SeqBench

For teaching labs

Sequence exercises you can assign as a link

Five exercises that cover the sequence work of a molecular cloning course — reading a plasmid map, restriction mapping, finding a gene, predicting a PCR product and deciding whether a clone is right. Each one is a single link that opens a SeqBench tool with the data already loaded, in a browser tab on any computer in the room. The questions go on your course page; the answer key under each exercise is the output of the same tool your students will use, on the same data.

  1. 1. Copy a link into your course page, learning platform or handout.
  2. 2. Students open it and work on the loaded data; their results are kept in their own browser.
  3. 3. Mark against the key below each exercise, computed by the tool itself.

Exercise 1 · Plasmid Annotator

Find the working parts of a plasmid

  • Plasmid anatomy
  • Origins and selection markers
  • Reading a map

Opens the Plasmid Annotator with the pUC19 sequence (2,686 bp, GenBank L09137.2) and no annotation.

Open the exercise →

Questions

  1. Which antibiotic would you select pUC19 transformants with, and which element on the map tells you so?
  2. Where is the origin of replication, and on which strand is it annotated?
  3. Which elements of the lac operon does pUC19 carry, and roughly where?
  4. Two of the features are binding sites for universal sequencing primers. Name them and give their coordinates.
Answer key
  • M13 fwd primer — 379–395, forward strand (primer)
  • MCS (pUC-style cluster) — 396–422, forward strand (mcs)
  • M13 rev primer — 465–481, reverse strand (primer)
  • lac operator — 487–507, reverse strand (operator)
  • lac promoter (-10) — 520–543, reverse strand (promoter)
  • CAP binding site — 558–579, reverse strand (operator)
  • ori (pMB1/ColE1) — 1,338–1,369, reverse strand (origin)
  • AmpR (bla) — 2,451–2,486, reverse strand (resistance)
  • AmpR promoter — 2,547–2,591, reverse strand (resistance)

Exercise 2 · Restriction Site Finder

Map the restriction sites of a cloning vector

  • Restriction mapping
  • Single cutters and the multiple cloning site
  • Predicting a digest

Opens the Restriction Site Finder with pUC19. Ask students to check EcoRI, BamHI, HindIII, PstI, PvuII.

Open the exercise →

Questions

  1. Which of EcoRI, BamHI, HindIII, PstI, PvuII cut pUC19 exactly once, and where?
  2. Why does a cloning vector want its single-cutter sites clustered in one short region?
  3. PvuII cuts pUC19 more than once. What fragment sizes would you see on a gel?
Answer key
  • EcoRI: 1 site, recognition sequence starting at 396
  • BamHI: 1 site, recognition sequence starting at 417
  • HindIII: 1 site, recognition sequence starting at 447
  • PstI: 1 site, recognition sequence starting at 435
  • PvuII: 2 sites, recognition sequence starting at 306 and 628
  • Single cutters: EcoRI, BamHI, HindIII, PstI — all within 396–447, the multiple cloning site, so an insert can be placed between any two of them.
  • PvuII cuts after 309 and 631: fragments of 2,364 bp and 322 bp.

Exercise 3 · ORF Finder

Find the gene that makes the plasmid selectable

  • Open reading frames
  • Six-frame translation
  • Strand and coordinates

Opens the ORF Finder with pUC19.

Open the exercise →

Questions

  1. What is the longest open reading frame in pUC19: its length in amino acids, its strand and its coordinates?
  2. Which protein does it encode, and what does that protein do for the bacterium?
  3. Why is this ORF read on the reverse strand when the map is numbered on the forward one?
Answer key
  • 286 aa, reverse strand, 1,626–2,486; it begins MSIQHFRVALIP…
  • β-lactamase (bla), which hydrolyses ampicillin — the ampicillin resistance used for selection; the Plasmid Annotator's AmpR (bla) signature sits inside this ORF (2451–2486).
  • A gene can be encoded on either strand; numbering is a convention of the map, not of the molecule.

Exercise 4 · In-Silico PCR

Predict a PCR product with the universal primers

  • Primer binding
  • Product size
  • Colony PCR screening

Opens In-Silico PCR with pUC19 as the template. Students enter the primers: M13 forward (-20) GTAAAACGACGGCCAGT and M13 reverse CAGGAAACAGCTATGAC.

Open the exercise →

Questions

  1. What size product do the two primers give on empty pUC19?
  2. Which restriction sites lie inside that product?
  3. A colony carrying a 500 bp insert in the cloning site is screened with the same primers. What size band do you expect?
Answer key
  • 103 bp, from 379 to 481.
  • EcoRI (396), BamHI (417), HindIII (447), PstI (435) — the multiple cloning site sits between the two primers.
  • About 603 bp: the empty-vector product plus the insert.

Exercise 5 · Sanger Trace Viewer — compare

Decide whether a clone is correct from its Sanger read

  • Sequence verification
  • Mismatches and their consequences
  • Reading a verdict critically

Opens the Sanger comparison with a read from "clone 3" and the annotated pUC19 map it should match. The read has already been base-called.

Open the exercise →

Questions

  1. How much of the map does the read cover, and which annotated features does it reach?
  2. List every difference between the read and the map.
  3. What does each difference do to the protein it falls in? Would you keep this clone?
Answer key
  • 99.87% identity over the stretch the read covers; the verdict is NEEDS REVIEW.
  • Substitution at map position 2306: C → A.
  • Introduces a STOP at codon 61 of bla (AmpR) — the protein is truncated (60 of 286 residues). — the identity alone (above 99%) would have looked like a pass. Discard the clone, or confirm with a second read first.

Using your own sequences

Any sequence becomes a link like these: add #seq=and the URL-encoded sequence (bare bases, FASTA or GenBank) to a tool's address — for example https://seqbench.com/tools/translate#seq=ATGGCCAAGTAA. Every link here uses pUC19, GenBank L09137.2, a public sequence.

Teaching with SeqBench? Tell us what your students got stuck on — exercises are added from what courses actually need.