Cloning Workspace

What the Cloning Workspace is

Cloning is the act of joining DNA fragments into a new molecule. Before you order primers or set up a reaction at the bench, it helps to know exactly what the assembled construct will be, where each fragment lands, and which oligos you need. The Cloning Workspace is the tool that answers those questions without leaving ResearchOS. It is a pure in-silico assembly engine. You describe the reaction, and it computes the product sequence, the features carried across the junctions, and the primers (when a chemistry needs them), all in your browser.

The Workspace opens from the Assemble button in the header of the Sequences library, not from an editor tab or the top navigation. Inside, a single method picker offers four assembly chemistries, and every one of them runs the same two-step flow. First you pick and order your fragments and set the method's options. Then you review the computed product and save it to the library.

The two-step flow

Every chemistry shares the same shape, so once you have done one assembly the rest feel familiar. The first step is the pick step. You choose the method, add the fragments or substrate molecules the reaction needs (from the library or pasted directly), order and orient them, and set whatever options the method exposes. The second step is the review step. The Workspace renders the assembled product with its features, the junctions it formed, and any warnings worth a second look (an internal cut site that the chosen enzyme would sever, an overlap that came out below target, an orientation-ambiguous ligation that yields more than one product). Saving the product creates a new sequence in the active collection, where it opens in the normal editor like any other sequence.

Overlap assembly (Gibson / NEBuilder HiFi)

Overlap assembly joins an ordered set of fragments that share short homologous ends. At the bench you amplify each fragment with primers whose 5' ends carry a homology tail matching the neighbouring fragment. The exonuclease chews back, the matching single strands anneal, and the fragments fuse with the shared homology present exactly once at each junction. This is the chemistry behind Gibson assembly and NEBuilder HiFi.

In the Workspace you add two or more fragments in the order you want them joined, choose whether the product is linear or circular, decide whether to carry the source fragments' annotations onto the product (on by default), and pick how the homology overlap is sized. You can fix the overlap to a length in base pairs (the default is 25 bp) or let the engine grow it until the homology reaches a target melting temperature (48 degrees C by default). From there the Workspace does two things. It computes the assembled product as the fragment bodies joined in order, with the homology living once at each seam. And it designs the per-fragment PCR primers, where each primer is an annealing region sized to a target Tm (about 60 degrees C, using the same nearest-neighbor model as the rest of the app) plus a 5' homology tail copied from the adjacent fragment's end, so the amplicons carry the overlap.

The designed oligos are yours to use directly. The review step offers them as a copyable oligo order list, or you can have them saved as primer_bind features on the product so the primers travel with the template they belong to. The junction report flags any overlap that came out shorter than you asked for (a flanking fragment was too short) or any two junctions that share the same overlap sequence (the assembly may be ambiguous).

Restriction and ligation

Restriction-ligation is the classic cut-and-paste of molecular cloning. A restriction enzyme cuts double-stranded DNA at a defined site, and when the two strands are cut at offset positions it leaves a single-stranded overhang (a sticky end). Two ends ligate when their overhangs are complementary and the same length, or when both ends are blunt. DNA ligase seals the strands and the overhang appears once, as the seam between the fragments.

In the Workspace you choose two or more sequences and pick from a fixed set of eight common cutters: EcoRI, BamHI, HindIII, PstI, KpnI, SmaI, XhoI, and NotI. This set covers the most widely-used single-cut and compatible-overhang combinations in subcloning work. For any site not in this set, use the full enzyme picker in the Restriction Digest tool to confirm cut positions, then work backwards to a compatible set from the eight available here. The engine digests each sequence on both strands, types every resulting end as blunt, a 5' overhang, or a 3' overhang, and then enumerates the products whose ends form a consistent ligation chain. Because a fragment can ligate in either orientation, a pair of identical overhangs is genuinely ambiguous and yields more than one product. The Workspace returns every distinct product rather than guessing, so you pick the intended one in the review step.

Golden Gate (Type IIS)

Golden Gate assembly is a one-pot, scarless method built on Type IIS enzymes. Unlike a classic restriction enzyme, a Type IIS enzyme such as BsaI, BsmBI, BbsI, or SapI cuts outside and downstream of its recognition site. That means the recognition sequence ends up on a flanking piece that gets discarded, and the central part keeps a custom four-base overhang (three bases for SapI) with no scar left behind. Design the overhangs to be unique and the parts only fit together one way.

In the Workspace you add your parts and choose one of the four supported Type IIS enzymes: BsaI, BsmBI, BbsI, or SapI. These cover the most common Golden Gate systems (MoClo, GoldenBraid, Loop, and SapI-based scarless cloning). The engine digests every part with the chosen enzyme, drops the pieces that still carry the recognition site (the flanks), and ligates the central parts by their custom overhangs into a seamless product. The review step warns you if a kept piece has a blunt end, which usually means a part is missing a flanking Type IIS site.

Gateway (BP and LR)

Gateway cloning moves a DNA segment between vectors by site-specific recombination, with no restriction digestion and no ligation. The reaction happens between short att sites that share a common core. BP Clonase recombines an attB-flanked insert with an attP donor to make an attL entry clone (and an attR byproduct), and LR Clonase recombines an attL entry clone with an attR destination to make an attB expression clone (and an attP byproduct). The att sites carry a specificity number, and a site 1 recombines only with the partner's site 1, which is what makes Gateway directional.

In the Workspace you pick the reaction (BP or LR) and the two substrate molecules. The engine locates the att sites on each substrate by matching the published site sequences on both strands, checks that each substrate presents one site 1 and one site 2, and then computes each product att site as the true crossover recombinant of the two inputs. The gene of interest transfers onto the partner backbone and the cassette transfers out, exactly as in the wet-lab reaction. The result is the desired clone plus the byproduct, each a circular molecule with its features carried across.

Reviewing and saving the product

Whichever chemistry you used, the review step is the same surface. It shows the assembled product sequence with its features, the junctions it formed, and any warnings. A compact header above the sequence displays the product name, topology, length in base pairs, and GC%, so you can spot a GC-extreme construct before you move to primer ordering. Features from each input fragment are rebased into the product coordinates, so a CDS or a promoter that started on an input fragment lands at the right place on the new construct. When a chemistry designs primers, those appear here too, ready to copy or to save as primer_bind features. When you accept the product, it is saved as a new sequence in the active collection and opens in the editor like any other sequence.

Where the products go

A product saved from the Cloning Workspace is a first-class sequence in the Sequences library, filed under the active collection. It opens in the same editor, renders the same circular and linear maps, carries the features the assembly rebased onto it, and goes through the same trash flow as every other record if you delete it. The primers you designed during an overlap assembly, if you saved them as features, travel with the construct as primer_bind annotations.