Details below on all constructs but after Gibson assembly the resulting assembly constructs will be in the following files:
Assembled_Ferritin_GS_linker_SpyCatcher003_6xHis.dna
Assembled_Ferritin_GS_linker_SpyTag002_6xHis.dna
Assembled_SpyCatcher003_GS_linker_Nanobody_6xHis.dna
Assembled_SpyTag002_GS_linker_Nanobody_6xHis.dna
ComponentPositionSequenceTranslationStart codon76-78ATGMHis6 tag~85-102CATCATCATCATCATCACHHHHHHThrombin site103-129AGCAGCGGCCTGGTGCCGCGCGGCAGCSSGLVPRGSSpyTag002130-171GTGCCTACTATCGTGATGGTGGACGCCTACAAGCGTTACVPTIVMVDAYKRYLinker172-189AAGGGTAGTGGTGAAAGTGGTKGSGEGVMBP start~190+AAAATCGAAGAAGGTAAACTGGTAATCTGG...KIEEG...
The SpyTag002 sequence translates to "VPTIVMVDAYKRY" when read in the correct frame, which is slightly different from the canonical "AHIVMVDAYKRYK" sequence - likely a variant of SpyTag002 optimized for this specific construct.
ComponentPositionSequenceSpyCatcher003 gene start155Position where the mature SpyCatcher003 coding sequence beginsSpyCatcher003 gene end589Position where the SpyCatcher003 sequence endsSpyCatcher003 length435 bpTotal length of the SpyCatcher003 gene
The SpyCatcher003 sequence starts with the codons for MVTTL (Methionine-Valine-Threonine-Threonine-Leucine), which is characteristic of the N-terminus of SpyCatcher003. This matches exactly with the information from Addgene, which indicates a 435 bp insert size.
Copy
atggtaaccaccttatcaggtttatcaggtgagcaaggtccgtccggtgatatgacaactgaagaagatagtgct
acccatattaaattctcaaaacgtgatgaggacggccgtgagttagctggtgcaactatggagttgcgtgattca
tctggtaaaactattagtacatggatttcagatggacatgtgaaggatttctacctgtatccaggaaaatataca
tttgtcgaaaccgcagcaccagacggttatgaggtagcaactccaattgaatttacagttaatgaggacggtcag
gttactgtagatggtgaagcaactgaaggtgacgctcatactggatccagtggtagctaaatggttgatgcttga
The sequence translates to a protein that aligns perfectly with the expected SpyCatcher003 protein sequence. At the C-terminus, we can see the GSSGS linker (GGA TCC AGT GGT AGC) that was mentioned in the Addgene information, followed by restriction sites that were used for cloning.
Near the end, we can identify the BamHI site (GGATCC) at position 523, which is likely part of the cloning strategy used to insert the SpyCatcher003 gene into the pDEST14 vector backbone.
The overall architecture of the construct is: