1. I'm guessing the rop gene's promoter just isn't annotated, most bacterial promoters aren't annotated. Honestly most of the time rop itself isn't annotated separately and gets lumped into a generic "ori" region. But yes, hypothetically if there's no transcriptional terminator between the lacI promoter and the rop gene then the lacI promoter could drive expression.

2. Kozak/IRES are eukaryotic ribosome binding sites, bacteria don't use them. E. coli's consensus sequence ribosome binding site is something like AGGAGG, usually located 5-7 basepairs upstream of the start codon, but it's rare for the binding site to match that sequence totally, like AGGAGA is a valid ribosome binding site for example.

3. This depends a lot on what the intervening sequence is exactly. I don't know the actual range but I've seen cases where the promoter is located ~1000 bp upstream of the translated gene and it still functioned. In E. coli transcription is typically terminated either by a rho-independent terminator that forms a RNA loop that halts the RNA polymerase or by rho-dependent termination where a Rho Factor protein binds to certain sequences and halts transcription. If any of these terminator sequences exist between the promoter and the downstream gene, the chance that transcriptional read-through will happen is much slimmer than it would be otherwise.

4. Plasmid replication is kind of a balancing act between copy number and host cell health. Not only does a plasmid use up host resources to replicate but it also increases gene dosage with copy number. pUC19 has the pBR322 ori with a couple point mutations and the rop protein removed, and can reach ~500 copies/cell compared to pBR322's ~20 copies. The pUC19 ori is almost never used for protein expression plasmids because at that huge copy number it can totally overwhelm the cell's transcription and translation systems and cause a lower overall yield of protein than a lower copy number origin. High copy number is good for greater DNA yields sometimes but it causes a whole range of issues when considering other factors. The reason natural plasmids evolved these copy control mechanisms was they decreased their host's fitness if they replicated too much, and since they're totally dependent on the host for replication this was detrimental to them.

1. I'm guessing the rop gene's promoter just isn't annotated, most bacterial promoters aren't annotated. Honestly most of the time rop itself isn't annotated separately and gets lumped into a generic "ori" region. But yes, hypothetically if there's no transcriptional terminator between the lacI promoter and the rop gene then the lacI promoter could drive expression.

2. Kozak/IRES are eukaryotic ribosome binding sites, bacteria don't use them. E. coli's consensus sequence ribosome binding site is something like AGGAGG, usually located 5-7 basepairs upstream of the start codon, but it's rare for the binding site to match that sequence totally, like AGGAGA is a valid ribosome binding site for example.

3. This depends a lot on what the intervening sequence is exactly. I don't know the actual range but I've seen cases where the promoter is located ~1000 bp upstream of the translated gene and it still functioned. In E. coli transcription is typically terminated either by a rho-independent terminator that forms a RNA loop that halts the RNA polymerase or by rho-dependent termination where a Rho Factor protein binds to certain sequences and halts transcription. If any of these terminator sequences exist between the promoter and the downstream gene, the chance that transcriptional read-through will happen is much slimmer than it would be otherwise.

4. Plasmid replication is kind of a balancing act between copy number and host cell health. Not only does a plasmid use up host resources to replicate but it also increases gene dosage with copy number. pUC19 has the pBR322 ori with a couple point mutations and the rop protein removed, and can reach ~500 copies/cell compared to pBR322's ~20 copies. The pUC19 ori is almost never used for protein expression plasmids because at that huge copy number it can totally overwhelm the cell's transcription and translation systems and cause a lower overall yield of protein than a lower copy number origin. High copy number is good for greater DNA yields sometimes but it causes a whole range of issues when considering other factors. The reason natural plasmids evolved these copy control mechanisms was they decreased their host's fitness if they replicated too much, and since they're totally dependent on the host for replication this was detrimental to them.

Thank you, Alexandra~ for your nice and detailed answers.

> You are right, Kozak/IRES for eukaryotic ribosome binding sites, the bacteria use SD sequence~. Sorry, I confused them.

> For rop, if I understand well what you mean, could I can say that, in general, low copy plasmid is better for protein synthesis, and high copy plasmid is preferred to plasmid amplification ?

I think you need to recognize that top and copy control are natural phenomenon such that plasmids in nature normally carry them so that their is not a strong negative selection against them. Laboratory created plasmids are different and don't need to compete in nature, so we often want high copy plasmids because that makes our lives easier.

While I agree with Alexandra that uncontrolled high level expression is not good (in general), you can still have a high copy number plasmid used for expression if the expression levels are tightly controlled.

It is clear, thank you all~