14.6.1 Sequence ordering, rules and assumptions
When the Golden Gate options are launched, or Auto-arrange is clicked, Geneious will detect existing type IIS restriction sites, overhang annotations, primer annotations and blunt ends on the selected sequences, and will try to produce the optimal arrangement based on the available overhangs. Geneious will only consider cut sites where the recognition site will be cut out, or primers that point towards each other. The ligation order and selected primers may be changed manually in the Construct Layout Panel.
Geneious will automatically arrange sequences using the following rules, in order of precedence:
- Existing type IIS cut site(s): If Geneious detects a pair of appropriately orientated type IIS sites with unique overhangs, these will be selected and no PCR primers will be designed. If only one type IIS cut site is detected, then Geneious will design a PCR primer that incorporates the site, and will design a second primer to introduce a compatible opposite orientation restriction site based on rules 3-6.
- Existing Overhang(s): If Geneious detects a pair of valid overhangs compatible with the speciﬁed type IIS site, then these will be selected. No PCR primers will be designed.
- Existing Primer Bind annotation(s) with valid type IIS cut site(s) on the extension: If Geneious detects a pair of inward facing Primer Bind annotations with extensions that produce valid, compatible, type IIS restriction sites, these will be used. New primers will not be designed. If Geneious detects a single Primer Bind annotation with a suitable type IIS site Geneious will use this, and will design a second primer to introduce a compatible opposite orientation restriction site based on rules 4-6.
- Existing Primer Bind annotations with other extensions: If Geneious detects a Primer Bind annotation with an extension which does not contain a valid type IIS site, this primer will be used with an addition to the primer extension to introduce a cut site for the selected type IIS restriction enzyme. This will be done by further extension to the 5’ terminus of the existing primer.
- Existing Primer Bind annotation(s) without extension(s): If a primer bind annotation without an extension is found, then an extension will be appended to introduce a cut site for the selected type IIS recognition enzyme, resulting in a new primer sequence.
- Blunt ends: If Geneious ﬁnds a blunt end, and no suitable type IIS sites or Primer Bind annotations are present, then a primer with an appropriate type IIS site extension will be designed. The fusion point will be the termini(us) of the blunt end fragment.
- Removal of unwanted internal Type IIS sites: If one or more sequences have cut sites for the speciﬁed type IIS restriction enzyme, then Geneious will assume that these sites should be used in the assembly process and design a strategy accordingly. If any of the sequences contain type IIS restriction sites that should not be used in the assembly then these will ﬁrst need to be engineered out of the fragment using PCR.