Gene 4
Sunday, April 30, 2023
3:36 PM
Phage Name: 
Stop Coordinate 
Direction (Forward or Reverse) 
Gap (Overlap) with Previous Gene 
Selected Start Coordinate 
Selected Function 
Annotation Decision 1 (Is this a gene?): 
Gathering Evidence 
Explain Your Rationale 
Was the gene called by an auto-annotation program (Glimmer, GeneMark) 
Yes, called by both Glimmer and GeneMark. This suggest strong coding potential in this region. 
"Original Glimmer call @bp 1530 has strength 6.04" 
Is there evidence for coding potential? 
Yes, GeneMarkS shows coding potential, as indicated below. For this long gene. Genemark also displays coding potential.  
Is the gene present in other annotated genomes? 
Yes – Zeina (4), Uzumaki (4), Gantchergoblin (3) align with Lewando Gene 4. To note, Lewando Gene 4 is labelled as 5 due to the reverse gene called by GeneMark that was later removed.  
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? 
1530, strength of 6.04 
"Original Glimmer call @bp 1530 has strength 6.04" 
Does the start site have an associated ribosome binding site with a high score? 
Yes, there was a Z value of 3.298 for the start site at 1530. This was the highest Z score by far out of all of the possible start site. The 1530 site also had the best final score, as well as a solid spacer distance.  
Is the predicted start codon the longest ORF? If not, does the longest ORF result in excessive overlap (>30bp)? 
No, it is not the longest ORF. Further upstream start sites could lead to a larger transcript.  
Is the start site conserved in other genomes as indicated by Starterator? 
Yes, same conserved start site in 53 other phages. This level of conservation supports the 1530 start site call.  
Is the start site conserved in other phage genomes as indicated by BLASTp? 
100% query cover with Uzumaki. This alignment with another AU6 subcluster phage shows that the correct start site was likely picked.  
BLASTp PhagesDB: Q1:S1 
BLASTp NCBI: Q1:S1 
BLASTp DNA Master: Q1:1 
1530 start site 
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 endolysis. The most of similar of them is the Uzumalo, which is a quite high scoring match (613 o NCBI). E value of NCBI is 2e-74. 
PhagesDB BLAST: e-value = 1e-173 
NCBI BLAST:  e-value = 0 
DNA Master BLAST:  e-value = 0 
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 endolysin. E value of 7.3e-12 and probability 99.46. Coverage appears appropriate.  
Database was PDBe 
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 gene is in an ordered region of the genome, however due to a large proportion of hypothetical proteins, it is unclear what the function of this region may be. 
Is the proposed function found on the SEA-PHAGES approved function list? 