Open Human Plasmid
3845bp plasmid optimized for gene expression in human cells. contains an open reading frame to clone in genes either through a 5'XhoI - 3' BamhI digest/ligation or using either of the unique sites for gibson or other assembly.
Working on human genetic engineering I noticed the lack of plasmid designs that incorporate all the best features from literature on achieving the best expression. I also saw that most plasmids had only half of their backbone annotated leaving me wondering if it was necessary to have the rest of the DNA sequence. To this end I decided to design and annotate a full plasmid that can be used for human genetic engineering and make it open source.
Descriptions of DNA elements and why I chose them can be found below.
Name Location Length
rop Promoter? 1..309 309
rop 310..501 192
?? 502..561 60
bom spacer??? 562..602 41
bom 603..745 143
Origin spacer??? 746..930 185
Bacterial Origin of Rep 931..1519 589
Origin Promoter? 1520..1579 60
These features above are all involved in bacterial replication of the plasmid DNA. In order to make lots of plasmid DNA for transfection, the plasmid needs to be replicated in bacteria. Some people use minimal origins but studies have shown that including bom and maybe even rop increase the copy number of the plasmid and that means lots more for transfection. expression levels are partially due to the amount that gets inside the cells and partially due to the DNA elements on the plasmid. The more you can make the more you can inject.
In order to identify parts in these origin regions I compared the sequences across many plasmids and found sequences that matched across many plasmids to try and determine what is necessary. All the sequences in this plasmid have matched multiple other plasmids but the exact function of some of these regions is still unclear to me hence all the "?".
pOpenHum_Amp These are Pretty standard
AmpR 1580..2440 861
AmpR promoter 2441..2545 105
These allow for expression of ampicillin resistant gene in bacteria for selection and purification of the plasmid DNA.
The expression region of the plasmid is the most complicated. Understand that the exact base location will be different for each plasmid due to the different selection regions.
CMV enhancer 2621..3000380
CMV promoter 3001..3204204
post CMV??? 3205..329086
HTLV-1 Region 3292..3519228
bglobin splice site? 3520..3678159
bglobin poly(A) 3773..382856
The CMV-pre sequence is found in a number of plasmids before what is normally deemed the CMV enhancer. I don't know if this is necessary or enhances gene expression?
The CMV enhancer and promoter are standard sequences and we are using them because they are generally considered the best known promoters for highest level gene expression.
This post CMV sequence is included in a number of plasmids and most likely includes an exon and splice site that increases gene expression though the exact identity has not been found by me yet.
The HTLV-1 region has been shown to increase gene expression 5x-10x!
The bglobin exon 2 and splice site has been shown to increase gene expression though the exact sequence here is unknown.
The ORF is where to insert the open reading frame between XhoI and BamHI unique sites.
The spacer region is of unknown function but found in many other expression plasmids. The bglobin poly(A) terminates transcription. This allows higher gene expression levels.
This product is not injectable or meant for direct human use. The DNA needs to replicated and purified before use in human cells.