Go to the NCBI database and search for pACYC184 in the nucelotide database; we are specifically interested in the one labeled as "Cloning vector pACYC184".

Question: How many nucleotides does this sequence have?
Answer: 4245

Question: What is its accession number?
Answer: X06403.1

Question: What are the first 10 nucleotides reported in its representation? (To be fair, this is actually a circular molecule, so the "start" is only by convention.)
Answer: GAATTCCGGA

Next, go to NCBI's ORF finder and enter the accession number for pACYC184, and have it compute all ORFs having minimum length of 150bp.

Question: How many such ORFs are found?
Answer: 16

Question: How many nucleotides are in the longest ORF?
Answer: 1191

Question: At what nucleotides does the longest ORF start and stop?
Answer: 1581..2771

By default, it reported all ORFs having length 75nt or longer. Redo the search with minimum ORF length of 150nt.

Question: How many ORFs have 150nt or more?
Answer: 16

Return to the search page. There is an option to "Ignore nested ORFs". Redo the search with this option and minimum length of 150nt.

Question: How many orfs are reported?
Answer: 9

Compare the results when including nested ORFs and the results when excluding them. Locate at least one specific ORF that is excluded in the latter search.

Question: What is the start..stop of such a nested orf that was excluded? What is the remaining ORF in which it was contained?

Click on the longest ORF to examine its details. Notice a box to the left that by default shows its amino acid sequence.

Question: What are the first four amino acid characters?
Answer: MKSN

You can switch to see the underlying nucelotide sequence by clicking on the "Display ORF as..." label.

Question: What are the first 12 nucleotides?
Answer: ATGAAATCTAAC

Question: Which of the stop codons ends this ORF?
Answer: TGA

Not every ORF is necessarily a gene. One way to suggest that an ORF is a gene is by comparing its sequence to a database of known genes from other genomes to look for similarity. BLAST is a popular such tool (and we will soon explore the underlying algorithm it uses for sequence alignment). The NCBI ORF Finder conveniently offers a button to perform a BLAST search for a selected ORF. (In fact, there is a "BLAST" button and a "SmartBLAST" button.) Let's use the SmartBLAST button.

Question: What conclusion is suggested by a SmartBLAST on this ORF?
Answer: Suggests its a Multidrug resistance protein, specifically tetracycline in humans

Let's go an examine the second longest of the identified ORFs.

Question: How many nucleotides are in this ORF?
Answer: 459

Question: At what nucleotides does this ORF start and stop?
Answer: 2578..2118

Question: How would you interpret the fact that its start index is larger than its stop index?
Answer: This is on the reverse strand

Question: What conclusion is suggested by a SmartBLAST on this ORF?
Answer: Best matched to a "hypothetical protein product"

Go back to the original database in which we found this plasmid. Within that view, there is a section labeled "FEATURES". Notice that two of those miscellaneous features are described as genes.

Question: give the start..end indices and descriptions for the two genes.
Answer:
219..3805 chloramphenicol resistence gene
1581..2771 tetracycline resistance gene

Question: Which of these corresponds to the longest ORF that we examined earlier?
Answer: 1581..2771 tetracycline resistance gene

Question: Can you find an ORF that corresponds to the other of these identified genes?
Answer: not exactly

Question: What if we remind you that this was actually a circular molecule? Can you find a pair of ORFs that are reported by the ORF finder that together form this gene?
Answer: ORF12 291..1
ORF8 4242..3805