Gene 33
28658
Direction (forward or backwards) 
Gap (overlap) with previous gene 
6bp gap
Selected start coordinate 
Membrane protein 
Is this a Gene? 
Gathering Evidence 
Was the gene called by an auto-annotation program (Glimmer, GeneMark)? 
Is there evidence for coding potential? 
There is evidence of a strong coding potential around 28350-28650bp 
Evidence of an earlier start site upstream? Coding potential seen before the black box  
Is this gene present in other annotated genomes? 
Present in DNA Master auto-annotated genome and Pharmerator genome (called gene 36) 
Pharmerator: E-value = 0.000e+0 
Does the gene violate any major guiding principles? 
What is the Best Possible Start Site for this Gene? 
Gathering evidence 
What start site do Glimmer and GeneMark suggest? 
Glimmer and GeneMark @28332bp 
Does the start site have an associated RBS with a high score? 
Start @28188bp: Z-score = 2.369; SD= -3.185; final score = -4.407 
ATG start codon 
Z-score >2, good 
Lower final score 
ORF = 471bp 
Start @28212bp: Z-score = 2.679; SD= -2.535; final score = -3.292 
ATG start codon 
Slightly larger Z-score and final score 
ORF = 447bp 
Start @28227bp: Z-score = 2.731; SD= -2.426; final score = -3.201 
TTG start codon -> guiding principles? 
Best/highest Z-score and final score 
ORF = 432bp 
Start @28332bp: Z-score = 2.016; SD= -3.925; final score = -4.970 
ATG start codon 
Good Z-score (>2) and final score, but not the best  
Is the predicted start codon the longest ORF? If not, does it result in excessive overlap (>30bp)? 
Only the first start sites would have overlap with the previous gene. 
Start @28188bp => 19bp overlap 
The rest of the start sites would result in a gap between gene 32 and 33  
Start @28212bp => 6bp gap 
Start @28227bp => 21bp gap 
Start @28332bp => 126bp 
Too big to be a gap (>120bp), violates guiding principles 
Sequences longer than 120bp may indicate the presence of another gene, but there is no evidence of an additional ORF, so therefore this likely means this is NOT the start site  
Is this start site conserved in other phage genomes as indicated by Starterator? 
Start @28188bp (start #8): 
Found in 76.8% of genes in pham 
Called 20.8% of time when present  
Conserved in other AM and AU1 phages  
Not seen to be conserved in other AU6 phages  
Start @28212bp (start #13): 
Found in 10.1% of genes in pham 
Called 74.1% of time when present  
Conserved in other AU6 phages (GantcherGoblin, Argan, Uzumaki) as well as some members of FK phage subcluster  
Start @28227bp (start #17): 
Found in 33.3% of genes in pham 
Called 8.7% of time when present  
Conserved in phages in the AM subcluster  
Start @28332 (start #32):  
Found in 11.6% of genes in pham 
Called 12.5% of time when present  
Only called in Lewando phage  
Is this start site conserved in other phage genomes as indicated by BLASTp? 
Weaker conservation of this start site (@28332bp), there are no BLASTp genes with 100% alignment to the start site. All search results have start sites upstream of the Glimmer predicted start.  
Present in BLASTp (NCBI) genomes: [top 2 hits] 
Zeina: E-value = 2e-52, subject start @60, query start @1 
Sequence identity = 91%, strong sequence conservation but not start site 
Uzumaki: E-value = 3e-52, subject start @41, query start @1 
Sequence identity = 90%, strong sequence conservation but not start site 
Start @28212bp 
What is the Function of the Putative Protein?  
Gathering evidence 
Explain rationale 
Does this protein align with a protein having a functional assignment in BLASTP (PhagesDB and/or GenBank) with alignment of 10-4 or smaller with appropriate coverage? 
Majority of hits for 'function unknown' 
Majority have E-values of 10-4 or smaller 
Top hits for membrane protein 
This for hypothetical protein  
Top hit (Zeina): membrane protein, E-value = 1e-63, 91.7% identity, 100% query cover  
DNA Master: 
Does this protein align with a protein having a functional assignment in the PDB or other databases in HHpred with a probability of 90% or greater with appropriate coverage?  
No hits with >90% probability  
Top hit: 38.7% probability for cytochrome b-245 light chain 
E-value = 240 
Applying PHROGS database: 
99.8% probability for membrane protein  
E-value = 1.7e-20 
99.8% probability for membrane protein 
E-value = 1.7e-17 
Is this gene located adjacent to genes of known function and in a region of the genome that shows high conservation of gene order? 
Deep TMHMM: 
No. of predicted TMRs = 3 
Pharmerator: 
Gene is conserved in other AU6 phages  
Length for this gene is shorter for Lewando than other AU6 phages  
No conserved/associated function with this gene 
Is the proposed function found on the SEA-PHAGES approved function list? 
Membrane protein 
NCBI BLASTp Search Results  