Codon optimization tool

Manufactured by Integrated DNA Technologies

Sourced in United States

The Codon Optimization Tool is a software application that assists in the optimization of DNA sequences for efficient gene expression. The tool analyzes the codon usage of a given DNA sequence and provides recommendations to enhance the expression of the encoded protein in a specific host organism. The core function of the Codon Optimization Tool is to optimize the codon usage without altering the amino acid sequence of the target protein.

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Lab products found in correlation

Plv ef1a ires puro vector, by Addgene (2 mentions) Maxiprep, by Qiagen (2 mentions) Sequence manipulation suite, by Integrated DNA Technologies (2 mentions) Stbl3 e coli cells, by Thermo Fisher Scientific (2 mentions) Eblocks, by Integrated DNA Technologies (2 mentions) Nebuilder hifi dna assembly, by New England Biolabs (1 mentions) Pflag cmv3, by Merck Group (1 mentions) Gensmart codon optimization, by GenScript (1 mentions) Plasmid maxiprep kit, by Thermo Fisher Scientific (1 mentions) 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions) Pmax plasmid, by Lonza (1 mentions) Pqe 80l, by Qiagen (1 mentions) Kanamycin, by Bio Basic (1 mentions) Clonnat, by Jena Biosciences (1 mentions) Nucleobond xtra maxi kit, by Macherey-Nagel (1 mentions) Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions) Freestyle293 cells, by Thermo Fisher Scientific (1 mentions) Gene fragments, by Integrated DNA Technologies (1 mentions) Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions) Gibson assembly master mix, by New England Biolabs (1 mentions)

Close spelling variants of this product

IDT codon optimization tool (4 citations)

21 protocols using codon optimization tool

Optimized Plasmid Construction for Alr2 Isoform Expression

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The plasmid backbone used for all constructs in this study was the pFLAG-CMV-3 (Sigma, E6783). Previously, it was determined that the N-terminal FLAG tag did not have an effect on the binding capability of Alr2 (Karadge et al., 2015 (link)). The Hydractinia Alr2 allele sequences were optimized for human expression using the Integrated DNA Technologies (IDT) Codon Optimization Tool (https://www.idtdna.com/CodonOpt). The full Alr2 sequence (domain 1 in the ectodomain through the cytoplasmic tail) for 111A06 and domain 1 sequences for Anc, 046B, Hap074, and 214 × 10⁶ were ordered as gBlocks Gene Fragments from IDT. All other mutant domain sequences were ordered from Twist Bioscience as Gene Fragments. Coding sequences for fluorescent proteins were cloned from vectors encoding eGFP and mRuby2 (gift from Michael Davidson, Addgene plasmid #54614 (Lam et al., 2012 (link))). Cloning was performed using the NEBuilder HiFi DNA Assembly (New England Biolabs, E2621S) with primers designed to amplify the vector and insert sequences with ≥20 bp overlap. The FLAG-111A06-eGFP/mRuby2 plasmids (pUP801, pUP746) were cloned first and then used as the template for cloning in the other domain 1 isoforms. Within the construct, linker sequences were used before (Leu-Ala-Ala-Ala) and after (Gly-Pro-Pro-Val-Glu-Lys) the Alr2 allele.

Huene A.L., Chen T, & Nicotra M.L. (2021). New binding specificities evolve via point mutation in an invertebrate allorecognition gene. iScience, 24(7), 102811.

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Codon Optimization and mRNA Structure Analysis

Cited 1 time

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For the vaccine mRNA to be efficiently translated by the host cells, codon optimization is important. Therefore, the codons of the final vaccine construct were optimized for efficient expression in human cells using several Codon Optimization Tools; JCat [72 (link)], GeneArt Instant Designer by Thermofisher, GenSmart™ Codon optimization by GenScript (GS), Codon Optimization Tool by Integrated DNA Technologies (IDT). The quality of the optimized codons was analyzed Using Rare Codon Analysis tools by GS. This tool can predict the efficiency of the translation of the mRNA expressed as the codon adaptation index (CAI) value. Also, the presence of any tandem unusual codons can be detected, shown as codon frequency distribution (CFD). Based on these parameters, the best-optimized sequence was chosen for further assessment.
The secondary structure of the mRNA construct was predicted using the RNAfold tool of ViennaRNA Package 2.0 [73 (link)]. Both the minimum free energy (MFE) structure and the centroid secondary structure of the mRNA were obtained from this tool along with their MFE.

Shahrear S, & Islam A.B. (2022). Modeling of MT. P495, an mRNA-based vaccine against the phosphate-binding protein PstS1 of Mycobacterium tuberculosis. Molecular Diversity.

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Optimized Cell-free Expression of synMinE

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The amino acid sequences of each synMinE variant were reverse-translated into DNA sequences using the Codon optimization tool from Integrated DNA Technologies (Coralville, IA USA) to optimize codon usage by referencing Escherichia coli K12. Then, the sequence of the first 30 bp (10 amino acids) were further altered by maximizing the frequency of A and T bases while keeping the translated amino acids to optimize the cell-free expression yield. Then, 5’ and 3’ additional sequences (Supplementary Table 1) coding T7 promoter, Ribosome binding site, T7 terminator etc. were further attached to the synMinE sequences. The resultant 48 sequences were synthesized using eBlocks Gene Fragments service (Integrated DNA Technologies).

Kohyama S., Frohn B.P., Babl L, & Schwille P. (2024). Machine learning-aided design and screening of an emergent protein function in synthetic cells. Nature Communications, 15, 2010.

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Optimized Bacterial Expression of Human FLG

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The human eighth FLG repeat unit (324 amino acids) was selected for this study (Figure 1A). The WT nucleotide sequence was optimized for E. coli codon usage bias (strain K12) using a codon optimization tool (Integrated DNA Technologies) (Supplementary Figure 1). The WT and E. coli codon-optimized FLG coding sequences (so-called “Codon Opt” further on) were synthesized (Integrated DNA Technologies) and cloned into plasmids pJL1 and pETBlue-1 by Gibson Assembly (Gibson et al., 2009 (link)) for CFPS and in vivo protein synthesis, respectively. 6xhistidine tag was added to the C-terminal end of FLG during PCR. E. coli DH5α competent cells were used for the cloning host. The sequences have confirmed by DNA Sanger-Sequencing using the 3130xl Genetic Analyzer (Applied Biosystems). The recombinant plasmids were isolated by plasmid maxiprep kit (Invitrogen, Waltham, MA). A schematic cloning workflow is described in the Supplementary Figure 2.

Kim J., Copeland C.E., Seki K., Vögeli B, & Kwon Y.C. (2020). Tuning the Cell-Free Protein Synthesis System for Biomanufacturing of Monomeric Human Filaggrin. Frontiers in Bioengineering and Biotechnology, 8, 590341.

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Cloning and Characterization of GFP-ENPL Fusion Protein

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Amino acid sequences of rat (Q66HD0) and mouse (P08113) ENPL were obtained from the UniProt database (https://www.uniprot.org/). The sequences were fit by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and Protein Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) accessed on 3 January 2020. Altogether, a similarity of 98.01% was found between the ENPL primary amino acid sequences in the two species (Supplementary Figure S1). The Q66HD0 rat ENPL sequence was used in our work. Based on the protein sequence, codon optimization was performed with Codon Optimization Tool of Integrated DNA Technologies, Inc. (https://eu.idtdna.com/) accessed on 3 January 2020. A GGSGGGSG linker was inserted between the GFP and ENPL sequences. The gene encoding the GFP-ENPL fusion protein was synthetized by Bio Basic Inc., (Markham, ON, Canada) and was inserted into a pMAX plasmid (Lonza, Basel, Switzerland) for in vitro expression. The DNA sequence of the pMAXGFP-ENPL is available in Supplementary Figure S2.

Koncz A., Turiák L., Németh K., Lenzinger D., Bárkai T., Lőrincz P., Zelenyánszki H., Vukman K.V., Buzás E.I, & Visnovitz T. (2023). Endoplasmin Is a Hypoxia-Inducible Endoplasmic Reticulum-Derived Cargo of Extracellular Vesicles Released by Cardiac Cell Lines. Membranes, 13(4), 431.

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Cloning and Expression of Fluorescent Fusion Proteins

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All constructs were generated using FX-cloning and FX-compatible vectors^{43 (link)}. Genes encoding full-length human TTYH proteins and the human AQP4 were codon-optimized for expression in human cell lines using the codon optimization tool from Integrated DNA Technologies and synthesized by GenScript. The genes were cloned into a pcDX vector containing a C-terminal Rhinovirus 3C Protease linker followed by Venus^{44 (link)} in case of TTYH genes and mCherry in case of AQP4 (ref. ^{45 (link)}), a Myc-tag, and a streptavidin-binding peptide (SBP)^{46 (link)}. For electrophysiological recordings, surface expression analysis, and liposome reconstitution, the TTYH genes were cloned into an analogous vector not containing Venus. Genes encoding murine LRRC8A and C cloned into the same vector were used for electrophysiological experiments^{28 (link)}. The Venus-only construct used in electrophysiology contained the Venus gene followed by a Myc-SBP tag.

Sukalskaia A., Straub M.S., Deneka D., Sawicka M, & Dutzler R. (2021). Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions. Nature Communications, 12, 4893.

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ACE2 Protein Expression and Purification

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The Homo sapiens angiotensin-converting enzyme 2 (ACE2), transcript variant 2 amino acid sequence (NCBI Reference Sequence: NM_021804.3) was reverse translated using the Sequence Manipulation Suite and codon optimized using Integrated DNA Technologies’ Codon Optimization Tool. This fragment was synthesized as a gene block (IDT), with 5’-TTTTCTTCCATTTCAGGTGTCGTGAGGATCC added to the 5’ end and 5’-TGAGAATTCCTCGAGGGCGGCCGCTCTAGAGTC added to the 3’ end. This product was then inserted into the pLV-EF1a-IRES-Puro vector (Addgene Plasmid #85132) that had been digested with EcoRI and BamHI using Gibson Assembly (NEB). The sequence of the resulting construct was confirmed by Sanger sequencing and propagated in Stbl3 E. coli cells (Life Technologies) at 30°C followed by MaxiPrep (Qiagen).

Puray-Chavez M., LaPak K.M., Schrank T.P., Elliott J.L., Bhatt D.P., Agajanian M.J., Jasuja R., Lawson D.Q., Davis K., Rothlauf P.W., Jo H., Lee N., Tenneti K., Eschbach J.E., Mugisha C.S., Vuong H.R., Bailey A.L., Hayes D.N., Whelan S.P., Horani A., Brody S.L., Goldfarb D., Major M.B, & Kutluay S.B. (2021). Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell. bioRxiv.

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Site-directed mutagenesis of Nef/Vpu

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Participant-derived Nef or Vpu clones were subjected to site-directed mutagenesis to evaluate the impact of specific polymorphisms on protein function. Individual mutations were introduced into the parental gene sequence, which was then codon optimized (Codon Optimization tool; Integrated DNA Technologies) and synthesized commercially (eBlocks; Integrated DNA Technologies). Products were cloned into pSELECT-GFPzeo, sequence-validated and assessed for function as described. Codon optimized parental clones were used as controls. Each mutant clone was tested at least three times in independent experiments, and representative results are shown.

Umviligihozo G., Mann J.K., Jin S.W., Mwimanzi F.M., Hsieh H.S., Sudderuddin H., Lee G.Q., Byakwaga H., Muzoora C., Hunt P.W., Martin J.N., Haberer J.E., Karita E., Allen S., Hunter E., Brumme Z.L, & Brockman M.A. (2022). Attenuated HIV-1 Nef But Not Vpu Function in a Cohort of Rwandan Long-Term Survivors. Frontiers in virology, 2, 917902.

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Site-directed mutagenesis of Nef/Vpu

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Synthetic Rab8a Silencing Resistance

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The sequence for the silencing resistant synthetic Rab8a construct (Rab8a-1^syn) was obtained by codon shuffling the sequence of Rab8a-1 through a combination of the Integrated DNA Technologies (IDT) Codon optimization tool and manual codon shuffling. The construct was then gene synthesized and inserted into the vector by Gibson assembly.

Pandey P., Leary A.Y., Tumtas Y., Savage Z., Dagvadorj B., Duggan C., Yuen E.L., Sanguankiattichai N., Tan E., Khandare V., Connerton A.J., Yunusov T., Madalinski M., Mirkin F.G., Schornack S., Dagdas Y., Kamoun S, & Bozkurt T.O. (2021). An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface. eLife, 10, e65285.

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