SPINE: Saturated Programmable INsertion Engineering
Protein domain insertion via programmed oligo libraries
Python script for generating oligo libraries and PCR primers for programmed domain insertion
Targeted genes must be in fasta format and include a minimum of 30 bases surrounding gene.
Entire plasmid sequence is advised to search for nonspecific amplification
Final output is fasta format. One file for oligo pools and one file for PCR primers
Gene start is defined as base number of first base in first codon and gene end is defined as base number of last base in last codon. (Program will subtract 1 from gene start for python numbering)
To define gene position within given fasta file, add start:# end:# to fasta description. (Otherwise use command line prompts)
'>geneA start:11 end:40'
Domain Insertion Scanning: python3 run_spine.py -wDir tests -geneFile combined_fasta.fa -oligoLen 230 -mutationType DIS
Deep Mutational Scanning: python3 run_spine.py -wDir tests -geneFile Kir.fa -oligoLen 230 -mutationType DMS -usage ecoli
optional arguments: -h, --help show this help message and exit -wDir WDIR Working directory for fasta files and output folder -geneFile GENEFILE Input all gene sequences including backbone in a fasta format. Place all in one fasta file. Name description can include start and end points (>gene1 start:1 end:2) -handle HANDLE Genetic handle for domain insertion. This is important for defining the linker. Currently uses BsaI (4 base overhang), but this can be swapped for SapI (3 base overhang). -matchSequences Find similar sequences between genes to avoid printing the same oligos multiple times. Default: No matching -oligoLen OLIGOLEN Synthesized oligo length -fragmentLen FRAGMENTLEN Maximum length of gene fragment -overlap OVERLAP Enter number of bases to extend each fragment for overlap. This could help with insertion coverage close to fragment boundary. Overlap does not add additional insertions and thus no additional oligos. -mutationType Run deep insertion scan "DIS" or deep mutation scan "DMS" -usage USAGE Default is "human". Or select "ecoli"