Cloning simulator
cloning_simulateRead-onlyIdempotent
Assemble fragments by Gibson/overlap, Golden Gate (Type IIS), restriction–ligation (sticky or blunt), TOPO/TA, LIC or SLIC (T4-polymerase chew-back) or In-Fusion/CPEC, returning the product, the junctions and — for the primer-design methods — the junction primers. Each method is modelled as its own chemistry rather than as one product model with different labels: LIC's chew-back stops at the first occurrence of the single dNTP supplied, so a tail carrying that base stops it early and a tail without one lets it run past the junction, and both are refused with the offending base and position named.
Input Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| names | No | Optional labels for each fragment. | |
| enzyme | No | Type IIS enzyme for Golden Gate — one of BsaI, BbsI, Esp3I (BsmBI); "BsmBI" also resolves to Esp3I. Any other name is rejected rather than substituted. | BsaI |
| insert | No | Insert sequence (restriction, topo and lic methods). | |
| method | Yes | Assembly method. gibson/goldengate design the junction primers for bare fragments; restriction and topo build a product from a vector and an insert; lic and slic model T4-polymerase chew-back (sequence-defined and time-stopped respectively); infusion is In-Fusion/CPEC, 15 bp terminal homology with no exonuclease, and works out the fragment ORDER for itself. | gibson |
| vector | No | Vector sequence (restriction, topo and lic methods; for lic, the LINEARISED vector). | |
| enzyme3 | No | Insert 3′ enzyme (restriction method). | BamHI |
| enzyme5 | No | Insert 5′ enzyme (restriction method). | EcoRI |
| circular | No | Produce a circular product. | |
| topoMode | No | TOPO chemistry (topo method): TA (Taq 3′-A), blunt, or directional (pENTR/D-TOPO, needs 5′-CACC on the insert). | ta |
| fragments | No | Fragments (5′→3′). Used by gibson/goldengate/slic/infusion. gibson and goldengate assemble them head-to-tail in the order given and add the junctions by primer; slic needs the homology already ON the fragments and keeps the order given; infusion needs it on the fragments too but derives the order from the homologies, so the order listed here is ignored. slic and infusion take at most 24. | |
| overlapLen | No | Gibson homology-arm length (bp). | |
| armTmTarget | No | Target annealing Tm (°C) for primer arms. | |
| chewBackLen | No | slic method: nucleotides removed from each 3′ end. A SLIC chew-back has no dNTP to arrest it and is stopped by time, so this is an input rather than a property of the sequence. 0 (default) models the intended reaction — chewed to the full homology and no further; any positive value is checked against each junction's own homology, and both overrunning it and falling short of meeting the chew-back coming the other way are reported. | |
| homologyLen | No | infusion method: terminal homology required at every junction, in bp. At least 15 — the junction is annealed and extended by a polymerase with no exonuclease to widen it and no ligase to seal it, so a shorter overlap is a different chemistry rather than a weaker version of this one. | |
| vectorEnzyme3 | No | Vector 3′ enzyme (restriction method); defaults to enzyme3. | |
| vectorEnzyme5 | No | Vector 5′ enzyme (restriction method); defaults to enzyme5. Set a different, compatible enzyme (e.g. BglII for a BamHI insert) to model heterologous-overhang cloning. | |
| insertNucleotide | No | lic method: the single dNTP the INSERT's chew-back was run with. Normally the COMPLEMENT of vectorNucleotide (the classic pair is dTTP on the vector, dATP on the insert) — with the same base for both, each junction's shared block would have to avoid that base and its complement at once. | |
| vectorNucleotide | No | lic method: the single dNTP the VECTOR's chew-back was run with (T for dTTP…). Required — T4 pol's exonuclease stops at the first occurrence of this base reading inward from each 3′ end, so there is no answer without it. | |
| mergePreAddedArms | No | Gibson only. When two fragments already share terminal sequence, count that share ONCE (the default — it is a homology arm the fragments already carry) or set false to concatenate it twice. The second reading is the right one when the shared block is a genuine tandem repeat, e.g. a 2x tag or enhancer split between its copies: nothing in the two fragments can tell the two apart, so the choice belongs to the caller. Either way the share is reported in preAddedArms and both readings are returned in `readings`. | |
| dephosphorylateVector | No | CIP/rSAP-dephosphorylate the vector to suppress self-ligation background (restriction method). |