The present invention relates to a nucleic acid molecule comprising one or multiple mutant IRES elements. Further, the present invention relates to methods of enhancing gene expression and to methods of differentially controlling expression of one or multiple gene(s) of interest. In addition, the present invention relates to a kit for studying interactions or any application requiring co-expression of multiple genes.

The invention refers to a library of bidirectional expression cassettes or expression vectors comprising a repertoire of bidirectional promoter sequences, each expression cassette comprising a promoter sequence operably linked to a first gene in one direction, and operably linked to an oppositely oriented second gene in the other direction which is different from the first gene, and bidirectional Pichia pastoris or CHO cells promoter sequences. The invention further refers to a method of screening or selecting a bidirectional promoter suitable for expressing at least two GOI in a host cell and a kit comprising a) an expression cassette consisting of the first and second genes and a stuffer sequence separating them, which stuffer sequence comprises a recognition site for a type IIS restriction enzyme at both ends; b) the type IIS restriction enzyme; c) and a repertoire of promoter, preferably a promoter library including bidirectional promoters.

Methods and compositions using populations of randomized modified FRT recombination sites to identify, isolate and/or characterize modified FRT recombination sites are provided. Kits comprising the library populations of FRT sites are also provided, as are methods to make a library of modified FRT recombination sites. The recombinogenic modified FRT recombination sites can be employed in a variety of methods for targeted recombination of polynucleotides of interest.

Provided are constructs and methods useful for the screening and identification of polynucleotide sequences of interest, in particular promoters, RNA stability modifying sequences and transcriptional modifying sequences.

A method for identifying and characterizing biological parts based on omics datasets and a dual-fluorescent reporter gene system, and a biological part library constructed thereon are provided, relating to a technical filed of biology. The method includes steps of: identifying the biological parts using the omics datasets; constructing a single-fluorescent reporter gene system using a shuttle vector pEZ15Asp as a skeleton for screening and determining fluorescent reporter genes; obtaining a dual-fluorescent reporter gene system skeleton; constructing recombinant plasmids, and finally transforming into competent cells for quantitative analysis of fluorescence intensities. The present invention is convenient and quick, and can screen and identify different biological parts such as RBS, UTRs, promoters, and terminators of different intensities in batch quantitatively in a relatively short time. Moreover, the present invention can quickly expand the biological part library of Z. mobilis, so as to be applied in metabolic engineering of different demands.

Compositions and methods are provided for rapid characterization of Cas endonuclease systems and the elements comprising such systems, including, but not limiting to, rapid characterization of PAM sequences, guide RNA elements and Cas endonucleases. Type II Cas9 endonuclease systems originating from Brevibacillus laterosporus, Lactobacillus reuteri Mlc3, Lactobacillus rossiae DSM 15814, Pediococcus pentosaceus SL4, Lactobacillus nodensis JCM 14932, Sulfurospirillum sp. SCADC, Bifidobacterium thermophilum DSM 20210, Loktanella vestfoldensis, Sphingomonas sanxanigenens NX02, Epilithonimonas tenax DSM 16811, Sporocytophaga myxococcoides are described herein. The present disclosure also describes methods for genome modification of a target sequence in the genome of a cell, for gene editing, and for inserting a polynucleotide of interest into the genome of a cell.

Provided are constructs and methods useful for the screening and identification of polynucleotide sequences of interest, in particular promoters, RNA stability modifying sequences and transcriptional modifying sequences.

Disclosed herein are libraries of reporter nucleic acids for functional regulatory elements as well as methods and kits for constructing and using such libraries. Exemplary libraries, methods, and kits can be used for high-throughput detection, identification, and/or quantitation of functional regulatory elements.

Methods and compositions using populations of randomized modified FRT recombination sites to identify, isolate and/or characterize modified FRT recombination sites are provided. Kits comprising the library populations of FRT sites are also provided, as are methods to make a library of modified FRT recombination sites. The recombinogenic modified FRT recombination sites can be employed in a variety of methods for targeted recombination of polynucleotides of interest.

A heterologous transcriptional repressor comprising a DNA targeting domain, preferably a catalytically inactive DNA targeting protein such as a CRISPR-Cas protein, and a KRAB domain selected from the group consisting of ZIM3-KRAB, ZIM2-KRAB, ZNF554-KRAB, ZNF264-KRAB, ZNF324-KRAB, ZNF354A-KRAB, ZFP82-KRAB, and ZNF669-KRAB. Also provided herein are expression constructs, vectors, and cells encoding or expressing said transcriptional repressor, as well as systems and methods for transcriptional repression of a target gene, and compositions, kits and reagents employed in the making and use thereof.