Your workspace
Bench tools
Nine tools you drive yourself, in the toolbar above every sequence. These are yours — no assistant involved unless you ask for one.
What is in the toolbar
| Tool | What it does |
|---|---|
| PCR Simulation | Simulates amplification with a primer pair and shows you the product. |
| Digest Simulation | Cuts with chosen enzymes and lists the fragments. |
| Primer Design | Designs primers for the region you have selected. |
| Enzyme Search | Finds restriction enzymes and where they cut this construct. |
| Sequence Alignment | Aligns two sequences and shows the differences. |
| Find & Replace | Searches for a pattern in the bases and replaces it. |
| Restriction Sites | Configures which enzyme sites are drawn on the map. |
| BLAST Search | Searches for similar sequences. |
| Cloning Planner | Works through a cloning strategy with assistance. |
Results open in a drawer below the map. Esc closes it. Everything here needs an account — the toolbar is disabled for guests.
Finding somewhere to cut
This is the tool most cloning starts with. Enzyme Search lists the enzymes that cut your construct and how often, which is the number that actually decides your strategy.
- Cuts once
- A candidate. A unique site is what makes directional cloning predictable.
- Cuts twice or more
- Usable if you want the fragment between them, and a problem otherwise — you will lose whatever sits in between.
- Does not cut
- Not usable on this construct, however standard the enzyme is elsewhere. This is the single most common reason a plan built from memory rather than from the sequence fails.
Restriction Sites is the display counterpart: it controls which enzymes are drawn on the map, so you can keep your working set visible without the map disappearing under three hundred labels.
Checking a digest before you run it
Digest Simulation takes your enzymes and returns the fragments with their sizes. Two things to do with that:
- Predict your gel
- You now know what a correct digest should look like. If your real gel does not match, you have learned something before you sequence.
- Pick a diagnostic digest
- Look for an enzyme combination whose fragment pattern differs clearly between the construct you want and the parent it came from. Two bands 80 bp apart will not tell you anything on an agarose gel.
Primers
Select the region you want to amplify, then open Primer Design. It designs against the actual sequence, so the primers come back with real melting temperatures and real positions rather than a template you have to adapt.
PCR Simulation is the check that follows: give it a pair and it tells you what product you would get. Run it before ordering. It is how you catch a primer that binds somewhere you did not intend, or a pair that produces nothing at all because one of them is on the wrong strand.
Primers you keep are drawn on the map as features. Clearing them is Remove Primers from the right-click menu.
Alignment and BLAST
Alignment: two sequences you already have
Use it to compare a sequencing read against your intended construct, or a variant against its parent. It shows you where they differ, which is usually the question you actually have.
BLAST: one sequence against a database
Use it when you do not know what something is — an unlabelled region, a fragment from a colleague, a construct with no history. Searches take time to come back; that is the database, not the interface.
Find & Replace
Searches the bases for a pattern and replaces it. Genuinely useful for a targeted change — removing an internal cut site, fixing a codon.
Cloning Planner
Works through a strategy with you — which sites, which fragments, what order — rather than answering one question at a time. Reach for it when you know what you want to end up with but not yet how to get there.
If you would rather describe the whole thing in a sentence and have a plan written for you, that is the chat instead: Designing a construct.
Getting there faster
Several tools have single-key shortcuts when you are not typing in a field: P primers, R restriction sites, D digest, B BLAST. ? shows the full list. Shortcuts has everything.