8 results about "PUC19" patented technology

The invention relates to a method for splitting, assembling and site-specific integration of large-fragment exogenous DNA (deoxyribonucleic acid) in Trichoderma reesei. Comprising the following steps: (1) constructing an arg:: Ptcu-Cas9-TtrpC-hph strain containing a Cas9 protein; (2) constructing a pUC19 <-> integration site: a pyr4 plasmid; (3) obtaining a plurality of target gene segments containing integration site homologous arms and having overlapping regions at the tail ends of adjacent DNA segments; (4) obtaining a gRNA expression cassette of a targeted genome integration site; (5) arg conversion: the arg is converted into a Ptcu-Cas9-TtrpC-hph strain; and (6) screening and verifying. According to the method, the CRISPR / Cas9 system is combined with the TAR in the trichoderma reesei for the first time, the exogenous large-fragment DNA is split into a plurality of small fragments, then the plurality of small-fragment exogenous DNA are assembled in the trichoderma reesei and are subjected to fixed-point integration, one-step editing of the large-fragment exogenous DNA is realized, and compared with a traditional genetics method, the gene editing efficiency in the trichoderma reesei is greatly improved.

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The invention discloses a replication-limited recombinant feline herpesvirus as well as a construction method and application thereof, and relates to the technical fields of genetic engineering and preventive veterinary medicine, exogenous feline parvovirus VP2 protein is expressed in the recombinant feline herpesvirus, and meanwhile, a gL gene in the feline herpesvirus is deleted. The construction method comprises the following steps: 1, constructing a QD-1 gL gene stable expression F81 cell line by using a lentivirus packaging three-plasmid system; 2, constructing a PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid, and carrying out GFP FPV VP2 recombinant plasmid construction; 3, an sgRNA expression vector lentiCRISPRv2-gL-sgRNA of the gL gene is constructed, and the sgRNA expression vector lentiCRISPRv2-gL-sgRNA of the And 4, transfecting the recombinant vectors in the step 2 and the step 3 to an F81-gL cell line to obtain the QD-1 gL-FPV VP2 recombinant feline herpesvirus. The recombinant feline herpesvirus can be used as a candidate vaccine to prepare a strain so as to resist feline herpesvirus and feline parvovirus infection.

The application discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is constructed by introducing an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, a MCS (multiple cloning site), a 3'-UTR sequence and a T7 terminator sequence on the basis of part of a sequence of an E. coli plasmid pUC19. The pSY1A plasmid vector has a MCS into which an exogenous gene can be inserted, and the MCS and a transcription assembly constitute a transcription module, so that the transcription module can be preserved by preserving the plasmid, and in-vitro transcription of the wheat germ cell-free expression system becomes more convenient and faster.

The invention discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is characterized in that on the basis of partial sequence of escherichia coli plasmid pUC19, an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, MCS multiple cloning sites, a 3 '-UTR sequence and a T7 terminator sequence are introduced to construct the plasmid vector pSY1A. According to the pSY1A plasmid vector provided by the invention, the vector has multiple cloning sites into which exogenous genes can be inserted, and the multiple cloning sites and a transcription component form a transcription module, so that the transcription module can be stored by storing plasmids, and the in-vitro transcription of a wheat germ cell-free expression system becomes more convenient and quicker.

The invention discloses a method for constructing a recombinant virus vector based on iridovirus PCNA protein. The method comprises the following steps: amplifying upstream and downstream fragments of an ORF068 gene by taking iridovirus as a template, cloning a puro gene fragment from a pLVX-puro plasmid, inserting the puro gene fragment into a pEGFP-N3 plasmid to construct a pEGFP-puro-GFP vector, respectively connecting the upstream and downstream fragments of the ORF068 with a CMV-puro-GFP fragment in the pEGFP-puro-GFP vector, and inserting the connected fragments into a pUC19 plasmid to construct a recombinant virus vector. The plasmid construction method provided by the invention is simple and strong in operability; and the constructed plasmid has strong stability in cells.

The invention belongs to the technical field of microbial genetic engineering and gene editing. The invention provides a Cas9 enzyme mediated cytosine base editing plasmid, and compared with a starting plasmid, the Cas9 enzyme mediated cytosine base editing plasmid has the following inserted sequences: a terminator Tcyc1; the base sequence of the terminator Tcyc1 is as shown in SEQ ID NO. 5; a promoter PgpdA; the base sequence of the promoter PgpdA is as shown in SEQ ID NO. 1; the preparation method comprises the following steps: preparing a mini-SDD7 deaminase; the amino acid sequence of the mini-SDD7 deaminase is as shown in SEQ ID NO. 2; an nCas9 (D10A) coding sequence is obtained; the amino acid sequence of the nCas9 (D10A) coding sequence is as shown in SEQ ID NO. 3; a UGI coding sequence; the amino acid sequence of the UGI coding sequence is as shown in SEQ ID NO. 4; the starting plasmid is a plasmid pUC19-AMA1-(P) Hyg, and the starting plasmid is a plasmid pUC19-AMA1-(P) Hyg. The base editing system provided by the invention can be used for gene editing in trichoderma koningii.

The invention provides a protein expression system and application thereof, the protein expression system at least comprises a recombinant expression vector and Escherichia fagenii for transforming the recombinant expression vector, and the recombinant expression vector comprises a target protein gene segment and a pUC57 vector segment. The research finds that the same type of plasmids have significant expression difference in the Escherichia fagenii, for example, the target protein amount expressed by the pUC57 plasmid is significantly higher than that expressed by the pUC19 plasmid, then the pUC57 plasmid and the Escherichia fagenii are combined to form a protein expression system, the protein expression system has a better expression effect on the target protein, and the expression efficiency of the protein expression system is improved. The method has the advantages of simple operation, no need of extra inducers, reduction of the production cost of recombinant proteins, substantial improvement of the yield and enzyme activity of target proteins, and provision of more choices for the production of biological pharmacy, diagnostic reagents and industrial enzymes.