Gene 1
Sunday, April 30, 2023
3:32 PM
Phage Name: 
Stop Coordinate 
Direction (Forward or Reverse) 
Gap (Overlap) with Previous Gene 
Selected Start Coordinate 
Selected Function 
Hypothetical Protein 
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 
"Original Glimmer call @bp 334 has strength 5.71" 
Is there evidence for coding potential? 
Yes, GeneMarkS shows coding potential. This  is pictured below. GeneMark also called this gene as well. 
Is the gene present in other annotated genomes? 
Yes, phamerator match to phage Uzumaki. This indicates that this phage gene has homologous genes within other phages in the AU6 sub cluster. Gene 1 in Lewando and Uzumaki are homologous. 
Does the gene violate any of the major guiding principles? 
No, there is a large gap though between this gene and the start codon, this may not be relevant though. Otherwise, this gene appears to follow the major guiding principles.  
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? 
Glimmer call at 334 
"Original Glimmer call @bp 334 has strength 5.71" 
Does the start site have an associated ribosome binding site with a high score? 
283 has the best final score, and is tied for the best z score. By in large, this indicates, statistically, that 283 is a good start site. The start site called by glimmer, at bp 334, has both a lower z score and final score. This indicates that 283 may be a better start site than the 334 start site. 
Is the predicted start codon the longest ORF? If not, does the longest ORF result in excessive overlap (>30bp)? 
No, there could be one further downstream start site, at bp277. This site does have a slightly worse final score than start site 283.  
Is the start site conserved in other genomes as indicated by Starterator? 
283 is the site with the most conservation in start site as indicated by starterator. This evidences the 283 start site, rather than the 334 start site. The 334 site does not appear to be conserved. This further suggests 283 as the start site. 
Is the start site conserved in other phage genomes as indicated by BLASTp? 
Not really, Zeina and other AU6 genes have start sites that are between 334 and 277 based on the queries, however not specifically at either position. Additionally, Uzumaki and Zeina genes 1 all appear to encode shorter proteins than Lewando_1. The highest scoring BLASTp matches do not align Q1:S1. 
BlastP PhagesDB: Q18:S1 
BlastP NCBI: Q18:S1 
BlastP DNA Master: Q1:18 
283 is the 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? 
The best match with a function attributed is the Zeina_1 protein, which appears to be a membrane protein. Matches are below 10^-4 in all databases. To note Zeina_1 does not have an assigned function in phages db . 
PhagesDB BLAST: e-value = 2e-33 
NCBI BLAST:  e-value = 2e-38 
DNA Master BLAST:  e-value = 9.4e-26 
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? 
No quality match 
None of the matches were particularily informative. The onlt match with 90% or greater was a family of unknow proteins. Database: NCBI conserved domains. 
To investigate whether this protein may be a membrane protein, a search with deepTMHMM was preformed (as per the functions guide). No transmembrane domains were found.  
Is the gene located adjacent to genes of known function and in a region of the genome that shows high conservation of gene order? 
There is syntenny in this region. HNH endonuclease, endolysin, and an uncharecterized protein al;l appear to be conserved in the AU6 subcluster. These proteins may be important in stting up replication and lysis.   
Is the proposed function found on the SEA-PHAGES approved function list? 
Hypothetical Protein 