Gene 3
Phage Name: 
No number yet 
Stop Coordinate 
Direction (Forward or Reverse) 
Gap (Overlap) with Previous Gene 
-10 overlap 
Selected Start Coordinate 
Selected Function 
HNH Endonuclease 
Annotation Decision 1 (Is this a gene?): 
Gathering Evidence 
Explain Your Rationale 
Was the gene called by an auto-annotation program (Glimmer, GeneMark) 
Yes on genemark – I could only see on the genemark page. This gene was not shown in DNA Master. A different, reverse, gene 3 was called.  
"Original Glimmer call @bp 1441 has strength 2.96 ** not called by GeneMark" 
Is there evidence for coding potential? 
Yes - although it is only order 2 coding potential as indicated by GeneMarkS. GeneMark shows little coding potential (likely due to GeneMark calling this region as a reverse gene) 
Is the gene present in other annotated genomes? 
Yes – in Zeina, Uzumaki as seen in phamerator. Genes 3 in Lewando, Uzumaki, and Zeina appear homologous 
Does the gene violate any of the major guiding principles 
 Yes, this is a gene 
Annotation Decision 2 (What is the best possible start site for this gene?): 
Gathering Evidence 
Explain Your Rationale 
What start site do Glimmer and GeneMark suggest? 
1100 has the second best Z value, 1094 is first, and the 1100 bp start site has the best final score, as the 1094 start has a relatively large spacer distance. Overall, this indicates that the 1100 bp start site is a viable translational start site.  
Does the start site have an associated ribosome binding site with a high score? 
No start site suggested in DNA master 
Is the predicted start codon the longest ORF? If not, does the longest ORF result in excessive overlap (>30bp)? 
No – it does not result in the largest ORF. Some upstream start sites would result in excessive overlaps but not all of them. This does not support the start site call.  
Is the start site conserved in other genomes as indicated by Starterator? 
Yes – 6 matching start sites. Homology in other phages for the start site supports this start site call. The 1100 start site was the most common start site.  
Is the start site conserved in other phage genomes as indicated by BLASTp? 
Yes – conserved start site relative to Zeina_3 and Uzumaki_3. This conservation supports the 1100 start site.  
BlastP PhagesDB: Q1:S1 
BlastP NCBI: Q1:S1 
BlastP DNA Master - No result (not originally called) 
Start site at 1100 
Annotation Decision 3 (What is the function of the putative protein?): 
Gathering Evidence 
Explain Your Rationale 
Does the protein align with a protein having functional assignment in BLASTp (phagesDB and/or GenBank) with an alignment of 10^-4 or smaller? 
Yes, the Lewando proteins aligns with many HNH endonucleases. The most of similar of them is the Zeina_3 HNH endonuclease, which itself has a alignment of 1e-61 on phages db. E value of NCBI is 2e-74. 
PhagesDB BLAST: e-value = 1e-61 
NCBI BLAST:  e-value = 2e-74 
DNA Master BLAST:  n/a 
Does this protein align with a protein having a functional assignment in the PDB or other database in HHPred with a probability of 90% or greater with appropriate coverage? 
Quality match 
Yes, match with a HNH endonuclease. E value of 7.8e-8 and probability 98.71. Coverage appears appropriate.  
Database was PDBe 
To note, in the HNH domain of this protien, the N residue is replaced by an F residue. This is a feature conserved in the AU6 subcluster, and seems to be a feature of the AU6 endonuclease. Results are shown below.  HNH signature at position 75-124 in 5H0M
Is the gene located adjacent to genes of known function and in a region of the genome that shows high conservation of gene order? 
Yes, this HNH endonuclease is located before a conserved endolysin gene in the AU6 subcluster and a conserved hypothetical protein. This indicates that the gene order here is conserved.  
Is the proposed function found on the SEA-PHAGES approved function list? 
HNH Endonuclease 