I'm currently having issues with a golden gate assembly I've been working on for months. The plasmid I'm trying to assemble consists of a backbone- promoter-intron-GOI-Terminator- antibiotic resistance gene which is then linked to the backbone. I've checked all the individual fragments through sequencing so I know the over hangs are correct and the sequences are correct. all the overhangs line up with the fragment they should ligate to. I'm having trouble with assembly when I use one specific GOI. I use the same backbone, promoter, intron, terminator and antibiotic resistance gene in all my golden gates and they work fine, but with this particular GOI it is not assembling correctly if at all. All the sequencing I've done when using this particular GOI usually is missing everything but the backbone and antibiotic resistance gene and I cant figure out why.
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