Verify a Construct - Did Your Cloning Actually Work?
Paste what you got back and the PCR you say made the insert. It re-derives the insert, finds it in either orientation, and gives you the exact mismatch positions.
🌐 Nothing you paste is logged or stored — every tool is also callable via REST & MCP, and in bulk from the batch tools
You cloned something, you sent it for sequencing, and now you have a sequence and a question. Paste the sequence you got back plus the template and two primers you say produced the insert, and this re-derives the insert from that PCR and looks for it in your construct - not by trusting your description of the insert, but by working out what those primers on that template would actually amplify. That distinction is the point: if the primers you wrote in your notebook do not produce what you think they produce, the discrepancy shows up here instead of being carried forward. Both orientations are searched, because a non-directional clone goes in backwards half the time and that is a different problem from a mutation. When the match is not exact you get the mismatch positions in the coordinates of the sequence you pasted, so they line up with what your viewer is showing. Declare a reading frame and it also checks for a premature stop, because a construct can be a perfect sequence match and still not express.
The insert is re-derived from these three rather than taken from you, which is the point: if the primers you recorded do not produce what you think they produce, that shows up here instead of being assumed.
Working on a whole construct rather than one insert check? Open SeqStudio — the full editor, with live feature annotation, plasmid maps, restriction and primer panels, undo/redo, multi-document tabs and GenBank / SnapGene import and export.
How to use the Verify Construct tool
- 1Paste the sequence you got back - a Sanger consensus, a whole-plasmid read, or whatever the sequencing service returned.
- 2Paste the template the insert was amplified from, and the two primers you used. These are what the insert gets re-derived from.
- 3If the insert should be in frame, give the 1-based position where the reading frame starts, and it will also check for a premature stop.
- 4Read the verdict, then the mismatch table. A single mismatch near a primer end is usually the primer; scattered mismatches through the middle are usually the template or the polymerase.
Frequently asked questions
Why does it want my primers instead of the insert sequence?
Because re-deriving the insert is a stronger check than comparing against what you say the insert is. If you paste an insert sequence, the tool can only tell you whether that sequence is present. If you paste the template and primers, it works out what that PCR actually produces - and a surprising number of failures are the primers not amplifying what the notebook says they amplify. When there is no product, or more than one, that is itself the finding and the tool says so rather than proceeding.
The insert is there but in the reverse-complement orientation. Is that a problem?
It depends entirely on your cloning strategy, which is why the tool reports the orientation rather than judging it. For a blunt ligation or a single-enzyme clone, either orientation is expected and roughly half your colonies will be backwards - fine if you only need the sequence present. For a directional clone, or anything that has to be in frame with a promoter or a tag in the backbone, the reverse orientation is the failure and you should pick another colony.
What does the identity percentage mean here?
It is the identity of the best local alignment of the re-derived insert against the sequence you pasted, over the part that aligned. Coverage is separately reported as how much of the insert's length that alignment spans, and the two answer different questions: 100% identity over 60% coverage means the insert is truncated, while 99% identity over 100% coverage means it is all there with a point difference. Both numbers matter and neither alone tells you what happened.
It says the insert is not present at all. What now?
Three ordinary explanations, in rough order of likelihood. The colony is wrong - empty vector or a different insert, which is what most failed clones are. The sequence you pasted is not the clone you think it is, which happens more often than people expect with mislabelled tubes and reused filenames. Or the primers do not amplify what you think, in which case the re-derived insert is not the insert you meant to look for; check the predicted product length against what you saw on your gel.
Why check for a premature stop separately?
Because a sequence match and an expressible construct are different claims. A clone can be 100% identical to the design and still carry a stop codon you did not intend - most often because the insert went in out of frame relative to the backbone, or because a linker or tag shifted the frame by one or two bases. Declaring where the reading frame starts lets the tool check the thing you actually care about rather than only the thing that is easy to check.
Does this check the backbone too?
No, and that limit is worth being clear about. This asks whether the insert you meant to clone is present, complete and exact. It says nothing about the rest of the plasmid, so a construct can pass here and still carry a deletion in the origin, a mutation in a resistance marker, or a rearrangement somewhere you did not look. For the whole-sequence question, compare your full read against the full expected sequence instead.
Can I verify a whole assembly recipe rather than just the insert?
Yes, though not from this page. verify_assembly takes the same method and parts a cloning simulation would - restriction-ligation, Gibson or Golden Gate, optionally deriving a part by PCR first - re-derives the whole expected product and diffs it against your claimed sequence. It needs you to declare the entire recipe, which is a heavier input than this page asks for, so it lives on the REST API and the MCP server where a script or an agent can supply it.
Is my data stored, and can I run this from code?
The sequences are aligned server-side and nothing is persisted. verify_construct is also callable from the REST API and the MCP server, which is the practical way to check a whole plate of clones against their designs in one pass rather than pasting them one at a time.
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