design_fusion_site_primers
Design the PCR primers that ADD Type-IIS fusion sites to a part: enzyme site, spacer and the four-base overhang you chose, on a Tm-matched annealing footprint. This is how a sequence that is not yet a Golden Gate part becomes one, and it is the other half of check_golden_gate_parts — that says which parts are unusable, this makes one usable.
Input Schema
| Name | Required | Description | Default |
|---|---|---|---|
| _files | No | The project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name. | |
| enzyme | No | the Type-IIS enzyme the assembly will use. | BsaI |
| _subject | No | The record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several. | |
| sequence | No | the template to amplify — wire a record, name a library document, or leave unset to use the open record. | |
| target_tm | No | target annealing temperature for the template-binding footprint. In °C. | |
| buffer_bases | No | 5' buffer bases outside the recognition site, so the enzyme has somewhere to sit (NEB recommends 6 nt). Advanced. | AAAAAA |
| left_overhang | Yes | the top-strand overhang to leave at the START of the released part — its length must match the enzyme's own overhang. | |
| right_overhang | Yes | the top-strand overhang to leave at the END of the released part. |
Output Schema
| Name | Required | Description | Default |
|---|---|---|---|
| forward | Yes | the forward primer, 5'→3': buffer, recognition site, spacer, your overhang, then the annealing footprint | |
| product | Yes | what the pair amplifies, tails included | |
| reverse | Yes | the reverse primer, same construction on the other end | |
| forwardTm | Yes | melting temperature of the forward primer's ANNEALING footprint — the tail does not bind the template on the first cycle | |
| reverseTm | Yes | the same for the reverse primer; the pair is matched, so a gap between these two is the thing to notice | |
| releasedPart | Yes | what the enzyme cuts OUT of that product — the part that actually enters the assembly, carrying your two overhangs |