be cut (via DpnI) whereas PCR product DNA is not. Such a trick is useful for some site directed mutagenesis protocols.

Miscellaneous

  • BaeI is an enzyme whose is exact cut position is unknown. It is explicitly included in some pSB plasmid replications origins so that the plasmid can optionally be destroyed via another enzyme. You may want to eliminate this site from your part if you intend to use this feature of the BioBricks plasmids.

Source: http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication