Snapgene codon optimization7/31/2023 Further, repeats in the cloned fragments can lead to intramolecular recombination and prevent successful cloning (Joska et al. The DNA must first be isolated from the yeast before it can be propagated in Escherichia coli in a second step. Another disadvantage is that a further cloning step is necessary. However, the length of the oligonucleotides can negatively influence the product yield, even completely suppress amplification. This is frequently done by amplifying these fragments using long oligonucleotides that carry the homologous sequences in their 5′ region. However, the homologous regions need to be introduced into the DNA fragments used for recombination. With this approach 25 or even more DNA fragments can be combined (Gibson et al. For yeast recombination, the baker’s yeast Saccharomyces cerevisiae is utilized to merge DNA fragments with homologous regions that can be as short as 30 bp (Hua et al. Beside the classical restriction and ligation-based cloning, further techniques like yeast recombination or In-Fusion® (Takara Bio) cloning were established enabling faster and-even more important-seamless cloning. Here, we describe Golden Gate vector sets for fast and efficient generation of gene deletions and gene fusions primarily intended for fungi, but with modifications applicable to a broader range of organisms.Ī number of cloning techniques is available to generate plasmids. However, the initial steps for characterization of a gene, namely the cloning of vectors for expression or localization of a gene product, are still time-consuming or expensive. With transition into the postgenomic era and the drastic increase in techniques within the field of transcriptomics and proteomics the identification of interesting genes has drastically sped up. The deletion of genes and the generation of gene fusions are important steps for the functional analysis of genes. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi. We thus expect these vectors to be beneficial for other fungi as well. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/ FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. For gene deletion, we provide five different donor vectors for selection marker cassettes. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. All rights reserved.The cloning of plasmids can be time-consuming or expensive. Supplementary data are available at Bioinformatics online. User-defined DNA sequences can also be compared against the COOL optimized sequences to show the extent by which the user's sequences can be further improved.ĬOOL is free to academic and non-commercial users and licensed to others for a fee by the National University of Singapore. In addition, users can visualize and compare the optimal synthetic sequences with respect to various fitness measures. COOL supports a simple and flexible interface for customizing various codon optimization parameters such as codon adaptation index, individual codon usage and codon pairing. Hence, we have developed Codon Optimization OnLine (COOL), which is the first web tool that provides the multi-objective codon optimization functionality to aid systematic synthetic gene design. However, most of the existing codon optimization tools consider a single design criterion and/or implement a rather rigid user interface to yield only one optimal sequence, which may not be the best solution. Codon optimization has been widely used for designing synthetic genes to improve their expression in heterologous host organisms.
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