Part 1: Ferritin-linker-spytag

Goal: Make a final sequence which will contain the original plasmid of ferritin https://www.addgene.org/122652/

that will be combined with a flexible linker and spytag002 gene which should result in:

Ferritin (1 subunit of 24)-linker-spytag002

GGAGGCGGCGGCAGCTCTGGC | GTGCCTACTATCGTGATGGTGGACGCCTACAAGCGTTACAAG | AAGCTTGCGGCCGCACTCGAGCACCACCACCACCACC

in black there are overhangs which will combine with the ferritin construct after PCR amplification in Gibson assembly; in red spytag002 (plus maybe short linker 3 amino acids different)

After some struggles (in red highlight) and a lot of help from Claude AI I was able to get simulation properly from snapgene viewer in action>Gibson Assembly> Multiple Fragments

PCR_spy.dna

PCR_ferritinL.dna

Assembled_gibson_ferritin_linker_spytag.dna

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His-tag

We'll use Gibson Assembly with two fragments:

  1. The ferritin plasmid backbone (opened up at the junction between ferritin and His-tag)