CN115976079A - Novel coronavirus replicon plasmids, construction and methods for drug screening - Google Patents

Abstract

A new coronavirus replicon plasmid, construction and method for drug screening, the new coronavirus replicon plasmid mainly comprises 5 'and 3' non-coding sequences of a new coronavirus genome, replicase genes ORF1a and ORF1b, a nucleocapsid protein gene, a neomycin phosphotransferase gene, a Nanoluc luciferase reporter gene, and a pBeloBAC11 vector containing a CMV eukaryotic promoter, an HDV ribozyme and a BGH termination signal. The operation of the plasmid does not involve and generate new live coronavirus particles, and is safer, more convenient and lower in cost than the operation involving the live virions. Moreover, the replicon plasmid contains a luciferase reporter gene, so that the novel crown replicon is more quickly applied and is suitable for large-scale drug screening.

Description

Novel coronavirus replicon plasmid, construction method and method for drug screening

Technical Field

The invention relates to the technical field of replicons, in particular to a novel coronavirus replicon plasmid, and a construction method and a method for drug screening.

Background

The new coronavirus is a single-stranded RNA virus with an envelope, the RNA length is nearly 30kb, proteins related to virus replication comprise replicase protein and N protein, and a plurality of compounds are researched and newly designed to aim at the replication and transcription processes of the new coronavirus.

A safe and efficient high-throughput compound screening and testing method is a problem to be solved urgently at present. At present, the domestic screening and testing of the medicine aiming at the new coronavirus are mostly based on live virus, and professional personnel are required to operate in a biosafety level 3 laboratory, but most laboratories do not have the experimental conditions. Moreover, live virus-based drug screening and testing are tedious, low in throughput and high in cost.

Disclosure of Invention

In order to solve the problems, the invention provides a new coronavirus replicon plasmid and a construction method and a working method applied to drug screening thereof, which effectively overcome the defects of high experimental conditions for drug screening and testing of new coronavirus, complicated operation of live virus-based drug screening and testing, low flux and high cost in the prior art.

In order to overcome the defects in the prior art, the invention provides a new coronavirus replicon plasmid and a solution for constructing and applying the new coronavirus replicon plasmid to a working method for drug screening, which comprises the following specific steps:

a novel coronavirus replicon plasmid comprising:

the new coronavirus replicon plasmid is a cDNA clone constructed on a BAC vector, and the cloning diagram is shown in FIG. 1. The clone comprises non-coding sequences at the 5 'end and the 3' end of the genome of the new coronavirus, ORF1a and ORF1b genes, and a nucleocapsid protein (N) gene; in addition, neomycin phosphotransferase gene (Neo) and Nanoluc luciferase (nLuc) reporter genes were inserted downstream of Transcription Regulatory Sequences (TRS) of the envelope protein (E) gene and the membrane protein (M) gene, respectively. The vector also includes a CMV eukaryotic promoter, a HDV ribozyme, and a BGH termination signal. The plasmid sequence is shown as SEQ ID NO.1.

A method of constructing a novel coronavirus replicon plasmid, said method of construction comprising the steps of: dividing a non-coding region at the 5' end of the new coronavirus genome and the sequence of ORF1a/ORF1b genes into 6 continuous fragments (A-F), and cloning each fragment into a pGEM-T Easy vector; the G fragment was synthesized in the following order: a transcription-regulating sequence (transcription-regulating sequence) of the E gene, a TRS-Nanoluc luciferase (nLuc) reporter gene of a neomycin phosphotransferase gene (Neo) -M gene and cloned into a pUC57 vector; the H fragment was amplified in the following order: TRS-N gene of N gene-non-coding sequence-poly (A) sequence of 3' end of genome, introducing II-type restriction enzyme cutting sites at two ends of fragment, and cloning to pGEM-T Easy vector. Each of the A-H fragments was cleaved from each plasmid using a class II restriction enzyme, and inserted into the cleaved pBeloBAC11 vector by in vitro ligation.

Use of the novel coronavirus replicon plasmid for in vitro monitoring of novel coronavirus prophylactic and therapeutic agents. Namely the application of the new coronavirus replicon plasmid in screening or monitoring anti-new coronavirus preventive or therapeutic drugs in vitro.

A method of using a neocoronavirus replicon plasmid for in vitro screening or monitoring of prophylactic or therapeutic drugs against neocoronavirus, comprising: transfecting the new corona replicon plasmid into cells, adding preventive and therapeutic drugs to the cells for treatment, and monitoring the chemiluminescence intensity after the treatment.

The invention has the beneficial effects that: the invention provides a safe and efficient system and method for screening and testing drugs, which can be used for screening and testing drugs targeting viral proteins and host factors involved in the replication and transcription processes of new coronavirus genomes. The system can be operated in a laboratory of low biosafety class, since no live virus particles are involved and not produced during the experiment. The system comprises luciferase reporter genes, is convenient and rapid to test, is standardized in result analysis, and can be applied to high-throughput screening. In addition, the system is theoretically applicable to any cell line. The invention has important application value for screening antiviral drugs of the new coronavirus. The defects of high experimental conditions for screening and testing the new coronavirus, complex operation, low flux and high cost of screening and testing the live virus-based medicine in the prior art are effectively overcome.

Drawings

FIG. 1 is a schematic of the cloning of the novel crown replicon cDNA of the present invention.

FIG. 2 is a map of the novel coronavirus replicon plasmid digested with the class II restriction enzyme BglII.

FIG. 3 is a replication curve of the novel coronavirus replicon plasmid after transfection into BHK21 cells.

Figure 4 is the result of the reed-seivir drug test based on the new coronavirus replicon plasmid.

FIG. 5 is a flow chart of drug testing based on the novel coronavirus replicon plasmid.

Detailed Description

The invention will be further described with reference to the following figures and examples.

As shown in fig. 1-5, the novel coronavirus replicon plasmid comprises: the novel coronavirus replicon plasmid is a cDNA clone constructed on a BAC vector, and the cloning diagram is shown in FIG. 1. The novel coronavirus replicon plasmid comprises genome 5 'and 3' non-coding sequences, replicase genes (ORF 1a and ORF1 b) and nucleocapsid protein (N) of the novel coronavirus, as well as a neomycin phosphotransferase gene (Neo) and a Nanoluc luciferase (nLuc) reporter gene, which are inserted into a pBeloBAC11 vector containing a CMV eukaryotic promoter, an HDV ribozyme and a BGH termination signal, and has a plasmid sequence shown as SEQ ID No.1.

The plasmid has a sequence shown as SEQ ID NO.1, and the specific sequence is as follows:

gcggccgcaaggggttcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattgtaatacgactcactatagggcgaattcgagctcggtacccgggGATCCCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTgCTAGGTGAACCGTATTAAAGGTTTATACCTTCCCAGGTAACAAACCAACCAACTTTCGATCTCTTGTAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTGGCTGTCACTCGGCTGCATGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAACACACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGACGTGCTCGTACGTGGCTTTGGAGACTCCGTGGAGGAGGTCTTATCAGAGGCACGTCAACATCTTAAAGATGGCACTTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCCTCAACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGtGGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGGCGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGTAATAAAGGAGCTGGTGGCCATAGTTACGGCGCCGATCTAAAGTCATTTGACTTAGGCGACGAGCTTGGCACTGATCCTTATGAAGATTTTCAAGAAAACTGGAACACTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCGTGAGCTTAACGGAGGGGCATACACTCGCTATGTCGATAACAACTTCTGTGGCCCTGATGGCTACCCTCTTGAGTGCATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTCATGCACTTTGTCCGAACAACTGGACTTTATTGACACTAAGAGGGGTGTATACTGCTGCCGTGAACATGAGCATGAAATTGCTTGGTACACGGAACGTTCTGAAAAGAGCTATGAATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAAATTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATTTCCCTTAAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAATTCGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGCACTGAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCCAAAATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAATTCAGAAGTAGGACCTGAGCATAGTCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTATGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGTTGTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTTCTTGAAATACTCCAAAAAGAGAAAGTCAACATCAATATTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATTATTTTGGCATCTTTTTCTGCTTCCACAAGTGCTTTTGTGGAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAACAAATTGTTGAATCCTGTGGTAATTTTAAAGTTACAAAAGGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAACAGAAATCAATACTGAGTCCTCTTTATGCATTTGCATCAGAGGCTGCTCGTGTTGTACGATCAATTTTCTCCCGCACTCTTGAAACTGCTCAAAATTCTGTGCGTGTTTTACAGAAGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTCACTGAGACTCATTGATGCTATGATGTTCACATCTGATTTGGCTACTAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCAGTTGACTTCGCAGTGGCTAACTAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCGTCCTTGATTGGCTTGAAGAGAAGTTTAAGGAAGGTGTAGAGTTTCTTAGAGACGGTTGGGAAATTGTTAAATTTATCTCAACCTGTGCTTGTGAAATTGTCGGTGGACAAATTGTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGACATTCTTTAAGCTTGTAAATAAATTTTTGGCTTTGTGTGCTGACTCTATCATTATTGGTGGAGCTAAACTTAAAGCCTTGAATTTAGGTGAAACATTTGTCACGCACTCAAAGGGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAACTGGCCTACTCATGCCTCTAAAAGCCCCAAAAGAAATTATCTTCTTAGAGGGAGAAACACTTCCCACAGAAGTGTTAACAGAGGAAGTTGTCTTGAAAACTGGTGATTTACAACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGCTCCATTGGTTGGTACACCAGTTTGTATTAACGGGCTTATGTTGCTCGAAATCAAAGACACAGAAAAGTACTGTGCCCTTGCACCTAATATGATGGTAACAAACAATACCTTCACACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGGTGATGACACTGTGATAGAAGTGCAAGGTTACAAGAGTGTGAATATCACTTTTGAACTTGATGAAAGGATTGATAAAGTACTTAATGAGAAGTGCTCTGCCTATACAGTTGAACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTGGCAGATGCTGTCATAAAAACTTTGCAACCAGTATCTGAATTACTTACACCACTGGGCATTGATTTAGATGAGTGGAGTATGGCTACATACTACTTATTTGATGAGTCTGGTGAGTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCCTCCAGATGAGGATGAAGAAGAAGGTGATTGTGAAGAAGAAGAGTTTGAGCCATCAACTCAATATGAGTATGGTACTGAAGATGATTACCAAGGTAAACCTTTGGAATTTGGTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCAAGAAGAAGATTGGTTAGATGATGATAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTAGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTAGTGGTTATTTAAAACTTACTGACAATGTATACATTAAAAATGCAGACATTGTGGAAGAAGCTAAAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAATGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCCTTAAATAAGGCTACTAACAATGCCATGCAAGTTGAATCTGATGATTACATAGCTACTAATGGACCACTTAAAGTGGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTAAACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAAGGTGAAGACATTCAACTTCTTAAGAGTGCTTATGAAAATTTTAATCAGCACGAAGTTCTACTTGCACCATTATTATCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTTAAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACTTAGCTGTCTTTGATAAAAATCTCTATGACAAACTTGTTTCAAGCTTTTTGGAAATGAAGAGTGAAAAGCAAGTTGAACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAAGCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAGAGAAAACAAGATGATAAGAAAATCAAAGCTTGTGTTGAAGAAGTTACAACAACTCTGGAAGAAACTAAGTTCCTCACAGAAAACTTGTTACTTTATATTGACATTAATGGCAATCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATTGACATCACTTTCTTAAAGAAAGATGCTCCATATATAGTGGGTGATGTTGTTCAAGAGGGTGTTTTAACTGCTGTGGTTATACCTACTAAAAAGGCTGGTGGCACTACTGAAATGCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAATTATATAACCACTTACCCGGGTCAGGGTTTAAATGGTTACACTGTAGAGGAGGCAAAGACAGTGCTTAAAAAGTGTAAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAATGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATTTGCGAGAAATGCTTGCACATGCAGAAGAAACACGCAAATTAATGCCTGTCTGTGTGGAAACTAAAGCCATAGTTTCAACTATACAGCGTAAATATAAGGGTATTAAAATACAAGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTTACACCAGTAAAACAACTGTAGCGTCACTTATCAACACACTTAACGATCTAAATGAAACTCTTGTTACAATGCCACTTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGCTGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACAGTTTCTGTTTCTTCACCTGATGCTGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCTGAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAAGATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTTCTTAAGAGAGGTGATAAAAGTGTATATTACACTAGTAATCCTACCACATTCCACCTAGATGGTGAAGTTATCACCTTTGACAATCTTAAGACACTTCTTTCTTTGAGAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGACAACATTAACCTCCACACGCAAGTTGTGGACATGTCAATGACATATGGACAACAGTTTGGTCCAACTTATTTGGATGGAGCTGATGTTACTAAAATAAAACCTCATAATTCACATGAAGGTAAAACATTTTATGTTTTACCTAATGATGACACTCTACGTGTTGAGGCTTTTGAGTACTACCACACAACTGATCCTAGTTTTCTGGGTAGGTACATGTCAGCATTAAATCACACTAAAAAGTGGAAATACCCACAAGTTAATGGTTTAACTTCTATTAAATGGGCAGATAACAACTGTTATCTTGCCACTGCATTGTTAACACTCcaaCAAATAGAGTTGAAGTTTAATCCACCTGCTCTACAAGATGCTTATTACAGAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGCACTTATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCTTATGAACAATTTAAGAAAGGTGTTCAGATACCTTGTACGTGTGGTAAACAAGCTACAAAATATCTAGTACAACAGGAGTCACCTTTTGTTATGATGTCAGCACCACCTGCTCAGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGTGAGTACACTGGTAATTACCAGTGTGGTCACTATAAACATATAACTTCTAAAGAAACTTTGTATTGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGTTACACAACAACCATAAAACCAGTTACTTATAAATTGGATGGTGTTGTTTGTACAGAAATTGACCCTAAGTTGGACAATTATTATAAGAAAGACAATTCTTATTTCACAGAGCAACCAATTGATCTTGTACCAAACCAACCATATCCAAACGCAAGCTTCGATAATTTTAAGTTTGTATGTGATAATATCAAATTTGCTGATGATTTAAACCAGTTAACTGGTTATAAGAAACCTGCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGACTTAAATGGTGATGTGGTGGCTATTGATTATAAACACTACACACCCTCTTTTAAGAAAGGAGCTAAATTGTTACATAAACCTATTGTTTGGCATGTTAACAATGCAACTAATAAAGCCACGTATAAACCAAATACCTGGTGTATACGTTGTCTTTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTTGATGTACTGAAGTCAGAGGACGCGCAGGGAATGGATAATCTTGCCTGCGAAGATCTAAAACCAGTCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAAGAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTCATGGTTTAGCTGCTGTTAATAGTGTCCCTTGGGATACTATAGCTAATTATGCTAAGCCTTTTCTTAACAAAGTTGTTAGTACAACTACTAACATAGTTACACGGTGTTTAAACCGTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTATTGCTACAATTGTGTACTTTTACTAGAAGTACAAATTCTAGAATTAAAGCATCTATGCCGACTACTATAGCAAAGAATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGAGGCTTCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACTGATAAATATTATAATTTGGTTTTTACTATTAAGTGTTTGCCTAGGTTCTTTAATCTACTCAACCGCTGCTTTAGGTGTTTTAATGTCTAATTTAGGCATGCCTTCTTACTGTACTGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCACTATTGCAACCTACTGTACTGGTTCTATACCTTGTAGTGTTTGTCTTAGTGGTTTAGATTCTTTAGACACCTATCCTTCTTTAGAAACTATACAAATTACCATTTCATCTTTTAAATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGGTTTTTGGCATATATTCTTTTCACTAGGTTTTTCTATGTACTTGGATTGGCTGCAATCATGCAATTGTTTTTCAGCTATTTTGCAGTACATTTTATTAGTAATTCTTGGCTTATGTGGTTAATAATTAATCTTGTACAAATGGCCCCGATTTCAGCTATGGTTAGAATGTACATCTTCTTTGCATCATTTTATTATGTATGGAAAAGTTATGTGCATGTTGTAGACGGTTGTAATTCATCAACTTGTATGATGTGTTACAAACGTAATAGAGCAACAAGAGTCGAATGTACAACTATTGTTAATGGTGTTAGAAGGTCCTTTTATGTCTATGCTAATGGAGGTAAAGGCTTTTGCAAACTACACAATTGGAATTGTGTTAATTGTGATACATTCTGTGCTGGTAGTACATTTATTAGTGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAAAGACCAATAAATCCTACTGACCAGTCTTCTTACATCGTTGATAGTGTTACAGTGAAGAATGGTTCCATCCATCTTTACTTTGATAAAGCTGGTCAAAAGACTTATGAAAGACATTCTCTCTCTCATTTTGTTAACTTAGACAACCTGAGAGCTAATAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTTTTGATGGTAAATCAAAATGTGAAGAATCATCTGCAAAATCAGCGTCTGTTTACTACAGTCAGCTTATGTGTCAACCTATACTGTTACTAGATCAGGCATTAGTGTCTGATGTTGGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATGCTTACGTTAATACGTTTTCATCAACTTTTAACGTACCAATGGAAAAACTCAAAACACTAGTTGCAACTGCAGAAGCTGAACTTGCAAAGAATGTGTCCTTAGACAATGTCTTATCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGATTCAGATGTAGAAACTAAAGATGTTGTTGAATGTCTTAAATTGTCACATCAATCTGACATAGAAGTTACTGGCGATAGTTGTAATAACTATATGCTCACCTATAACAAAGTTGAAAACATGACACCCCGTGACCTTGGTGCTTGTATTGACTGTAGTGCGCGTCATATTAATGCGCAGGTAGCAAAAAGTCACAACATTGCTTTGATATGGAACGTTAAAGATTTCATGTCATTGTCTGAACAACTACGAAAACAAATACGTAGTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACATGTGCAACTACTAGACAAGTTGTTAATGTTGTAACAACAAAGATAGCACTTAAGGGTGGTAAAATTGTTAATAATTGGTTGAAGCAGTTAATTAAAGTTACACTTGTGTTCCTTTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCATGTCATGTCTAAACATACTGACTTTTCAAGTGAAATCATAGGATACAAGGCTATTGATGGTGGTGTCACTCGTGACATAGCATCTACAGATACTTGTTTTGCTAACAAACATGCTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGTTATACTAATGACAAAGCTTGCCCATTGATTGCTGCAGTCATAACAAGAGAAGTGGGTTTTGTCGTGCCTGGTTTGCCTGGCACGATATTACGCACAACTAATGGTGACTTTTTGCATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACATCTGTTACACACCATCAAAACTTATAGAGTACACTGACTTTGCAACATCAGCTTGTGTTTTGGCTGCTGAATGTACAATTTTTAAAGATGCTTCTGGTAAGCCAGTACCATATTGTTATGATACCAATGTACTAGAAGGTTCTGTTGCTTATGAAAGTTTACGCCCTGACACACGTTATGTGCTCATGGATGGCTCTATTATTCAATTTCCTAACACCTACCTTGAAGGTTCTGTTAGAGTGGTAACAACTTTTGATTCTGAGTACTGTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGTGTTTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATGATTATTACAGATCTTTACCAGGAGTTTTCTGTGGTGTAGATGCTGTAAATTTACTTACTAATATGTTTACACCACTAATTCAACCTATTGGTGCTTTGGACATATCAGCATCTATAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGCCTTGCCTACTATTTTATGAGGTTTAGAAGAGCTTTTGGTGAATACAGTCATGTAGTTGCCTTTAATACTTTACTATTCCTTATGTCATTCACTGTACTCTGTTTAACACCAGTTTACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGTACTTGACATTTTATCTTACTAATGATGTTTCTTTTTTAGCACATATTCAGTGGATGGTTATGTTCACACCTTTAGTACCTTTCTGGATAACAATTGCTTATATCATTTGTATTTCCACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAAGAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTTTTGAAGAAGCTGCGCTGTGCACCTTTTTGTTAAATAAAGAAATGTATCTAAAGTTGCGTAGTGATGTGCTATTACCTCTTACGCAATATAATAGATACTTAGCTCTTTATAATAAGTACAAGTATTTTAGTGGAGCAATGGATACAACTAGCTACAGAGAAGCTGCTTGTTGTCATCTCGCAAAGGCTCTCAATGACTTCAGTAACTCAGGTTCTGATGTTCTTTACCAACCACCACAAACCTCTATCACCTCAGCTGTTTTGCAGAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAAGTTGAGGGTTGTATGGTACAAGTAACTTGTGGTACAACTACACTTAACGGTCTTTGGCTTGATGACGTAGTTTACTGTCCAAGACATGTGATCTGCACCTCTGAAGACATGCTTAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTAATCATAATTTCTTGGTACAGGCTGGTAATGTTCAACTCAGGGTTATTGGACATTCTATGCAAAATTGTGTACTTAAGCTTAAGGTTGATACAGCCAATCCTAAGACACCTAAGTATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTCAGTGTTAGCTTGTTACAATGGTTCACCATCTGGTGTTTACCAATGTGCTATGAGGCCCAATTTCACTATTAAGGGTTCATTCCTTAATGGTTCATGTGGTAGTGTTGGTTTTAACATAGATTATGACTGTGTCTCTTTTTGTTACATGCACCATATGGAATTACCAACTGGAGTTCATGCTGGCACAGACTTAGAAGGTAACTTTTATGGACCTTTTGTTGACAGGCAAACAGCACAAGCAGCTGGTACGGACACAACTATTACAGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAAATGGAGACAGGTGGTTTCTCAATCGATTTACCACAACTCTTAATGACTTTAACCTTGTGGCTATGAAGTACAATTATGAACCTCTAACACAAGACCATGTTGACATACTAGGACCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATATGTGTGCTTCATTAAAAGAATTACTGCAAAATGGTATGAATGGACGTACCATATTGGGTAGTGCTTTATTAGAAGATGAATTTACACCTTTTGATGTTGTTAGACAATGCTCAGGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCAAGGGTACACACCACTGGTTGTTACTCACAATTTTGACTTCACTTTTAGTTTTAGTCCAGAGTACTCAATGGTCTTTGTTCTTTTTTTTGTATGAAAATGCCTTTTTACCTTTTGCTATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGTTTGTCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGTTACCTTCTCTTGCCACTGTAGCTTATTTTAATATGGTCTATATGCCTGCTAGTTGGGTGATGCGTATTATGACATGGTTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGCTAAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCCTTATGACAGCAAGAACTGTGTATGATGATGGTGCTAGGAGAGTGTGGACACTTATGAATGTCTTGACACTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATCAAGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTTCTAACTACTCAGGTGTAGTTACAACTGTCATGTTTTTGGCCAGAGGTATTGTTTTTATGTGTGTTGAGTATTGCCCTATTTTCTTCATAACTGGTAATACACTTCAGTGTATAATGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTTACTTTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTCTTGGTGTTTATGATTACTTAGTTTCTACACAGGAGTTTAGATATATGAATTCACAGGGACTACTCCCACCCAAGAATAGCATAGATGCCTTCAAACTCAACATTAAATTGTTGGGTGTTGGTGGCAAACCTTGTATCAAAGTAGCCACTGTACAGTCTAAAATGTCAGATGTAAAGTGCACATCAGTAGTCTTACTCTCAGTTTTGCAACAACTCAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTACACAATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTTGAAAAAATGGTTTCACTACTTTCTGTTTTGCTTTCCATGCAGGGTGCTGTAGACATAAACAAGCTTTGTGAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTATAGCCTCAGAGTTTAGTTCCCTTCCATCATATGCAGCTTTTGCTACTGCTCAAGAAGCTTATGAGCAGGCTGTTGCTAATGGTGATTCTGAAGTTGTTCTTAAAAAGTTGAAGAAGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGATGCAGCCATGCAACGTAAGTTGGAAAAGATGGCTGATCAAGCTATGACCCAAATGTATAAACAGGCTAGATCTGAGGACAAGAGGGCAAAAGTTACTAGTGCTATGCAGACAATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGCACTCAACAACATTATCAACAATGCAAGAGATGGTTGTGTTCCCTTGAACATAATACCTCTTACAACAGCAGCCAAACTAATGGTTGTCATACCAGACTATAACACATATAAAAATACGTGTGATGGTACAACATTTACTTATGCATCAGCATTGTGGGAAATCCAACAGGTTGTAGATGCAGATAGTAAAATTGTTCAACTTAGTGAAATTAGTATGGACAATTCACCTAATTTAGCATGGCCTCTTATTGTAACAGCTTTAAGGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCTTAGTCCTGTTGCACTACGACAGATGTCTTGTGCTGCCGGTACTACACAAACTGCTTGCACTGATGACAATGCGTTAGCTTACTACAACACAACAAAGGGAGGTAGGTTTGTACTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGCTAGATTCCCTAAGAGTGATGGAACTGGTACTATCTATACAGAACTGGAACCACCTTGTAGGTTTGTTACAGACACACCTAAAGGTCCTAAAGTGAAGTATTTATACTTTATTAAAGGATTAAACAACCTAAATAGAGGTATGGTACTTGGTAGTTTAGCTGCCACAGTACGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTGTTAAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACCGGAAGCCAATATGGATCAAGAATCCTTTGGTGGTGCATCGTGTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTAAAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTGTTGTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTTGTAGTTAAGAGACACACTTTCTCTAACTACCAACATGAAGAAACAATTTATAATTTACTTAAGGATTGTCCAGCTGTTGCTAAACATGACTTCTTTAAGTTTAGAATAGACGGTGACATGGTACCACATATATCACGTCAACGTCTTACTAAATACACAATGGCAGACCTCGTCTATGCTTTAAGGCATTTTGATGAAGGTAATTGTGACACATTAAAAGAAATACTTGTCACATACAATTGTTGTGATGATGATTATTTCAATAAAAAGGACTGGTATGATTTTGTAGAAAACCCAGATATATTACGCGTATACGCCAACTTAGGTGAACGTGTACGCCAAGCTTTGTTAAAAACAGTACAATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGTGTACTGACATTAGATAATCAAGATCTCAATGGTAACTGGTATGATTTCGGTGATTTCATACAAACCACGCCAGGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGCCTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTATTTTAAATATTGGGATCAGACATACCACCCAAATTGTGTTAACTGTTTGGATGACAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTATTCTCTACAGTGTTCCCACCTACAAGTTTTGGACCACTAGTGAGAAAAATATTTGTTGATGGTGTTCCATTTGTAGTTTCAACTGGATACCACTTCAGAGAGCTAGGTGTTGTACATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGTTTTAAGGAATTACTTGTGTATGCTGCTGACCCTGCTATGCACGCTGCTTCTGGTAATCTATTACTAGATAAACGCACTACGTGCTTTTCAGTAGCTGCACTTACTAACAATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTATGACTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAATTAAAACACTTCTTCTTTGCTCAGGATGGTAATGCTGCTATCAGCGATTATGACTACTATCGTTATAATCTAcCAACAATGTGtGATATCAGACAACTACTATTTGTAGTTGAAGTTGTTGATAAGTACTTTGATTGTTACGATGGTGGCTGTATTAATGCTAACCAAGTCATCGTCAACAACCTAGACAAATCAGCTGGTTTTCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGATTCAATGAGTTATGAGGATCAAGATGCACTTTTCGCATATACAAAACGTAATGTCATCCCTACTATAACTCAAATGAATCTTAAGTATGCCATTAGTGCAAAGAATAGAGCTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAATAGACAGTTTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGTAGTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAACTGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCTAAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGTTCTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATAGATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATGTTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTCCGCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGTTGACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCTCAATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTATGCATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTATTATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTGACCTTACTAAAGGACCTCATGAATTTTGCTCTCAACATACAATGCTAGTTAAACAGGGTGATGATTATGTGTACCTTCCTTACCCAGATCCATCAAGAATCCTAGGGGCCGGCTGTTTTGTAGATGATATCGTAAAAACAGATGGTACACTTATGATTGAACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTTACTAAACATCCTAATCAGGAGTATGCTGATGTCTTTCATTTGTACTTACAATACATAAGAAAGCTACATGATGAGTTAACAGGACACATGTTAGACATGTATTCTGTTATGCTTACTAATGATAACACTTCAAGGTATTGGGAACCTGAGTTTTATGAGGCTATGTACACACCGCATACAGTCTTACAGGCTGTTGGGGCTTGTGTTCTTTGCAATTCACAGACTTCATTAAGATGTGGTGCTTGCATACGTAGACCATTCTTATGTTGTAAATGCTGTTACGACCATGTCATATCAACATCACATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCAATGCTCCAGGTTGTGATGTCACAGATGTGACTCAACTTTACTTAGGAGGTATGAGCTATTATTGTAAATCACATAAACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAAGTTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAATGTTACTGACTTTAATGCAATTGCAACATGTGACTGGACAAATGCTGGTGATTACATTTTAGCTAACACCTGTACTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTCAAAGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTGCTACTGTACGTGAAGTGCTGTCTGACAGAGAATTACATCTTTCATGGGAAGTTGGTAAACCTAGACCACCACTTAACCGAAATTATGTCTTTACTGGTTATCGTGTAACTAAAAACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAAAAGGTGACTATGGTGATGCTGTTGTTTACCGAGGTACAACAACTTACAAATTAAATGTTGGTGATTATTTTGTGCTGACATCACATACAGTAATGCCATTAAGTGCACCTACACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAACACTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCAAAAGGTTGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGGTACTGGTAAGAGTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTGCTCGCATAGTGTATACAGCTTGCTCTCATGCCGCTGTTGATGCACTATGTGAGAAGGCATTAAAATATTTGCCTATAGATAAATGTAGTAGAATTATACCTGCACGTGCTCGTGTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATTAGAACAGTATGTCTTTTGTACTGTAAATGCATTGCCTGAGACGACAGCAGATATAGTTGTCTTTGATGAAATTTCAATGGCCACAAATTATGATTTGAGTGTTGTCAATGCCAGATTACGTGCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATTACCTGCACCACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCTCAATGCTTTAAAATGTTTTATAAGGGTGTTATCACGCATGATGTTTCATCTGCAATTAACAGGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACGTAACCCTGCTTGGAGAAAAGCTGTCTTTATTTCACCTTATAATTCACAGAATGCTGTAGCCTCAAAGATTTTGGGACTACCAACTCAAACTGTTGATTCATCACAGGGCTCAGAATATGACTATGTCATATTCACTCAAACCACTGAAACAGCTCACTCTTGTAATGTAAACAGATTTAATGTTGCTATTACCAGAGCAAAAGTAGGCATACTTTGCATAATGTCTGATAGAGACCTTTATGACAAGTTGCAATTTACAAGTCTTGAAATTCCACGTAGGAATGTGGCAACTTTACAAGCTGAAAATGTAACAGGACTCTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTACACAGGCACCTACACACCTCAGTGTTGACACTAAATTCAAAACTGAAGGTTTATGTGTTGACATACCTGGCATACCTAAGGACATGACCTATAGAAGACTCATCTCTATGATGGGTTTTAAAATGAATTATCAAGTTAATGGTTACCCTAACATGTTTATCACCCGCGAAGAAGCTATAAGACATGTACGTGCATGGATTGGCTTCGATGTCGAGGGGTGTCATGCTACTAGAGAAGCTGTTGGTACCAATTTACCTTTACAGCTAGGTTTTTCTACAGGTGTTAACCTAGTTGCTGTACCTACAGGTTATGTTGATACACCTAATAATACAGATTTTTCCAGAGTTAGTGCTAAACCACCGCCTGGAGATCAATTTAAACACCTCATACCACTTATGTACAAAGGACTTCCTTGGAATGTAGTGCGTATAAAGATTGTACAAATGTTAAGTGACACACTTAAAAATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCACATGGCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAATAGGACCTGAGCGCACCTGTTGTCTATGTGATAGACGTGCCACATGCTTTTCCACTGCTTCAGACACTTATGCCTGTTGGCATCATTCTATTGGATTTGATTACGTCTATAATCCGTTTATGATTGATGTTCAACAATGGGGTTTTACAGGTAACCTACAAAGCAACCATGATCTGTATTGTCAAGTCCATGGTAATGCACATGTAGCTAGTTGTGATGCAATCATGACTAGGTGTCTAGCTGTCCACGAGTGCTTTGTTAAGCGTGTTGACTGGACTATTGAATATCCTATAATTGGTGATGAACTGAAGATTAATGCGGCTTGTAGAAAGGTTCAACACATGGTTGTTAAAGCTGCATTATTAGCAGACAAATTCCCAGTTCTTCACGACATTGGTAACCCTAAAGCTATTAAGTGTGTACCTCAAGCTGATGTAGAATGGAAGTTCTATGATGCACAGCCTTGTAGTGACAAAGCTTATAAAATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTCACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATATCCTGCTAATTCCATTGTTTGTAGATTTGACACTAGAGTGCTATCTAACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTATGTAAATAAACATGCATTCCACACACCAGCTTTTGATAAAAGTGCTTTTGTTAATTTAAAACAATTACCATTTTTCTATTACTCTGACAGTCCATGTGAGTCTCATGGAAAACAAGTAGTGTCAGATATAGATTATGTACCACTAAAGTCTGCTACGTGTATAACACGTTGCAATTTAGGTGGTGCTGTCTGTAGACATCATGCTAATGAGTACAGATTGTATCTCGATGCTTATAACATGATGATCTCAGCTGGCTTTAGCTTGTGGGTTTACAAACAATTTGATACTTATAACCTCTGGAACACTTTTACAAGACTTCAGAGTTTAGAAAATGTGGCTTTTAATGTTGTAAATAAGGGACACTTTGATGGACAACAGGGTGAAGTACCAGTTTCTATCATTAATAACACTGTTTACACAAAAGTTGATGGTGTTGATGTAGAATTGTTTGAAAATAAAACAACATTACCTGTTAATGTAGCATTTGAGCTTTGGGCTAAGCGCAACATTAAACCAGTACCAGAGGTGAAAATACTCAATAATTTGGGTGTGGACATTGCTGCTAATACTGTGATCTGGGACTACAAAAGAGATGCTCCAGCACATATATCTACTATTGGTGTTTGTTCTATGACTGACATAGCCAAGAAACCAACTGAAACGATTTGTGCACCACTCACTGTCTTTTTTGATGGTAGAGTTGATGGTCAAGTAGACTTATTTAGAAATGCCCGTAATGGTGTTCTTATTACAGAAGGTAGTGTTAAAGGTTTACAACCATCTGTAGGTCCCAAACAAGCTAGTCTTAATGGAGTCACATTAATTGGAGAAGCCGTAAAAACACAGTTCAATTATTATAAGAAAGTTGATGGTGTTGTCCAACAATTACCTGAAACTTACTTTACTCAGAGTAGAAATTTACAAGAATTTAAACCCAGGAGTCAAATGGAAATTGATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAATTAGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTCATAGTCAGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAACGTTTTAAGGAATCACCTTTTGAATTAGAAGATTTTATTCCTATGGACAGTACAGTTAAAAACTATTTCATAACAGATGCGCAAACAGGTTCATCTAAGTGTGTGTGTTCTGTTATTGATTTATTACTTGATGATTTTGTTGAAATAATAAAATCCCAAGATTTATCTGTAGTTTCTAAGGTTGTCAAAGTGACTATTGACTATACAGAAATTTCATTTATGCTTTGGTGTAAAGATGGCCATGTAGAAACATTTTACCCAAAATTACAATCTAGTCAAGCGTGGCAACCGGGTGTTGCTATGCCTAATCTTTACAAAATGCAAAGAATGCTATTAGAAAAGTGTGACCTTCAAAATTATGGTGATAGTGCAACATTACCTAAAGGCATAATGATGAATGTCGCAAAATATACTCAACTGTGTCAATATTTAAACACATTAACATTAGCTGTACCCTATAATATGAGAGTTATACATTTTGGTGCTGGTTCTGATAAAGGAGTTGCACCAGGTACAGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGCTGCTTGTCGATTCAGATCTTAATGACTTTGTCTCTGATGCAGATTCAACTTTGATTGGTGATTGTGCAACTGTACATACAGCTAATAAATGGGATCTCATTATTAGTGATATGTACGACCCTAAGACTAAAAATGTTACAAAAGAAAATGACTCTAAAGAGGGTTTTTTCACTTACATTTGTGGGTTTATACAACAAAAGCTAGCTCTTGGAGGTTCCGTGGCTATAAAGATAACAGAACATTCTTGGAATGCTGATCTTTATAAGCTCATGGGACACTTCGCATGGTGGACAGCCTTTGTTACTAATGTGAATGCGTCATCATCTGAAGCATTTTTAATTGGATGTAATTATCTTGGCAAACCACGCGAACAAATAGATGGTTATGTCATGCATGCAAATTACATATTTTGGAGGAATACAAATCCAATTCAGTTGTCTTCCTATTCTTTATTTGACATGAGTAAATTTCCCCTTAAATTAAGGGGTACTGCTGTTATGTCTTTAAAAGAAGGTCAAATCAATGATATGATTTTATCTCTTCTTAGTAAAGGTAGACTTATAATTAGAGAAAACAACAGAGTTGTTATTTCTAGTGATGTTCTTGTTAACAACTAAGCACAAGCTGATGAGTACGAACTTATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGATCGTGTTAAAAATCTGAATTCTTCTAGAGTTCCTGATCTTCTGGTCTAAACGAACTAAATATTATATTAGTTTTTCTGTTTGGAACTTTAATTTTAGCCATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAAAGACTTTTTAGAGTATCATGACGTTCGTGTTGTTTTAGATTTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGATGTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAGTGTGTAACATTAGGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTACGATCGAGTGTACAGTGAACAATGCTAGGGAGAGCTGCCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAGTGCTATCCCCATGTGATTTTAATAGCTTCTTAGGAGAATGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCGCGCCgggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAagcttgagtattctatagtgtcacctaaatagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgaaccccttgcggccgcccgggccgtcgaccaattctcatgtttgacagcttatcatcgaatttctgccattcatccgcttattatcacttattcaggcgtagcaaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacggaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcgcgataagctcatggagcggcgtaaccgtcgcacaggaaggacagagaaagcgcggatctgggaagtgacggacagaacggtcaggacctggattggggaggcggttgccgccgctgctgctgacggtgtgacgttctctgttccggtcacaccacatacgttccgccattcctatgcgatgcacatgctgtatgccggtataccgctgaaagttctgcaaagcctgatgggacataagtccatcagttcaacggaagtctacacgaaggtttttgcgctggatgtggctgcccggcaccgggtgcagtttgcgatgccggagtctgatgcggttgcgatgctgaaacaattatcctgagaataaatgccttggcctttatatggaaatgtggaactgagtggatatgctgtttttgtctgttaaacagagaagctggctgttatccactgagaagcgaacgaaacagtcgggaaaatctcccattatcgtagagatccgcattattaatctcaggagcctgtgtagcgtttataggaagtagtgttctgtcatgatgcctgcaagcggtaacgaaaacgatttgaatatgccttcaggaacaatagaaatcttcgtgcggtgttacgttgaagtggagcggattatgtcagcaatggacagaacaacctaatgaacacagaaccatgatgtggtctgtccttttacagccagtagtgctcgccgcagtcgagcgacagggcgaagccctcgagtgagcgaggaagcaccagggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccacttatccacggggatatttttataattattttttttatagtttttagatcttcttttttagagcgccttgtaggcctttatccatgctggttctagagaaggtgttgtgacaaattgccctttcagtgtgacaaatcaccctcaaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctcagaagaagctgttttttcacaaagttatccctgcttattgactcttttttatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtcatggcggaaacagcggttatcaatcacaagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatatagtctctcccgggatcaaaaacgtatgctgtatctgttcgttgaccagatcagaaaatctgatggcaccctacaggaacatgacggtatctgcgagatccatgttgctaaatatgctgaaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagtttcgcggggaaggaagtggttttttatcgccctgaagaggatgccggcgatgaaaaaggctatgaatcttttccttggtttatcaaacgtgcgcacagtccatccagagggctttacagtgtacatatcaacccatatctcattcccttctttatcgggttacagaaccggtttacgcagtttcggcttagtgaaacaaaagaaatcaccaatccgtatgccatgcgtttatacgaatccctgtgtcagtatcgtaagccggatggctcaggcatcgtctctctgaaaatcgactggatcatagagcgttaccagctgcctcaaagttaccagcgtatgcctgacttccgccgccgcttcctgcaggtctgtgttaatgagatcaacagcagaactccaatgcgcctctcatacattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacaggatagtctgagggttatctgtcacagatttgagggtggttcgtcacatttgttctgacctactgagggtaatttgtcacagttttgctgtttccttcagcctgcatggattttctcatactttttgaactgtaatttttaaggaagccaaatttgagggcagtttgtcacagttgatttccttctctttcccttcgtcatgtgacctgatatcgggggttagttcgtcatcattgatgagggttgattatcacagtttattactctgaattggctatccgcgtgtgtacctctacctggagtttttcccacggtggatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttctttgcttcctcgccagttcgctcgctatgctcggttacacggctgcggcgagcgctagtgataataagtgactgaggtatgtgctcttcttatctccttttgtagtgttgctcttattttaaacaactttgcggttttttgatgactttgcgattttgttgttgctttgcagtaaattgcaagatttaataaaaaaacgcaaagcaatgattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgcataaacgctggtcatgaaatgacgaaggctatcgccattgcacagtttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggtttcttctcaggctatcagagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggttatacaattgaacaaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttgctgcccataaaggtggcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgttttgctcgtggaaggtaacgacccccagggaacagcctcaatgtatcacggatgggtaccagatcttcatattcatgcagaagacactctcctgcctttctatcttggggaaaaggacgatgtcacttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcaccgtattgaaactgagttaatgggcaaatttgatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtcatagttattgacagcgcgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtttgactacacctccgcactgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgcttaccaaatacagcaatagtaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacgtgaaacggatgaagttggtaaaggtcagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggagaaatgctctttctatttgggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgttattccaaaacatacgctcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtaggagtaatggctcgcggtaatgccattactttgcctgtatgtggtcgggatgtgaagtttactcttgaagtgctccggggtgatagtgttgagaagacctctcgggtatggtcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtcaacagacaccggcgttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtgattatcgtgttctggttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgattatcgcccaacaagtgcttatgaacgtggtcagcgttatgcaagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattacccgctgtatcaacaccgccaaattgcctaaatcagttgttgctcttttttctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaaaagcctttacagataaagaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaagaagttatcactcttttaacttctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgtataagggcgataaaatggtgcttaacctggacaggtctcgtgttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaagccagcaccctgatgcgaccacgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaatattctctctgggcccactgttccacttgtatcgtcggtctgataatcagactgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgataatcagactgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacgatcccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagcgtgagactacgattccatcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgcctgctgtggattgctgctgtgtcctgcttatccacaacattttgcgcacggttatgtggacaaaatacctggttacccaggccgtgccggcacgttaaccgggctgcatccgatgcaagtgtgtcgctgtcgacgagctcgcgagctcggacatgaggttgccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctctgaaaagaaaggaaacgacaggtgctgaaagcgagctttttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtccgagctcatcgctaataacttcgtatagcatacattatacgaagttatattcgat

the construction method of the novel coronavirus replicon plasmid comprises the following steps: dividing a non-coding region at the 5' end of a new coronavirus genome and a sequence of ORF1a/ORF1b genes into 6 continuous fragments (A-F), introducing class II restriction enzyme sites at two ends of each fragment when amplifying each fragment gene, and cloning each fragment to a pGEM-T Easy vector; the G fragment was synthesized in the following order: transcription-regulation sequence of E gene, TRS-Nanoluc luciferase (nLuc) reporter gene of neomycin phosphotransferase gene (Neo) -M gene, introducing II-type restriction enzyme cutting sites at two ends of the fragment, and cloning to pUC57 vector; the H fragment was amplified in the following order: TRS-N gene of N gene-non-coding sequence-poly (A) sequence of 3' end of genome, introducing II-type restriction enzyme cutting sites at two ends of fragment, and cloning to pGEM-T Easy vector.

Each of the A-H fragments was cleaved from each plasmid using a class II restriction enzyme, and inserted into the cleaved pBeloBAC11 vector by in vitro ligation. Cells infected with DH10B bacteria were electroporated and screened on agarose plates with chloramphenicol. Selecting a single clone from the plate, extracting plasmids, carrying out enzyme digestion on the plasmids by using a type II restriction enzyme BglII, identifying the plasmids through an enzyme digestion map, and carrying out sequencing verification on the plasmids.

The operation steps of the antiviral drug test method based on the new coronavirus replicon plasmid are as follows:

cells transfected with the novel coronavirus replicon plasmid (e.g., huh-7) by directional transfection were seeded into 96-well plates and placed at a concentration of 5% CO ₂ Culturing at 37 deg.C for 4 hr, removing cell culture solution containing plasmid and transfection reagent, adding cell culture solution containing drug or DMSO only, and placing in 5% CO ₂ And cultured in a cell culture chamber at a temperature of 37 ℃ for 20 hours. The cell culture fluid containing the drug was removed, the cells were washed once with PBS, and luciferase activity in the cells was determined using the Nanoluc luciferase substrate monitoring kit. The luciferase activity in the cells of the experimental group was compared with that of the control group, and the drug inhibition efficiency was calculated.

Calculating the formula:

inhibition efficiency (%) = (1-test group luciferase activity/control group luciferase activity) × 100%

Example 1: construction strategy for novel coronavirus replicon plasmids

Dividing a non-coding region at the 5' end of a new coronavirus genome and a sequence of ORF1a/ORF1b genes into 6 continuous fragments (A-F), introducing class II restriction enzyme sites at two ends of each fragment when amplifying each fragment gene, and cloning each fragment to a pGEM-T Easy vector; the G fragment was synthesized in the following order: transcription-regulating sequence (transcription-regulating sequence) of E gene, TRS-Nanoluc luciferase (nLuc) reporter gene of neomycin phosphotransferase gene (Neo) -M gene, introducing II-type restriction enzyme sites at two ends of the fragment, and cloning to pUC57 vector; the H fragment was amplified in the following order: TRS-N gene of N gene-non-coding sequence-poly (A) sequence of 3' end of genome, introducing II-type restriction enzyme cutting sites at two ends of fragment, and cloning to pGEM-T Easy vector.

Each of the A-H fragments was cleaved from each plasmid using a class II restriction enzyme, and inserted into the cleaved pBeloBAC11 vector by in vitro ligation. Cells infected with DH10B bacteria were electroporated and screened on agarose plates with chloramphenicol. Selecting a single clone from the plate, extracting plasmids, carrying out enzyme digestion on the plasmids by using a type II restriction enzyme BglII, identifying the plasmids through an enzyme digestion map, and carrying out sequencing verification on the plasmids.

Example 2: amplification and identification of the novel coronavirus replicon plasmids

The operation steps of amplification, extraction and identification of the plasmid pBAC-CMV-SARS-CoV-2Replicon are as follows:

(1) Replicon plasmid transformation: 100ng of plasmid pBAC-CMV-SARS-CoV-2Replicon was transformed into DH10B competent cells by electrotransformation, and then the competent cells were transferred to SOC medium and cultured in a shaker at 37 ℃ and 200rpm for 1 hour. Competent cells were collected by centrifugation at 3000g for 5 minutes, plated with a bacterial culture plate containing chloramphenicol, and cultured in an incubator at 37 ℃ for about 17 hours, whereby a clear single colony appeared on the plate.

(2) Replicon plasmid amplification: from the above plate, 1 single colony was picked up to 5mL of LB liquid medium containing chloramphenicol, and cultured on a shaker at 37 ℃ and 200rpm for 12 hours. Then, the bacterial suspension was inoculated into 250mL of LB medium containing chloramphenicol in accordance with 1.

(3) Replicon plasmid extraction: the plasmid was preliminarily extracted using Buffer T1, buffer T2 and Buffer T3 in BAC/PAC DNA Maxi Kit (Omega) Kit, and the DNA was precipitated using isopropanol. Then Plasmid extraction was performed using the Plasmid Mini Kit (Omega) to obtain a purified replicon Plasmid.

(4) Replicon plasmid identification: after the plasmid extraction, 200ng of plasmid is taken for enzyme digestion identification. The enzyme digestion system is as follows: 200ng replicon plasmid, 0.3. Mu.L BglII (NEB), 1. Mu.L 10 × Cutsmart Buffer, ddH2O make up to 10. Mu.L. Enzyme digestion is carried out for 1 hour at 37 ℃, and then the enzyme digestion system is carried out

Performing 1% agarose gel electrophoresis, comparing the restriction enzyme map, and obtaining the correct map as shown in figure 2.

(5) Replicon plasmid transfected cells:

the replicon transfects BHK21 cells, and the expression of Nanoluc luciferase in the cells is monitored at different time points after the transfection, and the monitoring result is shown in figure 3, which proves that the replicon plasmid can replicate.

Example 3: novel coronavirus replicon plasmids for drug screening and monitoring

The new coronavirus replicon plasmid is transfected into Huh-7 cells by reverse transfection, after the cells are cultured for 4h, the cell culture solution containing the plasmid is removed, gradient diluted Reidesvir is added, after the cells are cultured for 20h, the cell culture solution is removed, a luciferase substrate is added, the chemiluminescence value of each hole is read in a chemiluminescence instrument, and the inhibition effect of the drug is analyzed according to the data statistics, as shown in FIG. 4.

The present invention has been described above by way of examples, and it will be apparent to those skilled in the art that the present disclosure is not limited to the above-described examples, and various changes, modifications, and substitutions can be made without departing from the scope of the present invention.

Sequence listing

<110> Nanjing Keguan biomedical science and technology Co., ltd

<120> novel coronavirus replicon plasmid, construction and method for drug screening

<140> 202210650267X

<141> 2022-06-09

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 33080

<212> DNA

<213> Artificial Sequence

<400> 1

gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 60

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 120

gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg 180

aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 240

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 300

ccagtgaatt gtaatacgac tcactatagg gcgaattcga gctcggtacc cggggatccc 360

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 420

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 540

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 600

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 660

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 720

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 780

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 840

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 900

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctgct aggtgaaccg 960

tattaaaggt ttataccttc ccaggtaaca aaccaaccaa ctttcgatct cttgtagatc 1020

tgttctctaa acgaacttta aaatctgtgt ggctgtcact cggctgcatg cttagtgcac 1080

tcacgcagta taattaataa ctaattactg tcgttgacag gacacgagta actcgtctat 1140

cttctgcagg ctgcttacgg tttcgtccgt gttgcagccg atcatcagca catctaggtt 1200

tcgtccgggt gtgaccgaaa ggtaagatgg agagccttgt ccctggtttc aacgagaaaa 1260

cacacgtcca actcagtttg cctgttttac aggttcgcga cgtgctcgta cgtggctttg 1320

gagactccgt ggaggaggtc ttatcagagg cacgtcaaca tcttaaagat ggcacttgtg 1380

gcttagtaga agttgaaaaa ggcgttttgc ctcaacttga acagccctat gtgttcatca 1440

aacgttcgga tgctcgaact gcacctcatg gtcatgttat ggttgagctg gtagcagaac 1500

tcgaaggcat tcagtacggt cgtagtggtg agacacttgg tgtccttgtc cctcatgtgg 1560

gcgaaatacc agtggcttac cgcaaggttc ttcttcgtaa gaacggtaat aaaggagctg 1620

gtggccatag ttacggcgcc gatctaaagt catttgactt aggcgacgag cttggcactg 1680

atccttatga agattttcaa gaaaactgga acactaaaca tagcagtggt gttacccgtg 1740

aactcatgcg tgagcttaac ggaggggcat acactcgcta tgtcgataac aacttctgtg 1800

gccctgatgg ctaccctctt gagtgcatta aagaccttct agcacgtgct ggtaaagctt 1860

catgcacttt gtccgaacaa ctggacttta ttgacactaa gaggggtgta tactgctgcc 1920

gtgaacatga gcatgaaatt gcttggtaca cggaacgttc tgaaaagagc tatgaattgc 1980

agacaccttt tgaaattaaa ttggcaaaga aatttgacac cttcaatggg gaatgtccaa 2040

attttgtatt tcccttaaat tccataatca agactattca accaagggtt gaaaagaaaa 2100

agcttgatgg ctttatgggt agaattcgat ctgtctatcc agttgcgtca ccaaatgaat 2160

gcaaccaaat gtgcctttca actctcatga agtgtgatca ttgtggtgaa acttcatggc 2220

agacgggcga ttttgttaaa gccacttgcg aattttgtgg cactgagaat ttgactaaag 2280

aaggtgccac tacttgtggt tacttacccc aaaatgctgt tgttaaaatt tattgtccag 2340

catgtcacaa ttcagaagta ggacctgagc atagtcttgc cgaataccat aatgaatctg 2400

gcttgaaaac cattcttcgt aagggtggtc gcactattgc ctttggaggc tgtgtgttct 2460

cttatgttgg ttgccataac aagtgtgcct attgggttcc acgtgctagc gctaacatag 2520

gttgtaacca tacaggtgtt gttggagaag gttccgaagg tcttaatgac aaccttcttg 2580

aaatactcca aaaagagaaa gtcaacatca atattgttgg tgactttaaa cttaatgaag 2640

agatcgccat tattttggca tctttttctg cttccacaag tgcttttgtg gaaactgtga 2700

aaggtttgga ttataaagca ttcaaacaaa ttgttgaatc ctgtggtaat tttaaagtta 2760

caaaaggaaa agctaaaaaa ggtgcctgga atattggtga acagaaatca atactgagtc 2820

ctctttatgc atttgcatca gaggctgctc gtgttgtacg atcaattttc tcccgcactc 2880

ttgaaactgc tcaaaattct gtgcgtgttt tacagaaggc cgctataaca atactagatg 2940

gaatttcaca gtattcactg agactcattg atgctatgat gttcacatct gatttggcta 3000

ctaacaatct agttgtaatg gcctacatta caggtggtgt tgttcagttg acttcgcagt 3060

ggctaactaa catctttggc actgtttatg aaaaactcaa acccgtcctt gattggcttg 3120

aagagaagtt taaggaaggt gtagagtttc ttagagacgg ttgggaaatt gttaaattta 3180

tctcaacctg tgcttgtgaa attgtcggtg gacaaattgt cacctgtgca aaggaaatta 3240

aggagagtgt tcagacattc tttaagcttg taaataaatt tttggctttg tgtgctgact 3300

ctatcattat tggtggagct aaacttaaag ccttgaattt aggtgaaaca tttgtcacgc 3360

actcaaaggg attgtacaga aagtgtgtta aatccagaga agaaactggc ctactcatgc 3420

ctctaaaagc cccaaaagaa attatcttct tagagggaga aacacttccc acagaagtgt 3480

taacagagga agttgtcttg aaaactggtg atttacaacc attagaacaa cctactagtg 3540

aagctgttga agctccattg gttggtacac cagtttgtat taacgggctt atgttgctcg 3600

aaatcaaaga cacagaaaag tactgtgccc ttgcacctaa tatgatggta acaaacaata 3660

ccttcacact caaaggcggt gcaccaacaa aggttacttt tggtgatgac actgtgatag 3720

aagtgcaagg ttacaagagt gtgaatatca cttttgaact tgatgaaagg attgataaag 3780

tacttaatga gaagtgctct gcctatacag ttgaactcgg tacagaagta aatgagttcg 3840

cctgtgttgt ggcagatgct gtcataaaaa ctttgcaacc agtatctgaa ttacttacac 3900

cactgggcat tgatttagat gagtggagta tggctacata ctacttattt gatgagtctg 3960

gtgagtttaa attggcttca catatgtatt gttctttcta ccctccagat gaggatgaag 4020

aagaaggtga ttgtgaagaa gaagagtttg agccatcaac tcaatatgag tatggtactg 4080

aagatgatta ccaaggtaaa cctttggaat ttggtgccac ttctgctgct cttcaacctg 4140

aagaagagca agaagaagat tggttagatg atgatagtca acaaactgtt ggtcaacaag 4200

acggcagtga ggacaatcag acaactacta ttcaaacaat tgttgaggtt caacctcaat 4260

tagagatgga acttacacca gttgttcaga ctattgaagt gaatagtttt agtggttatt 4320

taaaacttac tgacaatgta tacattaaaa atgcagacat tgtggaagaa gctaaaaagg 4380

taaaaccaac agtggttgtt aatgcagcca atgtttacct taaacatgga ggaggtgttg 4440

caggagcctt aaataaggct actaacaatg ccatgcaagt tgaatctgat gattacatag 4500

ctactaatgg accacttaaa gtgggtggta gttgtgtttt aagcggacac aatcttgcta 4560

aacactgtct tcatgttgtc ggcccaaatg ttaacaaagg tgaagacatt caacttctta 4620

agagtgctta tgaaaatttt aatcagcacg aagttctact tgcaccatta ttatcagctg 4680

gtatttttgg tgctgaccct atacattctt taagagtttg tgtagatact gttcgcacaa 4740

atgtctactt agctgtcttt gataaaaatc tctatgacaa acttgtttca agctttttgg 4800

aaatgaagag tgaaaagcaa gttgaacaaa agatcgctga gattcctaaa gaggaagtta 4860

agccatttat aactgaaagt aaaccttcag ttgaacagag aaaacaagat gataagaaaa 4920

tcaaagcttg tgttgaagaa gttacaacaa ctctggaaga aactaagttc ctcacagaaa 4980

acttgttact ttatattgac attaatggca atcttcatcc agattctgcc actcttgtta 5040

gtgacattga catcactttc ttaaagaaag atgctccata tatagtgggt gatgttgttc 5100

aagagggtgt tttaactgct gtggttatac ctactaaaaa ggctggtggc actactgaaa 5160

tgctagcgaa agctttgaga aaagtgccaa cagacaatta tataaccact tacccgggtc 5220

agggtttaaa tggttacact gtagaggagg caaagacagt gcttaaaaag tgtaaaagtg 5280

ccttttacat tctaccatct attatctcta atgagaagca agaaattctt ggaactgttt 5340

cttggaattt gcgagaaatg cttgcacatg cagaagaaac acgcaaatta atgcctgtct 5400

gtgtggaaac taaagccata gtttcaacta tacagcgtaa atataagggt attaaaatac 5460

aagagggtgt ggttgattat ggtgctagat tttactttta caccagtaaa acaactgtag 5520

cgtcacttat caacacactt aacgatctaa atgaaactct tgttacaatg ccacttggct 5580

atgtaacaca tggcttaaat ttggaagaag ctgctcggta tatgagatct ctcaaagtgc 5640

cagctacagt ttctgtttct tcacctgatg ctgttacagc gtataatggt tatcttactt 5700

cttcttctaa aacacctgaa gaacatttta ttgaaaccat ctcacttgct ggttcctata 5760

aagattggtc ctattctgga caatctacac aactaggtat agaatttctt aagagaggtg 5820

ataaaagtgt atattacact agtaatccta ccacattcca cctagatggt gaagttatca 5880

cctttgacaa tcttaagaca cttctttctt tgagagaagt gaggactatt aaggtgttta 5940

caacagtaga caacattaac ctccacacgc aagttgtgga catgtcaatg acatatggac 6000

aacagtttgg tccaacttat ttggatggag ctgatgttac taaaataaaa cctcataatt 6060

cacatgaagg taaaacattt tatgttttac ctaatgatga cactctacgt gttgaggctt 6120

ttgagtacta ccacacaact gatcctagtt ttctgggtag gtacatgtca gcattaaatc 6180

acactaaaaa gtggaaatac ccacaagtta atggtttaac ttctattaaa tgggcagata 6240

acaactgtta tcttgccact gcattgttaa cactccaaca aatagagttg aagtttaatc 6300

cacctgctct acaagatgct tattacagag caagggctgg tgaagctgct aacttttgtg 6360

cacttatctt agcctactgt aataagacag taggtgagtt aggtgatgtt agagaaacaa 6420

tgagttactt gtttcaacat gccaatttag attcttgcaa aagagtcttg aacgtggtgt 6480

gtaaaacttg tggacaacag cagacaaccc ttaagggtgt agaagctgtt atgtacatgg 6540

gcacactttc ttatgaacaa tttaagaaag gtgttcagat accttgtacg tgtggtaaac 6600

aagctacaaa atatctagta caacaggagt caccttttgt tatgatgtca gcaccacctg 6660

ctcagtatga acttaagcat ggtacattta cttgtgctag tgagtacact ggtaattacc 6720

agtgtggtca ctataaacat ataacttcta aagaaacttt gtattgcata gacggtgctt 6780

tacttacaaa gtcctcagaa tacaaaggtc ctattacgga tgttttctac aaagaaaaca 6840

gttacacaac aaccataaaa ccagttactt ataaattgga tggtgttgtt tgtacagaaa 6900

ttgaccctaa gttggacaat tattataaga aagacaattc ttatttcaca gagcaaccaa 6960

ttgatcttgt accaaaccaa ccatatccaa acgcaagctt cgataatttt aagtttgtat 7020

gtgataatat caaatttgct gatgatttaa accagttaac tggttataag aaacctgctt 7080

caagagagct taaagttaca tttttccctg acttaaatgg tgatgtggtg gctattgatt 7140

ataaacacta cacaccctct tttaagaaag gagctaaatt gttacataaa cctattgttt 7200

ggcatgttaa caatgcaact aataaagcca cgtataaacc aaatacctgg tgtatacgtt 7260

gtctttggag cacaaaacca gttgaaacat caaattcgtt tgatgtactg aagtcagagg 7320

acgcgcaggg aatggataat cttgcctgcg aagatctaaa accagtctct gaagaagtag 7380

tggaaaatcc taccatacag aaagacgttc ttgagtgtaa tgtgaaaact accgaagttg 7440

taggagacat tatacttaaa ccagcaaata atagtttaaa aattacagaa gaggttggcc 7500

acacagatct aatggctgct tatgtagaca attctagtct tactattaag aaacctaatg 7560

aattatctag agtattaggt ttgaaaaccc ttgctactca tggtttagct gctgttaata 7620

gtgtcccttg ggatactata gctaattatg ctaagccttt tcttaacaaa gttgttagta 7680

caactactaa catagttaca cggtgtttaa accgtgtttg tactaattat atgccttatt 7740

tctttacttt attgctacaa ttgtgtactt ttactagaag tacaaattct agaattaaag 7800

catctatgcc gactactata gcaaagaata ctgttaagag tgtcggtaaa ttttgtctag 7860

aggcttcatt taattatttg aagtcaccta atttttctaa actgataaat attataattt 7920

ggtttttact attaagtgtt tgcctaggtt ctttaatcta ctcaaccgct gctttaggtg 7980

ttttaatgtc taatttaggc atgccttctt actgtactgg ttacagagaa ggctatttga 8040

actctactaa tgtcactatt gcaacctact gtactggttc tataccttgt agtgtttgtc 8100

ttagtggttt agattcttta gacacctatc cttctttaga aactatacaa attaccattt 8160

catcttttaa atgggattta actgcttttg gcttagttgc agagtggttt ttggcatata 8220

ttcttttcac taggtttttc tatgtacttg gattggctgc aatcatgcaa ttgtttttca 8280

gctattttgc agtacatttt attagtaatt cttggcttat gtggttaata attaatcttg 8340

tacaaatggc cccgatttca gctatggtta gaatgtacat cttctttgca tcattttatt 8400

atgtatggaa aagttatgtg catgttgtag acggttgtaa ttcatcaact tgtatgatgt 8460

gttacaaacg taatagagca acaagagtcg aatgtacaac tattgttaat ggtgttagaa 8520

ggtcctttta tgtctatgct aatggaggta aaggcttttg caaactacac aattggaatt 8580

gtgttaattg tgatacattc tgtgctggta gtacatttat tagtgatgaa gttgcgagag 8640

acttgtcact acagtttaaa agaccaataa atcctactga ccagtcttct tacatcgttg 8700

atagtgttac agtgaagaat ggttccatcc atctttactt tgataaagct ggtcaaaaga 8760

cttatgaaag acattctctc tctcattttg ttaacttaga caacctgaga gctaataaca 8820

ctaaaggttc attgcctatt aatgttatag tttttgatgg taaatcaaaa tgtgaagaat 8880

catctgcaaa atcagcgtct gtttactaca gtcagcttat gtgtcaacct atactgttac 8940

tagatcaggc attagtgtct gatgttggtg atagtgcgga agttgcagtt aaaatgtttg 9000

atgcttacgt taatacgttt tcatcaactt ttaacgtacc aatggaaaaa ctcaaaacac 9060

tagttgcaac tgcagaagct gaacttgcaa agaatgtgtc cttagacaat gtcttatcta 9120

cttttatttc agcagctcgg caagggtttg ttgattcaga tgtagaaact aaagatgttg 9180

ttgaatgtct taaattgtca catcaatctg acatagaagt tactggcgat agttgtaata 9240

actatatgct cacctataac aaagttgaaa acatgacacc ccgtgacctt ggtgcttgta 9300

ttgactgtag tgcgcgtcat attaatgcgc aggtagcaaa aagtcacaac attgctttga 9360

tatggaacgt taaagatttc atgtcattgt ctgaacaact acgaaaacaa atacgtagtg 9420

ctgctaaaaa gaataactta ccttttaagt tgacatgtgc aactactaga caagttgtta 9480

atgttgtaac aacaaagata gcacttaagg gtggtaaaat tgttaataat tggttgaagc 9540

agttaattaa agttacactt gtgttccttt ttgttgctgc tattttctat ttaataacac 9600

ctgttcatgt catgtctaaa catactgact tttcaagtga aatcatagga tacaaggcta 9660

ttgatggtgg tgtcactcgt gacatagcat ctacagatac ttgttttgct aacaaacatg 9720

ctgattttga cacatggttt agccagcgtg gtggtagtta tactaatgac aaagcttgcc 9780

cattgattgc tgcagtcata acaagagaag tgggttttgt cgtgcctggt ttgcctggca 9840

cgatattacg cacaactaat ggtgactttt tgcatttctt acctagagtt tttagtgcag 9900

ttggtaacat ctgttacaca ccatcaaaac ttatagagta cactgacttt gcaacatcag 9960

cttgtgtttt ggctgctgaa tgtacaattt ttaaagatgc ttctggtaag ccagtaccat 10020

attgttatga taccaatgta ctagaaggtt ctgttgctta tgaaagttta cgccctgaca 10080

cacgttatgt gctcatggat ggctctatta ttcaatttcc taacacctac cttgaaggtt 10140

ctgttagagt ggtaacaact tttgattctg agtactgtag gcacggcact tgtgaaagat 10200

cagaagctgg tgtttgtgta tctactagtg gtagatgggt acttaacaat gattattaca 10260

gatctttacc aggagttttc tgtggtgtag atgctgtaaa tttacttact aatatgttta 10320

caccactaat tcaacctatt ggtgctttgg acatatcagc atctatagta gctggtggta 10380

ttgtagctat cgtagtaaca tgccttgcct actattttat gaggtttaga agagcttttg 10440

gtgaatacag tcatgtagtt gcctttaata ctttactatt ccttatgtca ttcactgtac 10500

tctgtttaac accagtttac tcattcttac ctggtgttta ttctgttatt tacttgtact 10560

tgacatttta tcttactaat gatgtttctt ttttagcaca tattcagtgg atggttatgt 10620

tcacaccttt agtacctttc tggataacaa ttgcttatat catttgtatt tccacaaagc 10680

atttctattg gttctttagt aattacctaa agagacgtgt agtctttaat ggtgtttcct 10740

ttagtacttt tgaagaagct gcgctgtgca cctttttgtt aaataaagaa atgtatctaa 10800

agttgcgtag tgatgtgcta ttacctctta cgcaatataa tagatactta gctctttata 10860

ataagtacaa gtattttagt ggagcaatgg atacaactag ctacagagaa gctgcttgtt 10920

gtcatctcgc aaaggctctc aatgacttca gtaactcagg ttctgatgtt ctttaccaac 10980

caccacaaac ctctatcacc tcagctgttt tgcagagtgg ttttagaaaa atggcattcc 11040

catctggtaa agttgagggt tgtatggtac aagtaacttg tggtacaact acacttaacg 11100

gtctttggct tgatgacgta gtttactgtc caagacatgt gatctgcacc tctgaagaca 11160

tgcttaaccc taattatgaa gatttactca ttcgtaagtc taatcataat ttcttggtac 11220

aggctggtaa tgttcaactc agggttattg gacattctat gcaaaattgt gtacttaagc 11280

ttaaggttga tacagccaat cctaagacac ctaagtataa gtttgttcgc attcaaccag 11340

gacagacttt ttcagtgtta gcttgttaca atggttcacc atctggtgtt taccaatgtg 11400

ctatgaggcc caatttcact attaagggtt cattccttaa tggttcatgt ggtagtgttg 11460

gttttaacat agattatgac tgtgtctctt tttgttacat gcaccatatg gaattaccaa 11520

ctggagttca tgctggcaca gacttagaag gtaactttta tggacctttt gttgacaggc 11580

aaacagcaca agcagctggt acggacacaa ctattacagt taatgtttta gcttggttgt 11640

acgctgctgt tataaatgga gacaggtggt ttctcaatcg atttaccaca actcttaatg 11700

actttaacct tgtggctatg aagtacaatt atgaacctct aacacaagac catgttgaca 11760

tactaggacc tctttctgct caaactggaa ttgccgtttt agatatgtgt gcttcattaa 11820

aagaattact gcaaaatggt atgaatggac gtaccatatt gggtagtgct ttattagaag 11880

atgaatttac accttttgat gttgttagac aatgctcagg tgttactttc caaagtgcag 11940

tgaaaagaac aatcaagggt acacaccact ggttgttact cacaattttg acttcacttt 12000

tagttttagt ccagagtact caatggtctt tgttcttttt tttgtatgaa aatgcctttt 12060

taccttttgc tatgggtatt attgctatgt ctgcttttgc aatgatgttt gtcaaacata 12120

agcatgcatt tctctgtttg tttttgttac cttctcttgc cactgtagct tattttaata 12180

tggtctatat gcctgctagt tgggtgatgc gtattatgac atggttggat atggttgata 12240

ctagtttgtc tggttttaag ctaaaagact gtgttatgta tgcatcagct gtagtgttac 12300

taatccttat gacagcaaga actgtgtatg atgatggtgc taggagagtg tggacactta 12360

tgaatgtctt gacactcgtt tataaagttt attatggtaa tgctttagat caagccattt 12420

ccatgtgggc tcttataatc tctgttactt ctaactactc aggtgtagtt acaactgtca 12480

tgtttttggc cagaggtatt gtttttatgt gtgttgagta ttgccctatt ttcttcataa 12540

ctggtaatac acttcagtgt ataatgctag tttattgttt cttaggctat ttttgtactt 12600

gttactttgg cctcttttgt ttactcaacc gctactttag actgactctt ggtgtttatg 12660

attacttagt ttctacacag gagtttagat atatgaattc acagggacta ctcccaccca 12720

agaatagcat agatgccttc aaactcaaca ttaaattgtt gggtgttggt ggcaaacctt 12780

gtatcaaagt agccactgta cagtctaaaa tgtcagatgt aaagtgcaca tcagtagtct 12840

tactctcagt tttgcaacaa ctcagagtag aatcatcatc taaattgtgg gctcaatgtg 12900

tccagttaca caatgacatt ctcttagcta aagatactac tgaagccttt gaaaaaatgg 12960

tttcactact ttctgttttg ctttccatgc agggtgctgt agacataaac aagctttgtg 13020

aagaaatgct ggacaacagg gcaaccttac aagctatagc ctcagagttt agttcccttc 13080

catcatatgc agcttttgct actgctcaag aagcttatga gcaggctgtt gctaatggtg 13140

attctgaagt tgttcttaaa aagttgaaga agtctttgaa tgtggctaaa tctgaatttg 13200

accgtgatgc agccatgcaa cgtaagttgg aaaagatggc tgatcaagct atgacccaaa 13260

tgtataaaca ggctagatct gaggacaaga gggcaaaagt tactagtgct atgcagacaa 13320

tgcttttcac tatgcttaga aagttggata atgatgcact caacaacatt atcaacaatg 13380

caagagatgg ttgtgttccc ttgaacataa tacctcttac aacagcagcc aaactaatgg 13440

ttgtcatacc agactataac acatataaaa atacgtgtga tggtacaaca tttacttatg 13500

catcagcatt gtgggaaatc caacaggttg tagatgcaga tagtaaaatt gttcaactta 13560

gtgaaattag tatggacaat tcacctaatt tagcatggcc tcttattgta acagctttaa 13620

gggccaattc tgctgtcaaa ttacagaata atgagcttag tcctgttgca ctacgacaga 13680

tgtcttgtgc tgccggtact acacaaactg cttgcactga tgacaatgcg ttagcttact 13740

acaacacaac aaagggaggt aggtttgtac ttgcactgtt atccgattta caggatttga 13800

aatgggctag attccctaag agtgatggaa ctggtactat ctatacagaa ctggaaccac 13860

cttgtaggtt tgttacagac acacctaaag gtcctaaagt gaagtattta tactttatta 13920

aaggattaaa caacctaaat agaggtatgg tacttggtag tttagctgcc acagtacgtc 13980

tacaagctgg taatgcaaca gaagtgcctg ccaattcaac tgtattatct ttctgtgctt 14040

ttgctgtaga tgctgctaaa gcttacaaag attatctagc tagtggggga caaccaatca 14100

ctaattgtgt taagatgttg tgtacacaca ctggtactgg tcaggcaata acagttacac 14160

cggaagccaa tatggatcaa gaatcctttg gtggtgcatc gtgttgtctg tactgccgtt 14220

gccacataga tcatccaaat cctaaaggat tttgtgactt aaaaggtaag tatgtacaaa 14280

tacctacaac ttgtgctaat gaccctgtgg gttttacact taaaaacaca gtctgtaccg 14340

tctgcggtat gtggaaaggt tatggctgta gttgtgatca actccgcgaa cccatgcttc 14400

agtcagctga tgcacaatcg tttttaaacg ggtttgcggt gtaagtgcag cccgtcttac 14460

accgtgcggc acaggcacta gtactgatgt cgtatacagg gcttttgaca tctacaatga 14520

taaagtagct ggttttgcta aattcctaaa aactaattgt tgtcgcttcc aagaaaagga 14580

cgaagatgac aatttaattg attcttactt tgtagttaag agacacactt tctctaacta 14640

ccaacatgaa gaaacaattt ataatttact taaggattgt ccagctgttg ctaaacatga 14700

cttctttaag tttagaatag acggtgacat ggtaccacat atatcacgtc aacgtcttac 14760

taaatacaca atggcagacc tcgtctatgc tttaaggcat tttgatgaag gtaattgtga 14820

cacattaaaa gaaatacttg tcacatacaa ttgttgtgat gatgattatt tcaataaaaa 14880

ggactggtat gattttgtag aaaacccaga tatattacgc gtatacgcca acttaggtga 14940

acgtgtacgc caagctttgt taaaaacagt acaattctgt gatgccatgc gaaatgctgg 15000

tattgttggt gtactgacat tagataatca agatctcaat ggtaactggt atgatttcgg 15060

tgatttcata caaaccacgc caggtagtgg agttcctgtt gtagattctt attattcatt 15120

gttaatgcct atattaacct tgaccagggc tttaactgca gagtcacatg ttgacactga 15180

cttaacaaag ccttacatta agtgggattt gttaaaatat gacttcacgg aagagaggtt 15240

aaaactcttt gaccgttatt ttaaatattg ggatcagaca taccacccaa attgtgttaa 15300

ctgtttggat gacagatgca ttctgcattg tgcaaacttt aatgttttat tctctacagt 15360

gttcccacct acaagttttg gaccactagt gagaaaaata tttgttgatg gtgttccatt 15420

tgtagtttca actggatacc acttcagaga gctaggtgtt gtacataatc aggatgtaaa 15480

cttacatagc tctagactta gttttaagga attacttgtg tatgctgctg accctgctat 15540

gcacgctgct tctggtaatc tattactaga taaacgcact acgtgctttt cagtagctgc 15600

acttactaac aatgttgctt ttcaaactgt caaacccggt aattttaaca aagacttcta 15660

tgactttgct gtgtctaagg gtttctttaa ggaaggaagt tctgttgaat taaaacactt 15720

cttctttgct caggatggta atgctgctat cagcgattat gactactatc gttataatct 15780

accaacaatg tgtgatatca gacaactact atttgtagtt gaagttgttg ataagtactt 15840

tgattgttac gatggtggct gtattaatgc taaccaagtc atcgtcaaca acctagacaa 15900

atcagctggt tttccattta ataaatgggg taaggctaga ctttattatg attcaatgag 15960

ttatgaggat caagatgcac ttttcgcata tacaaaacgt aatgtcatcc ctactataac 16020

tcaaatgaat cttaagtatg ccattagtgc aaagaataga gctcgcaccg tagctggtgt 16080

ctctatctgt agtactatga ccaatagaca gtttcatcaa aaattattga aatcaatagc 16140

cgccactaga ggagctactg tagtaattgg aacaagcaaa ttctatggtg gttggcacaa 16200

catgttaaaa actgtttata gtgatgtaga aaaccctcac cttatgggtt gggattatcc 16260

taaatgtgat agagccatgc ctaacatgct tagaattatg gcctcacttg ttcttgctcg 16320

caaacataca acgtgttgta gcttgtcaca ccgtttctat agattagcta atgagtgtgc 16380

tcaagtattg agtgaaatgg tcatgtgtgg cggttcacta tatgttaaac caggtggaac 16440

ctcatcagga gatgccacaa ctgcttatgc taatagtgtt tttaacattt gtcaagctgt 16500

cacggccaat gttaatgcac ttttatctac tgatggtaac aaaattgccg ataagtatgt 16560

ccgcaattta caacacagac tttatgagtg tctctataga aatagagatg ttgacacaga 16620

ctttgtgaat gagttttacg catatttgcg taaacatttc tcaatgatga tactctctga 16680

cgatgctgtt gtgtgtttca atagcactta tgcatctcaa ggtctagtgg ctagcataaa 16740

gaactttaag tcagttcttt attatcaaaa caatgttttt atgtctgaag caaaatgttg 16800

gactgagact gaccttacta aaggacctca tgaattttgc tctcaacata caatgctagt 16860

taaacagggt gatgattatg tgtaccttcc ttacccagat ccatcaagaa tcctaggggc 16920

cggctgtttt gtagatgata tcgtaaaaac agatggtaca cttatgattg aacggttcgt 16980

gtctttagct atagatgctt acccacttac taaacatcct aatcaggagt atgctgatgt 17040

ctttcatttg tacttacaat acataagaaa gctacatgat gagttaacag gacacatgtt 17100

agacatgtat tctgttatgc ttactaatga taacacttca aggtattggg aacctgagtt 17160

ttatgaggct atgtacacac cgcatacagt cttacaggct gttggggctt gtgttctttg 17220

caattcacag acttcattaa gatgtggtgc ttgcatacgt agaccattct tatgttgtaa 17280

atgctgttac gaccatgtca tatcaacatc acataaatta gtcttgtctg ttaatccgta 17340

tgtttgcaat gctccaggtt gtgatgtcac agatgtgact caactttact taggaggtat 17400

gagctattat tgtaaatcac ataaaccacc cattagtttt ccattgtgtg ctaatggaca 17460

agtttttggt ttatataaaa atacatgtgt tggtagcgat aatgttactg actttaatgc 17520

aattgcaaca tgtgactgga caaatgctgg tgattacatt ttagctaaca cctgtactga 17580

aagactcaag ctttttgcag cagaaacgct caaagctact gaggagacat ttaaactgtc 17640

ttatggtatt gctactgtac gtgaagtgct gtctgacaga gaattacatc tttcatggga 17700

agttggtaaa cctagaccac cacttaaccg aaattatgtc tttactggtt atcgtgtaac 17760

taaaaacagt aaagtacaaa taggagagta cacctttgaa aaaggtgact atggtgatgc 17820

tgttgtttac cgaggtacaa caacttacaa attaaatgtt ggtgattatt ttgtgctgac 17880

atcacataca gtaatgccat taagtgcacc tacactagtg ccacaagagc actatgttag 17940

aattactggc ttatacccaa cactcaatat ctcagatgag ttttctagca atgttgcaaa 18000

ttatcaaaag gttggtatgc aaaagtattc tacactccag ggaccacctg gtactggtaa 18060

gagtcatttt gctattggcc tagctctcta ctacccttct gctcgcatag tgtatacagc 18120

ttgctctcat gccgctgttg atgcactatg tgagaaggca ttaaaatatt tgcctataga 18180

taaatgtagt agaattatac ctgcacgtgc tcgtgtagag tgttttgata aattcaaagt 18240

gaattcaaca ttagaacagt atgtcttttg tactgtaaat gcattgcctg agacgacagc 18300

agatatagtt gtctttgatg aaatttcaat ggccacaaat tatgatttga gtgttgtcaa 18360

tgccagatta cgtgctaagc actatgtgta cattggcgac cctgctcaat tacctgcacc 18420

acgcacattg ctaactaagg gcacactaga accagaatat ttcaattcag tgtgtagact 18480

tatgaaaact ataggtccag acatgttcct cggaacttgt cggcgttgtc ctgctgaaat 18540

tgttgacact gtgagtgctt tggtttatga taataagctt aaagcacata aagacaaatc 18600

agctcaatgc tttaaaatgt tttataaggg tgttatcacg catgatgttt catctgcaat 18660

taacaggcca caaataggcg tggtaagaga attccttaca cgtaaccctg cttggagaaa 18720

agctgtcttt atttcacctt ataattcaca gaatgctgta gcctcaaaga ttttgggact 18780

accaactcaa actgttgatt catcacaggg ctcagaatat gactatgtca tattcactca 18840

aaccactgaa acagctcact cttgtaatgt aaacagattt aatgttgcta ttaccagagc 18900

aaaagtaggc atactttgca taatgtctga tagagacctt tatgacaagt tgcaatttac 18960

aagtcttgaa attccacgta ggaatgtggc aactttacaa gctgaaaatg taacaggact 19020

ctttaaagat tgtagtaagg taatcactgg gttacatcct acacaggcac ctacacacct 19080

cagtgttgac actaaattca aaactgaagg tttatgtgtt gacatacctg gcatacctaa 19140

ggacatgacc tatagaagac tcatctctat gatgggtttt aaaatgaatt atcaagttaa 19200

tggttaccct aacatgttta tcacccgcga agaagctata agacatgtac gtgcatggat 19260

tggcttcgat gtcgaggggt gtcatgctac tagagaagct gttggtacca atttaccttt 19320

acagctaggt ttttctacag gtgttaacct agttgctgta cctacaggtt atgttgatac 19380

acctaataat acagattttt ccagagttag tgctaaacca ccgcctggag atcaatttaa 19440

acacctcata ccacttatgt acaaaggact tccttggaat gtagtgcgta taaagattgt 19500

acaaatgtta agtgacacac ttaaaaatct ctctgacaga gtcgtatttg tcttatgggc 19560

acatggcttt gagttgacat ctatgaagta ttttgtgaaa ataggacctg agcgcacctg 19620

ttgtctatgt gatagacgtg ccacatgctt ttccactgct tcagacactt atgcctgttg 19680

gcatcattct attggatttg attacgtcta taatccgttt atgattgatg ttcaacaatg 19740

gggttttaca ggtaacctac aaagcaacca tgatctgtat tgtcaagtcc atggtaatgc 19800

acatgtagct agttgtgatg caatcatgac taggtgtcta gctgtccacg agtgctttgt 19860

taagcgtgtt gactggacta ttgaatatcc tataattggt gatgaactga agattaatgc 19920

ggcttgtaga aaggttcaac acatggttgt taaagctgca ttattagcag acaaattccc 19980

agttcttcac gacattggta accctaaagc tattaagtgt gtacctcaag ctgatgtaga 20040

atggaagttc tatgatgcac agccttgtag tgacaaagct tataaaatag aagaattatt 20100

ctattcttat gccacacatt ctgacaaatt cacagatggt gtatgcctat tttggaattg 20160

caatgtcgat agatatcctg ctaattccat tgtttgtaga tttgacacta gagtgctatc 20220

taaccttaac ttgcctggtt gtgatggtgg cagtttgtat gtaaataaac atgcattcca 20280

cacaccagct tttgataaaa gtgcttttgt taatttaaaa caattaccat ttttctatta 20340

ctctgacagt ccatgtgagt ctcatggaaa acaagtagtg tcagatatag attatgtacc 20400

actaaagtct gctacgtgta taacacgttg caatttaggt ggtgctgtct gtagacatca 20460

tgctaatgag tacagattgt atctcgatgc ttataacatg atgatctcag ctggctttag 20520

cttgtgggtt tacaaacaat ttgatactta taacctctgg aacactttta caagacttca 20580

gagtttagaa aatgtggctt ttaatgttgt aaataaggga cactttgatg gacaacaggg 20640

tgaagtacca gtttctatca ttaataacac tgtttacaca aaagttgatg gtgttgatgt 20700

agaattgttt gaaaataaaa caacattacc tgttaatgta gcatttgagc tttgggctaa 20760

gcgcaacatt aaaccagtac cagaggtgaa aatactcaat aatttgggtg tggacattgc 20820

tgctaatact gtgatctggg actacaaaag agatgctcca gcacatatat ctactattgg 20880

tgtttgttct atgactgaca tagccaagaa accaactgaa acgatttgtg caccactcac 20940

tgtctttttt gatggtagag ttgatggtca agtagactta tttagaaatg cccgtaatgg 21000

tgttcttatt acagaaggta gtgttaaagg tttacaacca tctgtaggtc ccaaacaagc 21060

tagtcttaat ggagtcacat taattggaga agccgtaaaa acacagttca attattataa 21120

gaaagttgat ggtgttgtcc aacaattacc tgaaacttac tttactcaga gtagaaattt 21180

acaagaattt aaacccagga gtcaaatgga aattgatttc ttagaattag ctatggatga 21240

attcattgaa cggtataaat tagaaggcta tgccttcgaa catatcgttt atggagattt 21300

tagtcatagt cagttaggtg gtttacatct actgattgga ctagctaaac gttttaagga 21360

atcacctttt gaattagaag attttattcc tatggacagt acagttaaaa actatttcat 21420

aacagatgcg caaacaggtt catctaagtg tgtgtgttct gttattgatt tattacttga 21480

tgattttgtt gaaataataa aatcccaaga tttatctgta gtttctaagg ttgtcaaagt 21540

gactattgac tatacagaaa tttcatttat gctttggtgt aaagatggcc atgtagaaac 21600

attttaccca aaattacaat ctagtcaagc gtggcaaccg ggtgttgcta tgcctaatct 21660

ttacaaaatg caaagaatgc tattagaaaa gtgtgacctt caaaattatg gtgatagtgc 21720

aacattacct aaaggcataa tgatgaatgt cgcaaaatat actcaactgt gtcaatattt 21780

aaacacatta acattagctg taccctataa tatgagagtt atacattttg gtgctggttc 21840

tgataaagga gttgcaccag gtacagctgt tttaagacag tggttgccta cgggtacgct 21900

gcttgtcgat tcagatctta atgactttgt ctctgatgca gattcaactt tgattggtga 21960

ttgtgcaact gtacatacag ctaataaatg ggatctcatt attagtgata tgtacgaccc 22020

taagactaaa aatgttacaa aagaaaatga ctctaaagag ggttttttca cttacatttg 22080

tgggtttata caacaaaagc tagctcttgg aggttccgtg gctataaaga taacagaaca 22140

ttcttggaat gctgatcttt ataagctcat gggacacttc gcatggtgga cagcctttgt 22200

tactaatgtg aatgcgtcat catctgaagc atttttaatt ggatgtaatt atcttggcaa 22260

accacgcgaa caaatagatg gttatgtcat gcatgcaaat tacatatttt ggaggaatac 22320

aaatccaatt cagttgtctt cctattcttt atttgacatg agtaaatttc cccttaaatt 22380

aaggggtact gctgttatgt ctttaaaaga aggtcaaatc aatgatatga ttttatctct 22440

tcttagtaaa ggtagactta taattagaga aaacaacaga gttgttattt ctagtgatgt 22500

tcttgttaac aactaagcac aagctgatga gtacgaactt atgattgaac aagatggatt 22560

gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 22620

gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 22680

ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 22740

atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 22800

gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 22860

tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 22920

tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 22980

gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 23040

agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 23100

ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 23160

cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 23220

tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 23280

cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgatcgtg 23340

ttaaaaatct gaattcttct agagttcctg atcttctggt ctaaacgaac taaatattat 23400

attagttttt ctgtttggaa ctttaatttt agccatggtc ttcacactcg aagatttcgt 23460

tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt 23520

gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag 23580

cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg 23640

cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg atgatcatca 23700

ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat 23760

cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt 23820

aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg 23880

ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat 23940

tctggcgtaa agacttttta gagtatcatg acgttcgtgt tgttttagat ttcatctaaa 24000

cgaacaaact aaaatgtctg ataatggacc ccaaaatcag cgaaatgcac cccgcattac 24060

gtttggtgga ccctcagatt caactggcag taaccagaat ggagaacgca gtggggcgcg 24120

atcaaaacaa cgtcggcccc aaggtttacc caataatact gcgtcttggt tcaccgctct 24180

cactcaacat ggcaaggaag accttaaatt ccctcgagga caaggcgttc caattaacac 24240

caatagcagt ccagatgacc aaattggcta ctaccgaaga gctaccagac gaattcgtgg 24300

tggtgacggt aaaatgaaag atctcagtcc aagatggtat ttctactacc taggaactgg 24360

gccagaagct ggacttccct atggtgctaa caaagacggc atcatatggg ttgcaactga 24420

gggagccttg aatacaccaa aagatcacat tggcacccgc aatcctgcta acaatgctgc 24480

aatcgtgcta caacttcctc aaggaacaac attgccaaaa ggcttctacg cagaagggag 24540

cagaggcggc agtcaagcct cttctcgttc ctcatcacgt agtcgcaaca gttcaagaaa 24600

ttcaactcca ggcagcagta ggggaacttc tcctgctaga atggctggca atggcggtga 24660

tgctgctctt gctttgctgc tgcttgacag attgaaccag cttgagagca aaatgtctgg 24720

taaaggccaa caacaacaag gccaaactgt cactaagaaa tctgctgctg aggcttctaa 24780

gaagcctcgg caaaaacgta ctgccactaa agcatacaat gtaacacaag ctttcggcag 24840

acgtggtcca gaacaaaccc aaggaaattt tggggaccag gaactaatca gacaaggaac 24900

tgattacaaa cattggccgc aaattgcaca atttgccccc agcgcttcag cgttcttcgg 24960

aatgtcgcgc attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc 25020

catcaaattg gatgacaaag atccaaattt caaagatcaa gtcattttgc tgaataagca 25080

tattgacgca tacaaaacat tcccaccaac agagcctaaa aaggacaaaa agaagaaggc 25140

tgatgaaact caagccttac cgcagagaca gaagaaacag caaactgtga ctcttcttcc 25200

tgctgcagat ttggatgatt tctccaaaca attgcaacaa tccatgagca gtgctgactc 25260

aactcaggcc taaactcatg cagaccacac aaggcagatg ggctatataa acgttttcgc 25320

ttttccgttt acgatatata gtctactctt gtgcagaatg aattctcgta actacatagc 25380

acaagtagat gtagttaact ttaatctcac atagcaatct ttaatcagtg tgtaacatta 25440

gggaggactt gaaagagcca ccacattttc accgaggcca cgcggagtac gatcgagtgt 25500

acagtgaaca atgctaggga gagctgccta tatggaagag ccctaatgtg taaaattaat 25560

tttagtagtg ctatccccat gtgattttaa tagcttctta ggagaatgac aaaaaaaaaa 25620

aaaaaaaaaa aaaaaaaaag gcgcgccggg tcggcatggc atctccacct cctcgcggtc 25680

cgacctgggc atccgaagga ggacgcacgt ccactcggat ggctaaggga gctgtgcctt 25740

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 25800

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 25860

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 25920

atagcaggca tgctggggat gcggtgggct ctatggaagc ttgagtattc tatagtgtca 25980

cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 26040

cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 26100

agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 26160

gtcgtgccag ctgcattaat gaatcggcca acgcgaaccc cttgcggccg cccgggccgt 26220

cgaccaattc tcatgtttga cagcttatca tcgaatttct gccattcatc cgcttattat 26280

cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataactgcct taaaaaaatt 26340

acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat 26400

ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc 26460

cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca 26520

cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct 26580

caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat 26640

atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt 26700

cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac 26760

cgtctttcat tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa 26820

taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat 26880

ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt 26940

ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt 27000

tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta 27060

tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa 27120

agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt 27180

gatcttccgt cacaggtatt tattcgcgat aagctcatgg agcggcgtaa ccgtcgcaca 27240

ggaaggacag agaaagcgcg gatctgggaa gtgacggaca gaacggtcag gacctggatt 27300

ggggaggcgg ttgccgccgc tgctgctgac ggtgtgacgt tctctgttcc ggtcacacca 27360

catacgttcc gccattccta tgcgatgcac atgctgtatg ccggtatacc gctgaaagtt 27420

ctgcaaagcc tgatgggaca taagtccatc agttcaacgg aagtctacac gaaggttttt 27480

gcgctggatg tggctgcccg gcaccgggtg cagtttgcga tgccggagtc tgatgcggtt 27540

gcgatgctga aacaattatc ctgagaataa atgccttggc ctttatatgg aaatgtggaa 27600

ctgagtggat atgctgtttt tgtctgttaa acagagaagc tggctgttat ccactgagaa 27660

gcgaacgaaa cagtcgggaa aatctcccat tatcgtagag atccgcatta ttaatctcag 27720

gagcctgtgt agcgtttata ggaagtagtg ttctgtcatg atgcctgcaa gcggtaacga 27780

aaacgatttg aatatgcctt caggaacaat agaaatcttc gtgcggtgtt acgttgaagt 27840

ggagcggatt atgtcagcaa tggacagaac aacctaatga acacagaacc atgatgtggt 27900

ctgtcctttt acagccagta gtgctcgccg cagtcgagcg acagggcgaa gccctcgagt 27960

gagcgaggaa gcaccaggga acagcactta tatattctgc ttacacacga tgcctgaaaa 28020

aacttccctt ggggttatcc acttatccac ggggatattt ttataattat tttttttata 28080

gtttttagat cttctttttt agagcgcctt gtaggccttt atccatgctg gttctagaga 28140

aggtgttgtg acaaattgcc ctttcagtgt gacaaatcac cctcaaatga cagtcctgtc 28200

tgtgacaaat tgcccttaac cctgtgacaa attgccctca gaagaagctg ttttttcaca 28260

aagttatccc tgcttattga ctctttttta tttagtgtga caatctaaaa acttgtcaca 28320

cttcacatgg atctgtcatg gcggaaacag cggttatcaa tcacaagaaa cgtaaaaata 28380

gcccgcgaat cgtccagtca aacgacctca ctgaggcggc atatagtctc tcccgggatc 28440

aaaaacgtat gctgtatctg ttcgttgacc agatcagaaa atctgatggc accctacagg 28500

aacatgacgg tatctgcgag atccatgttg ctaaatatgc tgaaatattc ggattgacct 28560

ctgcggaagc cagtaaggat atacggcagg cattgaagag tttcgcgggg aaggaagtgg 28620

ttttttatcg ccctgaagag gatgccggcg atgaaaaagg ctatgaatct tttccttggt 28680

ttatcaaacg tgcgcacagt ccatccagag ggctttacag tgtacatatc aacccatatc 28740

tcattccctt ctttatcggg ttacagaacc ggtttacgca gtttcggctt agtgaaacaa 28800

aagaaatcac caatccgtat gccatgcgtt tatacgaatc cctgtgtcag tatcgtaagc 28860

cggatggctc aggcatcgtc tctctgaaaa tcgactggat catagagcgt taccagctgc 28920

ctcaaagtta ccagcgtatg cctgacttcc gccgccgctt cctgcaggtc tgtgttaatg 28980

agatcaacag cagaactcca atgcgcctct catacattga gaaaaagaaa ggccgccaga 29040

cgactcatat cgtattttcc ttccgcgata tcacttccat gacgacagga tagtctgagg 29100

gttatctgtc acagatttga gggtggttcg tcacatttgt tctgacctac tgagggtaat 29160

ttgtcacagt tttgctgttt ccttcagcct gcatggattt tctcatactt tttgaactgt 29220

aatttttaag gaagccaaat ttgagggcag tttgtcacag ttgatttcct tctctttccc 29280

ttcgtcatgt gacctgatat cgggggttag ttcgtcatca ttgatgaggg ttgattatca 29340

cagtttatta ctctgaattg gctatccgcg tgtgtacctc tacctggagt ttttcccacg 29400

gtggatattt cttcttgcgc tgagcgtaag agctatctga cagaacagtt cttctttgct 29460

tcctcgccag ttcgctcgct atgctcggtt acacggctgc ggcgagcgct agtgataata 29520

agtgactgag gtatgtgctc ttcttatctc cttttgtagt gttgctctta ttttaaacaa 29580

ctttgcggtt ttttgatgac tttgcgattt tgttgttgct ttgcagtaaa ttgcaagatt 29640

taataaaaaa acgcaaagca atgattaaag gatgttcaga atgaaactca tggaaacact 29700

taaccagtgc ataaacgctg gtcatgaaat gacgaaggct atcgccattg cacagtttaa 29760

tgatgacagc ccggaagcga ggaaaataac ccggcgctgg agaataggtg aagcagcgga 29820

tttagttggg gtttcttctc aggctatcag agatgccgag aaagcagggc gactaccgca 29880

cccggatatg gaaattcgag gacgggttga gcaacgtgtt ggttatacaa ttgaacaaat 29940

taatcatatg cgtgatgtgt ttggtacgcg attgcgacgt gctgaagacg tatttccacc 30000

ggtgatcggg gttgctgccc ataaaggtgg cgtttacaaa acctcagttt ctgttcatct 30060

tgctcaggat ctggctctga aggggctacg tgttttgctc gtggaaggta acgaccccca 30120

gggaacagcc tcaatgtatc acggatgggt accagatctt catattcatg cagaagacac 30180

tctcctgcct ttctatcttg gggaaaagga cgatgtcact tatgcaataa agcccacttg 30240

ctggccgggg cttgacatta ttccttcctg tctggctctg caccgtattg aaactgagtt 30300

aatgggcaaa tttgatgaag gtaaactgcc caccgatcca cacctgatgc tccgactggc 30360

cattgaaact gttgctcatg actatgatgt catagttatt gacagcgcgc ctaacctggg 30420

tatcggcacg attaatgtcg tatgtgctgc tgatgtgctg attgttccca cgcctgctga 30480

gttgtttgac tacacctccg cactgcagtt tttcgatatg cttcgtgatc tgctcaagaa 30540

cgttgatctt aaagggttcg agcctgatgt acgtattttg cttaccaaat acagcaatag 30600

taatggctct cagtccccgt ggatggagga gcaaattcgg gatgcctggg gaagcatggt 30660

tctaaaaaat gttgtacgtg aaacggatga agttggtaaa ggtcagatcc ggatgagaac 30720

tgtttttgaa caggccattg atcaacgctc ttcaactggt gcctggagaa atgctctttc 30780

tatttgggaa cctgtctgca atgaaatttt cgatcgtctg attaaaccac gctgggagat 30840

tagataatga agcgtgcgcc tgttattcca aaacatacgc tcaatactca accggttgaa 30900

gatacttcgt tatcgacacc agctgccccg atggtggatt cgttaattgc gcgcgtagga 30960

gtaatggctc gcggtaatgc cattactttg cctgtatgtg gtcgggatgt gaagtttact 31020

cttgaagtgc tccggggtga tagtgttgag aagacctctc gggtatggtc aggtaatgaa 31080

cgtgaccagg agctgcttac tgaggacgca ctggatgatc tcatcccttc ttttctactg 31140

actggtcaac agacaccggc gttcggtcga agagtatctg gtgtcataga aattgccgat 31200

gggagtcgcc gtcgtaaagc tgctgcactt accgaaagtg attatcgtgt tctggttggc 31260

gagctggatg atgagcagat ggctgcatta tccagattgg gtaacgatta tcgcccaaca 31320

agtgcttatg aacgtggtca gcgttatgca agccgattgc agaatgaatt tgctggaaat 31380

atttctgcgc tggctgatgc ggaaaatatt tcacgtaaga ttattacccg ctgtatcaac 31440

accgccaaat tgcctaaatc agttgttgct cttttttctc accccggtga actatctgcc 31500

cggtcaggtg atgcacttca aaaagccttt acagataaag aggaattact taagcagcag 31560

gcatctaacc ttcatgagca gaaaaaagct ggggtgatat ttgaagctga agaagttatc 31620

actcttttaa cttctgtgct taaaacgtca tctgcatcaa gaactagttt aagctcacga 31680

catcagtttg ctcctggagc gacagtattg tataagggcg ataaaatggt gcttaacctg 31740

gacaggtctc gtgttccaac tgagtgtata gagaaaattg aggccattct taaggaactt 31800

gaaaagccag caccctgatg cgaccacgtt ttagtctacg tttatctgtc tttacttaat 31860

gtcctttgtt acaggccaga aagcataact ggcctgaata ttctctctgg gcccactgtt 31920

ccacttgtat cgtcggtctg ataatcagac tgggaccacg gtcccactcg tatcgtcggt 31980

ctgattatta gtctgggacc acggtcccac tcgtatcgtc ggtctgatta ttagtctggg 32040

accacggtcc cactcgtatc gtcggtctga taatcagact gggaccacgg tcccactcgt 32100

atcgtcggtc tgattattag tctgggacca tggtcccact cgtatcgtcg gtctgattat 32160

tagtctggga ccacggtccc actcgtatcg tcggtctgat tattagtctg gaaccacggt 32220

cccactcgta tcgtcggtct gattattagt ctgggaccac ggtcccactc gtatcgtcgg 32280

tctgattatt agtctgggac cacgatccca ctcgtgttgt cggtctgatt atcggtctgg 32340

gaccacggtc ccacttgtat tgtcgatcag actatcagcg tgagactacg attccatcaa 32400

tgcctgtcaa gggcaagtat tgacatgtcg tcgtaacctg tagaacggag taacctcggt 32460

gtgcggttgt atgcctgctg tggattgctg ctgtgtcctg cttatccaca acattttgcg 32520

cacggttatg tggacaaaat acctggttac ccaggccgtg ccggcacgtt aaccgggctg 32580

catccgatgc aagtgtgtcg ctgtcgacga gctcgcgagc tcggacatga ggttgccccg 32640

tattcagtgt cgctgatttg tattgtctga agttgttttt acgttaagtt gatgcagatc 32700

aattaatacg atacctgcgt cataattgat tatttgacgt ggtttgatgg cctccacgca 32760

cgttgtgata tgtagatgat aatcattatc actttacggg tcctttccgg tgatccgaca 32820

ggttacgggg cggcgacctc gcgggttttc gctatttatg aaaattttcc ggtttaaggc 32880

gtttccgttc ttcttcgtca taacttaatg tttttattta aaataccctc tgaaaagaaa 32940

ggaaacgaca ggtgctgaaa gcgagctttt tggcctctgt cgtttccttt ctctgttttt 33000

gtccgtggaa tgaacaatgg aagtccgagc tcatcgctaa taacttcgta tagcatacat 33060

tatacgaagt tatattcgat 33080

Claims (4)

1. A novel coronavirus replicon system, comprising:

the new coronavirus replicon plasmid cDNA clone comprises genome 5 'and 3' non-coding sequences, replicase genes (ORF 1a and ORF1 b) and nucleocapsid protein (N), and neomycin phosphotransferase gene (Neo) and luciferase reporter gene (Nanoluc) reporter genes inserted into pBeloBAC11 vector of CMV eukaryotic promoter, HDV ribozyme and BGH termination signal, and plasmid sequence as SEQ ID No.1.

2. A method for constructing a novel coronavirus replicon plasmid, comprising:

dividing the 5' non-coding region of the genome of the novel coronavirus and the sequence of ORF1a/ORF1b gene into 6 consecutive fragments (A-F); a synthetic G fragment comprising: transcription-regulating sequence of E gene, neomycin phosphotransferase gene (Neo), TRS of M gene and Nanoluc luciferase (nLuc) reporter gene; an amplified H fragment comprising: TRS of N Gene-non-coding sequence of 3' end of genome-poly (A) sequence, and each fragment of A-H was inserted into pBeloBAC11 vector by in vitro ligation.

3. Use of a plasmid containing the neocoronavirus replicon of claim 1 for in vitro screening or monitoring of prophylactic or therapeutic drugs against neocoronavirus.

4. The method of using the neocoronavirus replicon plasmid of claim 3 for in vitro screening or monitoring of prophylactic or therapeutic drugs against neocoronavirus, comprising: transfecting the new coronavirus replicon to cells, adding a drug treatment cell to be screened or monitored, and monitoring chemiluminescence intensity after treatment.

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