The Blast program (NCBI) was used for database search. Parameters of the oligonucleotides were examined using the OligoAnalyzer 3.1 (Integrated DNA Technologies, Coralville, IA, USA). The sequence alignments were performed using the software package Lasergene Version 10 (DNASTAR Inc., Madison, WI, USA). SecretomeP2 server (https://services.healthtech.dtu.dk/service.php?SecretomeP-2.0 , accessed on 18 June 2016) was used to predict the possible way of KYNU secretion and UniProt resources (https://www.uniprot.org/ , accessed on 19 June 2016) were used for the structural analyses. ENCODE (https://www.encodeproject.org/experiments/ENCSR329MHM/ , accessed on 19 June 2016) transcriptome analysis was used to estimate the KYNU expression level in HepG2, HeLa, U87MG, and A549 cells. Octopus topology program (Stockholm University, Stockholm Bioinformatics Center, Stockholm, Sweden) was used to estimate transmembrane topology.
Lasergene version 10
Manufactured by DNASTAR
Sourced in United States
Lasergene Version 10 is a comprehensive software suite for analysis of biological sequence data. The core function of Lasergene is to provide tools for tasks such as sequence assembly, alignment, and analysis.
Automatically generated - may contain errors
5 protocols using lasergene version 10
1
Comprehensive Bioinformatic Analysis of KYNU
2
Bioinformatic Sequence Alignment Pipeline
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The Basic Local Alignment Search Tool (BLAST) program from the National Center for Biotechnology Information (NCBI) [26 ], was used for search. Oligo analyzer 3.1 (Integrated DNA technologies, Coralville, IA, USA) was used to estimate parameters of the oligonucleotides. The sequence alignments were performed using software package Lasergene Version 10 (DNASTAR, Inc., Madison, WI, USA).
3
Bioinformatic Sequence Analysis Workflow
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The Blast program (NCBI) was used for database search. The sequence alignments and neighbour-joining phylogenetic analyses were performed using software package Lasergene Version 10 (DNASTAR, Inc. Madison, USA).
4
Cloning and Sequencing of PCR Amplicons
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PCR amplicons were cloned into the pCR2.1-TOPO vector according to the protocol of the supplier (Thermo Scientific, Waltham, MA, USA). JM109 cells (Promega, Corp., Madison, WI, USA) were transformed and plated on LB/ampicillin agar for 16 h at 37 °C. Selected colonies were grown in LB/amp overnight at 37 °C. The plasmids were isolated using the PureYield plasmid mini preps system (Promega), tested for the inserts by digestion with Eco RI and sequenced using BigDye terminator kit V.3.1. (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using LaserGene Version 10 (DNASTAR, Inc., Madison, WI, USA) and Geneious package software (Biomatters Ltd., Auckland, New Zealand).
5
Computational Bioinformatic Analyses of Protein
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The NCBI Blast program was used for the database search. Parameters of PCR primers were selected and analyzed using the OligoAnalyzer 3.1 (Integrated DNA technologies, Coralville, IA, USA). Sequences were aligned using Lasergene Version 10 (DNASTAR, Inc., Madison, WI, USA) and Geneious package (Biomatters Ltd., Auckland, New Zealand) software. The image processing program (ImageJ, US National Institutes of Health, Bethesda, MD, USA) was used to compare protein bands intensity and ExPasy NetNGlyc 1.0 package was used to predict N-glycosylation sites. The ProP 1.0 Server (https://services.healthtech.dtu.dk/ (accessed on 15 February 2020)) was used to predict furin cleavage sites.
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