Design
The HMA3 Sequence is with 2619 bp quite long, so we decided to get it synthesized by IDT. First option was to use the whole sequence (2631 bp). CAMV35S is used as promoter with 341 bp.
Our idea is to cut with EcoRI at G/AATTC (above in red). Since this codon happened to be two times in our cDNA sequence, we needed to change this codon two times to prevent a second (and third) cut with EcoRI. You can see that above in blue/red. Then to cut with PstI at CTGCA/G to make sure the sequence. Second option is to use 4 part fragments, you can see the fractionation in the table below.
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