Helping to your question number 1

you will get more information from this article link provided below (Nucleic acid journal)

Title of article---PrimerZ: streamlined primer design for promoters,

exons and human SNPs

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933185/pdf/gkm383.pdf 

Is this also applicable to bacterial (prokaryotic) promoters? I'm designing vector for bacterial expression.

Hi Edward,

This review should give you a good start on understanding bacterial promoters:

https://www.researchgate.net/publication/8666569_The_regulation_of_bacterial_transcription_initiation

Regarding terminators & stop codons: be careful not to confuse transcription with translation. Terminators stop the synthesis of mRNA by the RNA polymerase; stop codons stop the synthesis of protein by the ribosome. They do two entirely different jobs. In plasmids they are important as the polymerase can continue round the plasmid, producing mRNA for all the genes in that orientation. This read through from a resistance gene promoter can cause problems from unwanted expression before induction.

- Richard

Article The regulation of bacterial transcription initiation

@Richard Thank you! Great resource! I will definitely look in to it. Your explanation on terminators sums it all. That is really confusing to me, but thanks for reminding me. But I need to study more, any suggestion for a good Terminator prediction program for Gram-positive bacteria?

If you're designing a bacterial  expression vector, there are known bacterial promoters that you can use. They have been around for a while and some commercial plasmids have them. Unless you want express your protein using the native promoter?

Yes. I am designing an expression vector myself. And I've verified expression of some of the genes using my native promoters. However, I am just wondering if some promoters (let's say) of gene 2 is in the sequence of gene 1 (preceding gene), particularly if the genes are just 20 to 100 bp apart. Are there still room for promoters in that gap? Or are they in the sequence of the preceding gene (gene 1)?

I am just verifying if the logic is correct that virtually all promoters are -35/-10 upstream of the target gene regardless of the gap or distance with another upstream gene.

I've looked at several gene sequences in the genome of  D. discoideum, my  model organism and in all of the genes I've looked at, there are usually intronic sequences that spans more than 35 bps in between genes but then I can't say for all the genes in the genome since not all promoter sequences in all genomes have been characterized.  You can check the DNA sequences of organisms at NCBI and maybe look for gene clusters without intronic gaps and maybe one could argue that its possible. 

Thanks for your answer. Interesting, my model organism is Lactobacillus species. What I do is extract DNA sequences upstream of the several genes in the genome and find a good prokaryotic promoter predictor. And Neural Network and BPROM shows that there are possible promoters at 80% threshold. But I just want to know if there are others who have done this procedure. Nevertheless, thank you so much for your response @Dr. Romeo.

Thank you. I will check it out. In my readings, there's also concept of "co-expression". I should study more about gene expression and genes in the genome. I appreciate the assistance on my struggle about bacterial gene expression.