I am adding two genes into the same plasmid. I plan to make it as promoter1-gene1-terminator1--promoter2-gene2-terminator2. Does anyone know how many bp interspace should I leave between terminator1 and promoter2? thanks a lot!
Hi,
There is no strict rule to follow and it could go straight after the first terminator if you like.
If Im designing a vector with two units of expression, I prefer to place the promoters facing each other. So you can copy out all your "promoter2-gene2-terminator2" sequence, then derive its antiparallel complementary 5-3 sequence, and put it into your design. You can use online tools such as sequence massager to do so.
Then your vector will look like this "promoter1-gene1-terminator1-terminator 2-gene2-promoter2" Then your two terminators will be facing each other and you dont need to add any bases in between.
It is very important that you use the complementary strand so that the sequence you are inverting runs on the opposite DNA strand.
This also reduces the chances of promoter 1 influencing the expression of gene2 which can happen if they are on the same orientation, i.e., the same strand.
Good luck!
The distance between a terminator and the promoter for the next gene in a genetic construct can vary depending on several factors, including the specific regulatory elements used, the type of organism, and the intended purpose of the construct. There is no fixed or universally applicable distance between these elements.
In some cases, the distance between a terminator and a promoter can be relatively short, with only a few base pairs separating them. This arrangement is often seen in bacterial operons, where multiple genes are transcribed together as a single unit. In these cases, a single terminator is located at the end of the operon, and the promoter for the next gene in the operon is located immediately downstream of the terminator.
However, in other cases, there can be a larger distance between the terminator and the promoter of the next gene. This distance can vary from a few tens to thousands of base pairs. The specific requirements for spacing between the terminator and promoter can depend on factors such as the need for transcriptional termination efficiency, the presence of regulatory sequences or factors, and the potential for interference or read-through between adjacent genes.
It is worth noting that advances in synthetic biology and genetic engineering techniques have allowed for more precise control over the design of genetic constructs. With the use of customizable DNA synthesis and assembly methods, researchers can design constructs with specific distances and regulatory elements tailored to their experimental or application needs.
Therefore, when designing a genetic construct, it is important to consider the specific requirements of the genes and regulatory elements involved, taking into account factors such as transcriptional termination efficiency, potential interference, and overall construct functionality.
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