Gene 5
Phage Name: 
Stop Coordinate 
Direction (Forward or Reverse) 
Gap (Overlap) with Previous Gene 
Selected Start Coordinate 
Selected Function 
Membrane 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, Gene 5 was called by both Glimmer and Genemark 
"Original Glimmer call @bp 2510 has strength 7.13" 
Is there evidence for coding potential? 
Yes - GeneMarkS displays strong codon potential - as seen below.  Genemark also shows coding potential.   
Is the gene present in other annotated genomes? 
Yes, homologues exist in both Uzumaki (5) and Zeina (5). This indicates that Gene 5, labelled gene 6 in phamerator, is conserved, further suggesting that is a gene. 
Does the gene violate any of the major guiding principles 
Kind of – this gene is a little short, indicating that this may require further examination. However, otherwise this gene does not break any of 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? 
Both call at 2510, Glimmer score of 7.13 
"Original Glimmer call @bp 2510 has strength 7.13" 
Does the start site have an associated ribosome binding site with a high score? 
No – there is a low score for the position 2510 start site. This site has the lowest Z value and the lowest final score. However this phenomena could be due to posttranscriptional regulation, wherein a poor promotor is used to produce small amounts of some gene deliberately.  
Is the predicted start codon the longest ORF? If not, does the longest ORF result in excessive overlap (>30bp)? 
Yes, this supports that 2510 is the start site as the particular 2510 site maximizes coding potential.  
Is the start site conserved in other genomes as indicated by Starterator? 
Yes, the 2510-type start site is conserved in Zeina, Uzumaki, and Gantchergoblin. This supports that 2510 is indeed the Lewando_5 start site. 
Is the start site conserved in other phage genomes as indicated by BLASTp? 
100% query cover with an arthrobacteriophage Zeina protein. The start site conservation, along with identical gene length, support this start site call.  
BlastP PhagesDB: Q1:S1 
BlastP NCBI: Q1:S1 
BlastP DNA Master: Q1:1 
Start site is at the 2510 
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 Lewando protein appears homologous to Zeina_5, which is labelled as both a membrane and unknown protein.  
PhagesDB BLAST: e-value = 4e-19 
NCBI BLAST:  e-value = 3e-20 
DNA Master BLAST:  e-value = 2.3e-8 
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. Matches do seem to indicate that this is a glycosylated prodien, and potentially a glycophorin. A transmembrane domain was detected with Hhpred.  
Database was NCBI conserved domains 
DeepTmHmm calls one transmembrane domain that corresponds to the transmembrane domain called by Sousi that is around 17a.a 
Number of predicted TMHs = 1 
Not sufficient evidence, must look at another source  
Exp. Number of AAs in TMHs = 27.9 
Total probability of N-in = 86.7% 
Probability that the N-term is on the cytoplasmic side of the membrane  
No. TMH = 1 
Region = 21-43210 
TM sequence: VCGLILGIIGTYVSMFMIALIHI 
(a) Hydrophobicity plot  
(b) Net charge density 
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. The protein is here labeled Lewando 6.  
Is the proposed function found on the SEA-PHAGES approved function list? 
Membrane protein