title: SfiNX: a method for assembly of protein coding sequences with high success rates.
abstract: OBJECTIVE
  Concatenation of two NdeI-XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.
  
  
  RESULTS
  A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.
  
  
  CONCLUSIONS
  This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo-cho 3, Matsuyama, Ehime, 790-8577, Japan. takai.kazuyuki.mz@ehime-u.ac.jp.|The Venture Buisiness Laboratory, Ehime University, Bunkyo-cho 3, Matsuyama, Ehime, 790-8577, Japan. takai.kazuyuki.mz@ehime-u.ac.jp.
authors.forename: Kazuyuki
authors.identifier: http://orcid.org/0000-0001-7096-0312
authors.initials: K
authors.lastname: Takai
authors.affiliation: The Venture Buisiness Laboratory, Ehime University, Bunkyo-cho 3, Matsuyama, Ehime, 790-8577, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Hisamatsu
mesh_terms: D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D020648:Peptide Elongation Factor 1; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D014908:Triticum / Q000235:genetics
chemical_list: D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; C037106:endodeoxyribonuclease NdeI; C041749:endodeoxyribonuclease SfiI; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Escherichia coli; Fusion; Gene assembly; Polycistron; Rare cutter