Gensmart codon optimization tool

Manufactured by GenScript

Sourced in United States

The GenSmart Codon Optimization Tool is a software application designed to analyze and optimize the codon usage of DNA sequences. It is a bioinformatics tool that can be used to improve the expression of recombinant proteins in various host organisms. The core function of this tool is to analyze the codon usage of a given DNA sequence and provide recommendations for codon optimization to enhance protein production.

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Lab products found in correlation

In fusion cloning, by Takara Bio (2 mentions) Maxiprep kit, by GenScript (2 mentions) Rare codon analysis tool, by GenScript (2 mentions) Puc57, by GenScript (1 mentions) Expi293 cell, by Thermo Fisher Scientific (1 mentions) Ndei and xhoi, by New England Biolabs (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Histrap hp ni nta column, by GE Healthcare (1 mentions) Ss34 rotor, by GE Healthcare (1 mentions) Ppiczαa expression vector, by Thermo Fisher Scientific (1 mentions)

10 protocols using gensmart codon optimization tool

Cloning and Insertion of Antibody Cassettes

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Homology donors comprising sequences to be inserted and flanking homology arms were cloned into plasmid ITR-CMV-GFP (containing ITRs from AAV2; Cell Biolabs) or pVAX by Infusion cloning (Takara). The GFP expression cassette hPGK-eGFP-BGHpA was amplified by PCR from plasmid CCR5-PGK-GFP.³³ Antibody components (J3, A6, CD4-mD1.22 or PGT121-scFv sequences, synthesized as gBlocks (IDT) as described above) were combined with the EEK promoter amplified from plasmid CCR5-EEK-GFP³³ and the splice donor sequence from the IGHG1 CH1 exon.
All homology arms were symmetrical and of equal length, either 500 bp or 750 bp each. Homology arms to direct insertion downstream of the CH1 exon were amplified by PCR from human genomic DNA. Homology arms for insertion downstream of the CH2 exon were synthesized as gBlocks (IDT). Antibody cassettes to be inserted downstream of CH2 additionally contained sequences for the Hinge and CH2 exons of IgG1, which were codon optimized using GenSmart Codon Optimization tool (Genscript) to reduce homology with the endogenous sequences. The sequences of all homology donor plasmids and components are provided in Supplementary Table 3.

Rogers G.L., Huang C., Mathur A., Huang X., Chen H.Y., Stanten K., Morales H., Chang C.H., Kezirian E.J, & Cannon P.M. (2023). Reprogramming human B cells with custom heavy chain antibodies. Research Square.

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Expression of Engineered Human ACE2 Protein

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Example 6

The mature amino acid sequence of human ACE2 (18-740 amino acid) was generated containing a myc-tag (EQKLISEEDLLRKR) and linker (GSPGGA) sequence at its N-terminal end. The linear amino acid sequence was reverse translated to its corresponding DNA sequence using the free GenSmart™ Codon Optimization Tool by GenScript for expression in human cells (gensmart-free-gene-codon-optimization). This sequence was submitted for gene synthesis and inserted into the plasmid cloning vector pUC57. The insert was amplified and cloned into the mammalian cell expression vector pcDNA3(−) containing the myc-tag-Protein M-HRP sequence (see above) by replacing the myc-tag-HRP sequence with the above myc-tag-ACE2 sequence, upstream of the sequence encoding 3 sets of 4 glycine residues and 1 serine residue (e.g., GGGGS)3 linker followed by the mature amino acid sequence of Protein M (37-556 amino acid), producing a final armY-ACE2 construct containing (IL-2 leader sequence—myc tag—ACE2—linker—Protein M). The plasmid expression vector construct was verified by restriction enzyme analysis, amplified in E. coli and purified using a maxiprep kit (GenScript Inc. and Eton Bioscience, Inc.).

US11401309B2. Protein M fusion proteins and uses (2022-08-02). None [US]. Inventors: Jay Dela Cruz [US].

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Cloning and Expression of Protein M-HRP Fusion

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Example 3

The mature amino acid sequence of horseradish peroxidase HRP (31-338 amino acid) was generated containing a myc-tag (EQKLISEEDL) and linker (AAN) sequence at its N-terminal end. The amino acid sequence encoding 3 sets of 4 glycine residues and 1 serine residue (e.g., GGGGS)3 linker followed by the mature amino acid sequence of Protein M (37-556 amino acid) was added to its C-terminal end producing a final Protein M-HRP construct containing (IL-2 leader sequence—myc tag—HRP—linker—Protein M). The linear amino acid sequence was reverse translated to its corresponding DNA sequence using the free GenSmart™ Codon Optimization Tool by GenScript for expression in human cells (gensmart-free-gene-codon-optimization). This sequence was submitted for gene synthesis and inserted into the plasmid cloning vector pUC57 (GenScript USA Inc.). The insert was amplified and cloned into a mammalian cell expression vector, pcDNA3(−). The plasmid expression vector construct was verified by restriction enzyme analysis, amplified in E. coli and purified using a maxiprep kit (GenScript Inc. and Eton Bioscience, Inc.).

US11401309B2. Protein M fusion proteins and uses (2022-08-02). None [US]. Inventors: Jay Dela Cruz [US].

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SARS-CoV-2 and Common Human Coronavirus RBD Purification

Cited 1 time

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Plasmid encoding mammalian cell codon optimized sequences for the receptor binding domain (RBD) of SARS-CoV-2 was generously gifted from the lab of Dr. Florian Krammer (Icahn School of Medicine, NYC) [11] (link). The sequence for the HCoV-OC43 RBD was obtained from the UniProt Protein Database (P36334 SPIKE_CVHOC). This construct was engineered to contain an N-terminal 13 amino acid signal sequence and a C-terminal 6xHis-tag for downstream protein purification. Mammalian cell codon optimization was performed using the GenScript GenSmart Codon Optimization Tool. The RBD gene was synthesized by GenScript and cloned into the pcDNA3.1 plasmid between EcoRI and XhoI restriction enzyme sites. The constructs for HCoV-229E and HCoV-NL63 RBD were designed similarly using UniProt Protein Database. Proteins were produced in Expi293 cells (ThermoFisher) using the manufacturers’ instructions. Proteins were purified, concentrated, and analyzed by SDS-PAGE.

Chen A.T., Stacey H.D., Marzok A., Singh P., Ang J., Miller M.S, & Loeb M. (2021). Effect of inactivated influenza vaccination on human coronavirus infection: Secondary analysis of a randomized trial in Hutterite colonies. Vaccine, 39(48), 7058-7065.

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Codon Optimization for mRNA Vaccine

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The codon sequences in the designed mRNA vaccine construct were optimized to ensure efficient expression within human cells. For this purpose, the GenSmart Codon Optimization Tool (http://www.genscript.com/) provided by GenScript (G.S.) was used. After optimization, a quality assessment of the optimized sequence was performed using the Rare Codon Analysis tools (http://www.genscript.com/) also provided by GenScript. The efficiency of mRNA translation was determined using Codon Adaptation Index (CAI). In addition, any unusual tandem codons present in the optimized sequence were identified through codon frequency distribution analysis. By optimizing the codon sequences, the expression and efficacy of the mRNA vaccine construct can potentially be improved by researchers.

Asadinezhad M., Khoshnood S., Asadollahi P., Ghafourian S., Sadeghifard N., Pakzad I., Zeinivand Y., Omidi N., Hematian A, & Kalani B.S. (2024). Development of innovative multi-epitope mRNA vaccine against Pseudomonas aeruginosa using in silico approaches. Briefings in Bioinformatics, 25(1), bbad502.

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Codon Optimization for Peptide Vaccine

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The peptide vaccine construct needs to undergo a codon optimization for efficient expression within the human cells. Accordingly, we used the GenSmart Codon Optimization Tool (http://www.genscript.com/) by GenScript (GS). Quality assessment of the optimized sequence was performed using the Rare Codon Analysis tools (http://www.genscript.com/) by GenScript (GS). The efficiency of translation of the mRNA is expressed as Codon Adaptation Index (CAI). The existence of any tandem unusual codons is indicated as Codon Frequency Distribution (CFD).

, & Al Tbeishat H. (2022). Novel In Silico mRNA vaccine design exploiting proteins of M. tuberculosis that modulates host immune responses by inducing epigenetic modifications. Scientific Reports, 12, 4645.

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Optimized Homology-Directed Antibody Cassette Insertion

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Homology donors comprising sequences to be inserted and flanking homology arms were cloned into plasmid ITR-CMV-GFP (containing ITRs from AAV2; Cell Biolabs) or pVAX by Infusion cloning (Takara). The GFP expression cassette hPGK-eGFP-BGHpA was amplified by PCR from plasmid CCR5-PGK-GFP.^{33 (link)} Antibody components (J3, A6, CD4-mD1.22 or PGT121-scFv sequences, synthesized as gBlocks (IDT) as described above) were combined with the EEK promoter amplified from plasmid CCR5-EEK-GFP^{33 (link)} and the splice donor sequence from the IGHG1 CH1 exon.
All homology arms were symmetrical and of equal length, either 500 bp or 750 bp each. Homology arms to direct insertion downstream of the CH1 exon were amplified by PCR from human genomic DNA. Homology arms for insertion downstream of the CH2 exon were synthesized as gBlocks (IDT). Antibody cassettes to be inserted downstream of CH2 additionally contained sequences for the Hinge and CH2 exons of IgG1, which were codon optimized using GenSmart Codon Optimization tool (Genscript) to reduce homology with the endogenous sequences. The sequences of all homology donor plasmids and components are provided in Supplementary Table 3.

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E. coli Agmatinase Codon Optimization

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The E. coli agmatinase amino acid sequence (UniProt P60651) was first codon optimized for expression in an E. coli expression system using the GenSmart codon optimization tool (GenScript) (S1 Fig in S1 File). The gene was synthesized (GenScript) with a 5′ NdeI restriction endonuclease site and a stop codon followed by a XhoI restriction endonuclease site at the 3′ terminus, and delivered in a pUC57 vector (SPEB-pUC57). The gene was subcloned into the pTHT vector, a variant of pET-28 with a tobacco etch virus (TEV) protease site in place of the thrombin site. Briefly, SPEB-pUC57 and pTHT were separately digested with NdeI and XhoI (NEB). The digested gene and plasmid were separated on an agarose gel, gel purified, and then ligated using T4 DNA ligase (NEB). The ligated product was used to transform 5-alpha E. coli cells (NEB) and plated on LB agar supplemented with 50 μg/mL kanamycin. Selected colonies were cultured and the resulting plasmids (SPEB-THT) isolated for sequence verification by Sanger sequencing.

Chitrakar I., Ahmed S.F., Torelli A.T, & French J.B. (2021). Structure of the E. coli agmatinase, SPEB. PLoS ONE, 16(4), e0248991.

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Optimized Expression and Purification of Tc_5171

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DNA sequence encoding the putative secreted antigen Tc_5171 was codon optimized for expression in bacteria using the GenSmart codon optimization tool (GenScript, Piscataway, New Jersey, USA). The synthesized gene was cloned in pET26(b+) vector using NcoI and XhoI restrictions enzymes. The plasmid encoding the gene and the C-terminal His₆ tag-encoding sequence was transformed into the E. coli BL21 (DE3) STAR expression strain. For 1 L of bacterial cell culture in minimal media, protein expression was induced at 0.8 O.D.₆₂₀ with 0.8 mM isopropyl-β-d-thiogalactopyranoside. Protein expression was performed at 37 °C with the culture flask aerated at 220 rpm on an orbital shaker for 4 h. Bacterial cells were centrifuged at 23,419×g (Sorvall GSA rotor) and the cell pellet subjected to sonication and lysis using 20 ml of lysis buffer (25 mM CAPS, pH 11.0, 3% Sarkosyl, 25 mM NaCl). The lysate was centrifuged at 30,597×g (Sorvall SS-34 rotor) for 30 min and the supernatant loaded on a HisTRAP HP NiNTA column (GE Lifesciences). The column was washed with 25 ml of wash buffer (10 mM Imidazole, 25 mM CAPS, pH 11.0, 0.3% Sarkosyl, 25 mM NaCl) and bound histidine-tagged protein eluted with a gradient of 0–350 mM Imidazole in CAPS buffer. Purified protein was dialyzed into HBS buffer (25 mM HEPES, pH 7.5 buffer with 135 mM NaCl).

Nagarkatti R., Acosta D., Acharyya N., de Araujo F.F., Elói-Santos S.M., Martins-Filho O.A., Teixeira-Carvalho A, & Debrabant A. (2020). A novel Trypanosoma cruzi secreted antigen as a potential biomarker of Chagas disease. Scientific Reports, 10, 19591.

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Xyloglucanase Gene Optimization and Expression

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A gene fragment encoding mature xyloglucanase from T. terrestris NRRL 8126 (TtGH74, XP_003650520.1) was synthesized by GenScript (Nanjing, China) with optimized codons. The codons were optimized using GenSmart™ Codon Optimization tool (Version Beta 1.0, https://www.genscript.com.cn/gensmart-free-gene-codon-optimization.html; accessed on 12 September 2019). The gene fragment was linked to the pPICZαA expression vector (Invitrogen, Carlsbad, CA, USA), located between the EcoRI and NotI sites. The CBM1 fragment in TtGH74 was removed by PCR using the following primers (Table 4).
The sequences encoding signal peptide, linker peptide and CBM1 in selected genes were deleted, and only the catalytic domain (CD) was selected for sequence alignment.

Wang B., Chen K., Zhang P., Long L, & Ding S. (2022). Comparison of the Biochemical Properties and Roles in the Xyloglucan-Rich Biomass Degradation of a GH74 Xyloglucanase and Its CBM-Deleted Variant from Thielavia terrestris. International Journal of Molecular Sciences, 23(9), 5276.

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