design_ligation_independent_primers
Read-only
Design the PCR primers a ligation-independent reaction needs — LIC, SLIC, In-Fusion, CPEC or USER: a Tm-matched pair per amplified fragment, in the order given.
Input Schema
| Name | Required | Description | Default |
|---|---|---|---|
| eklic | No | LIC only: use the Ek/LIC vectors' own fixed extensions (Novagen TB163) instead of designing an arm. One insert only, and the vector is supplied cut. | |
| _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. | |
| arm_nt | No | the homology arm on ONE primer, which is the unit every one of these authorities states. Left unset, the method's own recommended arm. Does not apply to USER, which finds its extension instead. In nt. | |
| method | No | the cloning method, which decides what the tail is: LIC's arm is the Ek/LIC vectors' fixed overhang, SLIC's and CPEC's are ranges, In-Fusion widens its own for more than one insert, and USER's is a complementary dU pair whose length the search finds. Values: lic (LIC), slic (SLIC), in-fusion (In-Fusion), cpec (CPEC), user (USER). | in-fusion |
| _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. | |
| backbone | No | whether the first fragment is amplified with the others (pydna splits each junction across the two neighbouring primers, so the junction is twice the arm) or supplied already cut (the whole arm goes on its neighbour's primer, and the junction is the arm). Values: amplified (Amplified), linearized (Linearized). | amplified |
| circular | No | close the ring: the last fragment also overlaps the first. USER does not wrap on its own, so a USER ring needs a vector named below. | |
| eklic_tag | No | Ek/LIC only: keep the C-terminal tag, or close the insert with the in-frame stop TB163 gives for a construct that omits it. Only applies when eklic is true. Advanced. | |
| fragments | No | the fragments to join, IN ASSEMBLY ORDER, the first being the backbone — wire upstream `record` outputs here, or name library documents. | |
| target_tm | No | target annealing Tm for each primer's 3' footprint. In °C. | |
| conditions | No | salt, magnesium, dNTP and oligo concentrations plus the salt-correction method the target Tm is measured in. Left unset it is filled from your reaction-conditions settings, and falls back to pydna's Taq defaults when there are none. Advanced. | |
| user_vector | No | USER only: join the two outer ends to a vector that already carries fixed 3' extensions, which is how a USER construct closes a ring. pNEB206A is the vector the method's own paper uses. Values: none (None (linear chain)), pNEB206A (pNEB206A). Only applies when method is user. | none |
| min_footprint | No | shortest annealing footprint the designer may settle for (pydna's `limit`). In nt. Advanced. | |
| include_subject | No | put the node's subject first, as the backbone. | |
| user_vector_end | No | USER with a vector: which of its two DIFFERENT extensions the first fragment's forward primer matches, which is what sets the insert's orientation in the vector. Values: left (Left (GGAGACAU)), right (Right (GGGAAAGU)). Only applies when user_vector is pNEB206A. Advanced. | left |
Output Schema
| Name | Required | Description | Default |
|---|---|---|---|
| armNt | Yes | the arm on one primer | |
| primers | Yes | per amplified fragment: the tailed amplicon its primers make, that amplicon's length, and both primers with tail, footprint, length and footprint Tm | |
| summary | Yes | what was designed, naming the method and its chemistry | |
| reactants | Yes | the molecules that actually react, as sequence RECORDS: the amplicons where primers were designed, and any fragment supplied cut as itself. THIS is what to wire into `ligation_independent_clone.fragments`, not the untailed fragments | |
| armVerdict | Yes | whether that arm is one the method's authority stands behind, in its own words | |
| extensions | Yes | USER only: per junction, the two fragments it joins, the single-stranded 3' extension each side gets (which is also where the dU sits), both tails, and whether that length is one the authority stands behind | |
| junctionBp | Yes | the homology the design creates at each junction — the number `ligation_independent_clone.arm_nt` needs for the reaction to be predicted as designed | |
| vectorEnds | Yes | USER with a vector: the two FIXED extensions that close the ring, which tail went on which outer primer, and where they come from. Not designed, because the vector was manufactured with them. Null when no vector is named, since a linear chain has no fixed ends to close on | |
| primerCount | Yes | oligos to order (two per amplified fragment) |