272 results about "Multi gene" patented technology

The invention belongs to the technical field of molecular genetic breeding, and particularly relates to a molecular marker Zm00001eb181850 gene related to corn leukoderma resistance and application thereof, the invention provides a molecular marker related to the corn leukoderma resistance, the gene sequence of the molecular marker is shown as SEQ ID NO: 1 and / or SEQ ID NO: 2, and the molecular marker Zm00001eb181850 gene is shown as SEQ ID NO: 1 and / or SEQ ID NO: 2. Researches prove that corn white spot resistance QTL qMWS4-1 and qMWS4-5 intervals and resistance SNP: 4-118983592 have co-localization, the corn white spot resistance QTL has a high phenotypic interpretation rate, the associated gene Zm00001eb181850 (SEQ ID NO: 2) is highly expressed in corn leaves, it is proved that the associated gene Zm00001eb181850 (SEQ ID NO: 2) is associated with the corn leaf spot, the corn white spot resistance is a quantitative character jointly regulated by multiple genes, and the corn leaf spot resistance QTL has a good application prospect. According to the technical scheme provided by the invention, a new method is provided for cultivating a new variety of leukoderma-resistant corn by utilizing a modern molecular breeding technology.

The present invention relates to a method for constructing an eight-gene-edited xenogeneic organ transplantation donor pig, belonging to the field of animal biotechnology. In the wild-type porcine fetal fibroblast cell line, the GGTA1, β4GalNT2, and CMAH genes are knocked out by using the CRISPR / Cas9 gene editing technology, and the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transfected. Combining with somatic cell cloning technology, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene-edited cloned pigs are constructed. Further, through genotype, mRNA, protein expression identification and functional analysis, eight-gene-edited xenogeneic organ transplantation donor pigs are obtained. The present invention solves the technical problems of high production difficulty, low efficiency, and low survival rate of donor pigs for multi-gene-edited xenogeneic organ transplantation, and maximally solves the common problems of immune rejection reaction and complement dysregulation faced during xenogeneic organ transplantation, laying a foundation for more targeted development of donor pigs suitable for different tissue and organ xenotransplantation, and having important value for promoting the clinical transformation of xenogeneic organ transplantation.

The invention discloses a rhodosporidium toruloides strain with high homologous recombination efficiency as well as a construction method and application thereof, and belongs to the field of oil-producing yeast engineering bacteria. According to the rhodosporidium toruloides strain, the rhodosporidium toruloides strain NP11-SaCas9-1 is used as an original strain, and an RAD52 gene is over-expressed. The modified yeast engineering bacterium provided by the invention can realize high homologous recombination through CRISPR system mediation, the homologous recombination efficiency reaches 90-100%, a multi-gene editing platform can be constructed on the basis, and the modified yeast engineering bacterium can be applied to the fields of synthetic biology, metabolic engineering, genetic engineering and the like, so that the modified yeast engineering bacterium is simpler, quicker, more convenient and more accurate in the genetic modification process. And the homologous recombination activity of the rhodosporidium toruloides can be improved by expressing the homologous recombination related protein Rad52, and the genetic engineering strategy can be used for modifying other unconventional yeasts.

The present invention relates to a method for the ex vivo molecular diagnosis of gastrointestinal stromal tumors, comprising the steps of: providing at least one isolated tissue sample comprising at least one nucleic acid and the subsequent extraction of the at least one nucleic acid. The method according to the present invention further comprises a step of quantifying the at least one previously isolated nucleic acid. Furthermore, the method according to the present invention comprises preparing at least one amplicon library of said previously isolated nucleic acid, which comprises sequences obtained from the FGFR1, NF1, SDHA, SDHB, SDHC and SDHD genes. This step is followed by the step of purifying the at least one amplicon library and the step of simultaneously sequencing the amplicons themselves. The present invention also relates to a multi-gene panel for ex vivo molecular diagnosis of gastrointestinal stromal tumors and a kit comprising such a panel.

The invention relates to the technical field of assisted reproduction polygene detection, and discloses an oocyte occurrence and maturation disorder detection panel, a detection kit and application thereof, and the detection panel comprises mutation genes related to the oocyte occurrence and maturation disorder for detection, copy number variation genes and rearrangement event and deletion genes. According to the application, by detecting high-risk genes and mutation sites, the risk of occurrence and maturation disorder of the oocytes is predicted in combination with clinic, the development potential of the oocytes is evaluated, the in-vitro maturation strategy is optimized, and the clinical outcome of assisted reproduction is improved. The detection panel can be used for efficiently detecting gene mutation which has clinical diagnosis and treatment significance on oocyte occurrence and maturation disorders; the method has important guiding significance on genetic counseling, risk prediction, clinical diagnosis, precise treatment and potential personalized intervention measures of patients suffering from recurrent oocyte occurrence disorder, oocyte maturation disorder and low oocyte maturation rate and patients to be subjected to assisted reproduction technology treatment.

The invention provides a double-tandem base editor as well as a preparation method and application thereof, and belongs to the technical field of gene editing. The double tandem base editor disclosed by the invention is obtained by recombining a gRNA1-tRNA-gRNA2 unit component and an adenine base editor, the gRNA1 is designed for a target gene 1 and comprises a gRNA target specific spacer region and a gRNA skeleton of the target gene 1; the gRNA 2 is designed for a target gene 2 and comprises a gRNA target specific spacer region and a gRNA skeleton of the target gene 2. According to the double tandem base editor provided by the invention, different gene loci can be targeted only by changing 20bp gRNA, multi-gene editing on large animal cells is realized, accurate and safe multi-gene editing is realized under the condition of not introducing double-strand breakage, and the mutation efficiency is higher than that of a multi-plasmid co-transfection base editor.

The present invention relates to transfer DNAs which can be used to produce a genetically modified plant that only has DNA from that species of plant. The present invention also relates to genetically modified plants with a transfer DNA of the invention which has been processed during integration and methods of making such plants. Also provided is a DNA construct and methods of making a multigene construct comprising five genes which confer upon a plant enhanced resistance to a wide variety of fungal pathogens.

The invention discloses a corynebacterium glutamicum engineering strain produced by L-glutamine as well as a construction method and application of the corynebacterium glutamicum engineering strain. The corynebacterium glutamicum engineering strain is prepared by integrating a glutamine synthase encoding gene glnA which is sourced from saccharomyces cerevisiae and has a tyrosine residue mutation at the 99th site on the surface and an sRNA-MicC-EcHfq regulation module of a targeted adenylyltransferase gene glnE into a vector plasmid; the method comprises the following steps: constructing a recombinant plasmid for removing adenosine acylation of glutamine synthase; and transferring the recombinant plasmid into corynebacterium glutamicum of which the odhA gene expression is controlled by a growth stage specific promoter Pcg2705, so as to obtain a corynebacterium glutamicum engineering strain for producing L-glutamine. According to the invention, through multi-gene synergistic modification and space-time metabolic flux optimization, the problem of'growth-synthesis' tradeoff in the prior art is solved, the fermentation efficiency is improved, the fermentation process is simple, and the application prospect is good.

The invention discloses a genetically engineered bacterium for high yield of L-alanine as well as a construction method and application of the genetically engineered bacterium, a dominant strain is obtained by knocking out an alanine racemase gene dadX and integrating coding sequences of alanine dehydrogenase from different sources, and the shake flask yield is 10.8 g / L; the activity of a corresponding mutant M18L / E75A / A78S / I266V is 4.3 times that of a wild type; 6 by-product related genes are knocked out, meanwhile, a multi-copy strain integrated with alanine dehydrogenase is constructed, and the yield of E.coli A-LB6 in a 5L fermentation tank is increased to 133.3 g / L. Through a dynamic regulation and control strategy, an oxygen-sensitive promoter Pomp * is adopted to replace a phosphoenolpyruvate carboxylase promoter, so that succinic acid byproducts are reduced by 85.8%, and the yield reaches 143.5 g / L after 48 hours of aerobic and anaerobic two-stage fermentation. And finally, by regulating and controlling the ATP synthetase gene cluster atp promoter, the yield of L-alanine in a 50 L fermentation tank reaches 151.3 g / L, and the saccharic acid conversion rate reaches 95.2%. The combination of polygene knockout and dynamic metabolism regulation effectively enhances the biosynthesis ability of alanine, and provides an efficient engineering strain for industrial production.

The embodiment of the invention provides a library construction method, a universal joint and a kit, relates to the technical field of synthetic biology, and is used for constructing a mixed library in which multiple gene segments are directionally connected. The library construction method comprises the following steps: providing a plurality of first initial gene segments; introducing cutting sequences to two ends of each first initial gene segment to obtain a plurality of second initial gene segments; introducing universal joints to two ends of each second initial gene segment to obtain a plurality of target gene segments; wherein the universal joint comprises an identification sequence; performing cutting and connection reaction on the plurality of target gene segments to obtain a mixed library; wherein the plurality of target gene segments are connected according to a set sequence. The library construction method is used for constructing the DNA mixed library.

Disclosed herein is a polynucleotide construct comprising one or more primary endonuclease recognition sequences upstream and downstream of a multiple gene editing site that comprises a plurality of secondary endonuclease recognition sequences. The primary endonuclease recognition sequences facilitate insertion of the multiple gene editing site into a host cell genome. The secondary endonuclease recognition sequences facilitate insertion of one or more exogenous donor genes into the host cell.

The invention discloses a construction method and application of a toxoplasma gondii polygene deletion vaccine strain. According to the invention, a TgBFD1 gene and a TgDAHPS gene are respectively knocked out from an ME49 [delta] 6pgdh1 insect strain through a gene editing technology, and a polygene deleted insect strain [delta] 6pgdh1 [delta] bfd1 [delta] dahps is successfully constructed; the strain can be subcultured in vitro, has the characteristics of no toxicity and no formation of tissue cysts in a host body, has better immune protection after immunization and inoculation, and can resist lethal infection of highly toxic strains. The toxoplasma gondii polygene deletion strain provided by the invention has better safety than ME49 delta 6pgdh1 strain, the toxicity of the toxoplasma gondii polygene deletion strain is obviously reduced, tissue cysts are not formed, the toxoplasma gondii polygene deletion strain can effectively resist infection of wild poisonous strains, the toxoplasma gondii polygene deletion strain has good immune protection capability, the defects that ME49 delta 6pgdh1 still has toxicity and can form tissue cysts are overcome, and the toxoplasma gondii polygene deletion strain has good application prospects. A method is provided for developing a safe and efficient toxoplasma gondii vaccine.

The present application relates to the field of gene editing technology, bioengineering and application of microbial fermentation, and discloses a Pichia pastoris high-efficiency gene editing system, a construction method thereof and application in a high-yield cordycepin engineering strain.The construction of the Pichia pastoris CRISPR-Cas9 gene editing system mainly includes the following steps: the histidine HIS4 nutritional deficiency back complementation is used to realize the resistance-free assembly of KhCas9; the sgRNA-tRNA array and the IIS type restriction endonuclease combination are used to simplify the gene editing operation and realize the simultaneous and high-efficiency editing of multiple genes; and the Brex27 domain is fused with KhCas9 to strengthen the homologous recombination efficiency. By using the gene editing system, through the optimization of methanol assimilation, 3'-AMP and adenosine precursor supply, ATP / NADPH energy supply and the like, the construction of a cordycepin engineering strain without resistance gene residues is realized for the first time, the gene editing efficiency of Pichia pastoris is greatly improved, and a powerful tool is provided for the industrialized production of cordycepin.

The invention relates to the technical field of gene vectors, and discloses a multi-target multi-gene stable silencing vector and an application thereof in treating diabetes mellitus, and a multi-gene multi-target gene modification combined element is safely and efficiently integrated at a gene safety parking point of a second intron region of a CCR5 gene in a genome in a reverse fixed point manner by using a non-viral vector system; therefore, the effect of continuously silencing expression of a plurality of target genes such as MSTN, GRB10 and PTBP1 for a long time is achieved, and aging of mesenchymal stem cells cultured in vitro is remarkably inhibited. When the mesenchymal stem cells modified by the multi-target multi-gene stable silencing vector are infused, the activity is high, the chronic inflammatory microenvironment is effectively inhibited, and the senescence process is delayed; moreover, proliferation and differentiation of pancreatic beta cells are promoted, insulin secretion is enhanced, insulin resistance is effectively resisted, and diseases such as diabetes mellitus are prevented.

The invention discloses a method for cultivating a heat-resistant broad-spectrum durable rice blast-resistant rice breeding material based on polygene polymerization. According to the method, a high-heat-resistance and high-dominance indica-japonica hybrid indica rice restorer line is selected as a recurrent parent, a breeding intermediate material for polymerizing complementary broad-spectrum rice blast resistance genes Pigm and Pi-jx is used as a donor parent, and polymerization of the heat-resistance and broad-spectrum lasting rice blast resistance genes Pigm and Pi-jx is realized through a method of combining molecular breeding with conventional breeding. Meanwhile, by selecting representative strains to carry out artificial inoculation identification of seedling blast and panicle blast, natural induction identification of a rice blast retransmission area and heat resistance identification, and by combining main agronomic trait evaluation in the whole growth period, a heat-resistant broad-spectrum durable rice blast-resistant breeding material is obtained. The breeding material bred by using the method disclosed by the invention has relatively strong heat resistance and broad-spectrum and lasting rice blast resistance, and the breeding process of heat-resistant and rice blast-resistant rice varieties is accelerated.

This invention belongs to the field of biomedical technology, specifically relating to the application of DNA damage response gene-related SNPs in the treatment and prognosis of acute myeloid leukemia (AML). This invention is the first to discover that single nucleotide polymorphisms (SNPs) in the DNA damage response pathway (focusing on three major functional modules: damage sensing, signal transduction, and DNA repair) are closely related to the clinical characteristics, treatment response, and survival prognosis of AML. This lays the foundation for constructing a multi-gene risk model based on the DNA damage response pathway and provides theoretical support for integrating germline SNP maps into precision medicine strategies for AML.

The invention discloses a mouse multi-gene collaborative insertion and knockout system based on multi-data coupling, and relates to the technical field of bioengineering, and the mouse multi-gene collaborative insertion and knockout system comprises a multi-gene collaborative targeting vector, a high-fidelity Cas9 protein expression unit, a homologous recombination template library, a multi-modal efficiency prediction module, a dual-fluorescence Cre report unit and an off-target inhibition element. According to the method, vector silencing is avoided by connecting the nuclear matrix attachment region in series with the gRNA expression cassette, and the multi-gene collaborative editing efficiency is improved to gt by combining homologous arm optimization design and a machine learning driven efficiency prediction model; 80%; cas9 fusion deaminase and a glycosylase inhibitor are combined with miRNA-mediated off-target inhibition, so that off-target sites of a whole genome are reduced to be less than or equal to 1 / sample; real-time monitoring of tissue specific expression is achieved by means of a double-fluorescence Cre reporting system, space-time regulation and control application is supported, and a high-reliability tool is provided for complex gene interaction research.

Provided are a prime editing system-based method for achieving inversion editing of a large genomic fragment and a composition used. By means of the efficient multi-gene prime editing protein construct ePPEplus in plants, the inversion of large fragments in plants is achieved by means of designing paired pegRNAs (pegL and pegR), and then by means of processes such as reverse transcription, DNA double-stranded annealing, DNA strand synthesis, and repair. The pegL comprises RTT-L, and the pegR comprises RTT-R. The nucleotide sequence of RTT-L is identical to that of strand A at the right terminus of a double-stranded DNA to be inverted. The nucleotide sequence of RTT-R is identical to that of strand B at the left terminus of said DNA. By means of testing in the monocotyledonous plant wheat and dicotyledonous plants Nicotiana benthamiana and tomato, it is indicated that the method is an effective method for mediating precise inversion of large-fragment DNA in plants.

The application belongs to the technical field of molecular biology and medicine, and discloses a methylation marker for detecting the benignity and malignancy of thyroid nodules and an application. 780 new methylation sites are identified, 273 tumor occurrence and development related genes are associated, and the Fc gamma R-mediated phagocytosis signal pathway is involved. The methylation marker for detecting the benignity and malignancy of thyroid nodules comprises multiple genes and multiple methylation sites. The methylation level of multiple genes and multiple sites is detected, the problem of low and unstable methylation signal of single gene or single site is overcome, and thus the sensitivity and specificity of detection are improved.

The Quadruplet Expanded DNA (QED) eukaryote genetic code comprising twenty nondegenerate QED codons encode proteins (the protein-encoding codons), and thirty-five nondegenerate QED codons (the noncoding codons) being highly correlated with cis-regulatory elements control and regulate transcription, alternate splicing, and polymerization in eukaryotic protein synthesis using canonical amino acids. The QED eukaryote genetic code is an advancement to gene therapeutics that allows for the correction of dysfunctional proteins. Additionally, the QED eukaryote genetic code is further applicable for changing paradigms relating to identifying cures for monogenic rare, multigene cancer, and neurodegenerative diseases.

The application discloses a mutant Cas12i nuclease and application thereof, and belongs to the technical field of gene editing. The Cas mutant protein provided by the application is a mutant protein in which the 7th, 168th, 273rd, 332nd, 478th, 505th and 551st amino acids of SEQ ID NO:1 are all mutated into arginine, and other amino acid sequences remain unchanged. The application improves the editing efficiency by mutating multiple amino acids of the wild-type Cas12i protein, and can be used for multi-gene editing.

The invention relates to the technical field of rice molecular breeding, in particular to a rice seedling-stage cold-resistant molecular breeding system based on polygene dominant haplotype polymerization and application thereof. According to the invention, KASP molecular markers of 31 key SNP loci of 11 dominant haplotypes of 8 genes are developed, and a complete cold-resistant molecular breeding technical system is constructed. The cold-resistant new germplasm RY868 is successfully created by applying the system, and the seedling rate of the cold-resistant new germplasm RY868 under cold stress in the seedling stage reaches 84.40% and is obviously improved by 2.78 times compared with that of a receptor parent. According to the research, the genetic effect of the cold-resistant polygene haplotype combination in the rice seedling stage is systematically analyzed for the first time, a complete technical system from gene dominant haplotype mining to breeding application is established, a polygene dominant haplotype pyramiding breeding strategy is provided, and an innovative solution is provided for molecular design breeding with complex quantitative traits.

The invention provides a genome method and an SNP (Single Nucleotide Polymorphism) marker combination for a pig to adapt to related traits in a high-temperature and high-humidity environment, and belongs to the technical field of genome prediction, the method comprises the following steps: taking a local pig SNP marker combination as a prior fixing effect; standard miscalculation of a p value, a beta value and a beta value based on whole genome association analysis; performing interval dimension reduction based on linkage imbalance; searching an optimal parameter by using a genetic algorithm, and adaptively grouping the SNPs; performing weighted summation on the G matrix; the SNP marker combination is used as a fixed effect, the weighted G matrix is used as a random effect, a genetic evaluation model is configured, and genome prediction is carried out on target characters. According to the method, the large-effect SNP and the minor-effect multi-gene effect which are causally associated with the target prediction character can be accurately captured, redundant information is removed through dimension reduction in the interval, the optimal parameter combination is found through the genetic algorithm, and the calculation speed is greatly increased while the accuracy of genome prediction is improved.

The application discloses a gene combination for biosynthesis of paclitaxel and application thereof, and relates to the technical field of genetic engineering. The application breaks through the limitation of traditional paclitaxel extraction and synthesis technology, and for the first time adopts a transformation method of Physcomitrella patens to perform multi-gene co-expression on a paclitaxel synthesis pathway. The application has wider gene source compatibility, and opens up a brand-new technical path for paclitaxel synthesis. As a model plant, the Physcomitrella patens has the characteristics of rapid growth and easy cultivation. After a specific gene combination is introduced into the Physcomitrella patens, a metabolic pathway related to paclitaxel synthesis in the Physcomitrella patens cells can be fully activated, and the Physcomitrella patens is prompted to efficiently synthesize paclitaxel.

The present disclosure provides a construction method of a bacterial cellulose (BC)-enriched plant using multi-gene tandem and use thereof, and relates to the technical field of genetic engineering. In the present disclosure, an acsAB gene, an acsC gene, and an acsD gene are combined and then subjected to codon optimization according to a codon preference of the crop. Resulting optimized genes are fused with a 35S promoter and a nopaline synthase (NOS) terminator to obtain three gene expression cassettes. The three gene expression cassettes are ligated into a plant expression vector to obtain a multi-gene plant transformation vector containing the above three gene expression cassettes. The multi-gene plant transformation vector is then transformed into rice to obtain a transgenic rice plant capable of synthesizing the BC. It is determined that the transgenic rice plant has a BC content of 3.81%.

The invention discloses a synonymous mutation harmfulness prediction method based on a gene language model, and the method comprises the steps: (1) data obtaining and screening: employing a data set existing in a laboratory, and guaranteeing the data reliability and task pertinence; (2) carrying out feature annotation: respectively carrying out embedded feature extraction on the reference sequence and the mutation sequence by utilizing four representative gene language models GPN-MSA, Hyena DNA, SpliceBERT and Caduceus; (3) feature processing and fusion: performing further extraction on GPN-MSA and SpliceBERT embedding by adopting dynamic convolution and bidirectional LSTM (Long Short Term Memory); extracting the embedding of the Hyena DNA by adopting TextCNN (Convolutional Neural Network); linear dimensionality reduction is carried out on Caduceus embedding, and then splicing and dimensionality reduction are carried out on the four features; (4) model training: training in a five-fold cross validation mode, and preventing over-fitting through an early stop mechanism to obtain an optimal weight; and (5) model prediction: performing harmfulness prediction by taking 0.5 as a threshold value. According to the method, the complementary information of the multi-gene language model is fused, the prediction precision and robustness of the synonymous mutation harmfulness are remarkably improved, and a new technical support is provided for research on the synonymous mutation harmfulness.

The application provides a fat sea base gene and adulteration identification method based on multi-gene joint DNA barcoding, four gene joint barcode combinations are formed by adopting nuclear gene ITS2 and chloroplast genes matK, rbcL and psbA-trnH, at least two pairs of optimized alternative primers for each target gene and fat sea genus specific preliminary screening primer Ster-1 are matched, genus level rapid preliminary screening is realized to exclude non-fat sea genus samples and shorten the identification process, and the efficient amplification success rate of multi-gene fragments is ensured through the flexible use of alternative primers; fat sea and round fat sea, Sterculia and mixed adulterants can be accurately distinguished, the problems that traditional identification methods are subjective and species with close genetic relationship are difficult to distinguish are effectively solved; the method is not limited by sample morphology and fragmentation degree, can be widely applied to the true and false identification, quality control and traceability of traditional Chinese medicinal materials fat sea and its products, and provides technical support for standardizing market order and ensuring the safety and effectiveness of clinical medication.

The invention discloses a soybean bidirectional promoter, a recombinant vector and a construction method and application thereof, and belongs to the technical field of plant genetic engineering. The soybean bidirectional promoter can simultaneously drive gene expression in two directions, the front end and the rear end of the bidirectional promoter are respectively connected with three genes in series, and simultaneous expression of six genes can be driven by one promoter. The bidirectional promoter is combined with a ZQ6 enhancement sequence and a 35S: MdMYB10 expression cassette, a method for simultaneously and remarkably improving the expression level of more than six genes is developed, and a method for enabling a plant to emit obvious self-luminescence under a dark condition is developed based on the method. The soybean two-way promoter mediated multi-gene efficient synergistic transformation method provided by the invention can be applied to the aspects of constructing a soybean multi-gene synergistic transformation system, realizing rapid polymerization of excellent characters, cultivating new soybean germplasm with excellent characters and the like.

Disclosed herein are advanced RNA targeting (ARNATAR) oligomeric compounds, which are elaborately designed compounds, for the inhibition of gene expression by the RISC pathway. Such compounds are useful in methods for reducing the expression of certain genes, where many genes are associated with a variety of diseases and disorders.

The application discloses a food-like lactobacillus double-plasmid multi-gene editing system based on CRISPR / Cas9 technology and application thereof. The food-like lactobacillus double-plasmid multi-gene editing system comprises (1) a plasmid pCRI01 comprising the following elements: a Cas9 protein coding gene; a temperature-sensitive replicon pSC101; an sgRNA expression module targeting the replicon of the plasmid pCRI02, which is controlled by an inducible promoter Pnis; an ampicillin resistance; and (2) a plasmid pCRI02 comprising the following elements: at least one sgRNA expression module driven by a P23 promoter; a lambda-Red recombinase system comprising Exo, Beta and Gam genes; an erythromycin resistance marker gene; and a kanamycin resistance gene. The multi-gene editing system provided by the application is a small-size pCRI01 and pCRI02 double-plasmid system, and the number of transformed cells after resistance screening is significantly improved; and the gene editing efficiency mediated by the pCRI plasmid is greatly improved with the assistance of the lambda-Red recombinase system.