Plasmid elimination module based on inducible promoter

Abstract

The invention relates to the field of gene engineering, in particular to a plasmid elimination module based on an inducible promoter, which comprises two types of mutually independent expression regions, one type of region is an inducible factor transcription region, and the inducible factor transcription region is composed of a constitutive promoter carried by an inducible transcription factor fragment, and the other type of region is an inducible transcription factor fragment. The kit comprises an RNA polymerase binding site, a transcription start site, a ribosome binding site, an induced transcription factor translation region and a terminator, the other region is an antisense RNA expression region and comprises an inducible factor promoter corresponding to an inducible transcription factor, an antisense RNA and a terminator; the antisense RNA is the antisense RNA of the metabolic key gene mRNA, and can be combined with the metabolic key gene mRNA to hinder the translation of the metabolic key gene mRNA. The method is high in elimination efficiency, elimination objects can be freely set according to needs, and selective or synchronous elimination of various plasmids can be realized.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to a plasmid elimination module based on an inducible promoter. Background Art

[0002] Episomal plasmids are a common platform for genetic manipulation of animal, plant, and microbial cells, and can be used for purposes such as genome editing, heterologous protein expression, and gene circuit construction. However, after using genome editing or gene circuit plasmids to achieve genome editing or gene circuit design purposes, the genome editing tools or gene circuits carried on the plasmids become unstable factors in the cell genome. At this time, it is necessary to eliminate the plasmids to obtain plasmid-free cells. The current plasmid elimination techniques are mainly divided into three types, namely temperature-sensitive replicon plasmid elimination, sacB plasmid elimination, and CRISPR protein-related plasmid elimination techniques.

[0003] The elimination efficiency of temperature-sensitive replicon plasmids is relatively low and is commonly used for low-copy plasmid elimination, but it has poor effects on medium- and high-copy plasmid elimination. Moreover, generally, the same replicon in the same cell can only serve one plasmid at the same time. If a temperature-sensitive replicon is used to serve multiple plasmids, it may exacerbate the spontaneous loss of plasmids, which is not conducive to the development of related experiments or production work.

[0004] Plasmid elimination based on the sacB gene can serve multiple plasmids simultaneously. However, when multiple plasmids are present in the cell, the plasmid elimination method based on the sacB gene cannot accurately eliminate specific types of plasmids, and its tolerance to mutations is weak, and it is prone to failure.

[0005] The plasmid elimination technique based on CRISPR proteins can eliminate multiple plasmids simultaneously and freely select the types of plasmids to be eliminated. However, there is a risk of CRISPR protein gene mutation failure or CRISPR protein cleavage off-target. Moreover, the gene sequence of CRISPR-related protein elements is relatively long, which is not conducive to use in chassis microorganisms that are difficult to transfer large plasmids. When there is no natural CRISPR protein available in the chassis cells, it is necessary to additionally transfer a plasmid carrying the CRISPR protein, and the self-targeting elimination failure rate of the plasmid carrying the CRISPR protein is relatively high. Summary of the Invention

[0006] The present invention proposes a plasmid elimination technique based on antisense RNA, and designs a plasmid elimination module. This module can promote the loss of episomal plasmids in cells, can efficiently achieve the selective elimination or synchronous elimination of various episomal plasmids, can be applied to the selective elimination or synchronous elimination of different types (copy numbers) of episomal plasmids in eukaryotic and prokaryotic cells, and has better tolerance to sequence mutations than the plasmid elimination technique based on CRISPR proteins. The gene sequence length of the related protein elements is less than that of CRISPR, and it is still applicable to chassis cells that are difficult to transfer large plasmids.

[0007] Antisense RNAs designed for key metabolic genes can reduce the expression of these key metabolic genes, thereby affecting the cell growth rate. During cell amplification and subculture, plasmids are randomly distributed to progeny cells. The number of progeny cells containing plasmids with this elimination module will become fewer and fewer, while the number of progeny cells without this elimination module will become more and more due to rapid growth, thus achieving the purpose of enriching plasmid-free cells and eliminating plasmids.

[0008] For the plasmid elimination module designed in the present invention, except that the antisense RNA expression region must be deployed on the plasmid to be eliminated, the remaining regions can be deployed within the cells where the plasmid to be eliminated is located. After completion of the deployment, the plasmid elimination module designed in the present invention can efficiently achieve plasmid elimination within the deployment range.

[0009] The significance of the present invention lies in proposing a general idea for constructing a plasmid elimination module that is convenient to use, programmable, widely adaptable, highly stable, and requires a relatively short sequence. Under the premise of meeting the preconditions, this plasmid elimination module can be used for plasmid elimination after the construction of genetically engineered bacteria and for the elimination of artificially constructed plasmids.

[0010] To achieve the above objectives, the present invention adopts the following technical solutions:

[0011] According to an embodiment of the present invention, a plasmid elimination module based on an inducible promoter includes two independent expression regions. One type of region is the inducer transcription region, which consists of a constitutive promoter (including an RNA polymerase binding site and a transcription start site), a ribosome binding site (RBS), an inducer transcription factor translation region, and a terminator carried by the inducer transcription factor fragment; the other type of region is the antisense RNA expression region, which includes an inducer promoter corresponding to the inducer transcription factor, an antisense RNA, and a terminator; the antisense RNA is the antisense RNA of the key metabolic gene mRNA and can bind to the key metabolic gene mRNA to hinder the translation of the key metabolic gene mRNA.

[0012] According to an embodiment of the present invention, the inducer transcription factor is the arabinose transcription factor.

[0013] According to an embodiment of the present invention, the inducer promoter is the arabinose promoter.

[0014] According to an embodiment of the present invention, the antisense RNA expression region is carried on the plasmid to be eliminated.

[0015] According to an embodiment of the present invention, the plasmid to be eliminated is a circular or linear DNA that can replicate independently outside the genome and be stably free in the cell, or replicate with the genome and be passed on to progeny cells.

[0016] According to an embodiment of the present invention, the plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and yeast 2μ plasmid.

[0017] According to an embodiment of the present invention, the gene for antisense RNA interference is a key metabolic gene.

[0018] According to an embodiment of the present invention, the key metabolic genes include genes related to the TCA cycle, protein synthesis, aerobic respiration, anaerobic respiration, lipid synthesis, cell wall synthesis, sugar transport pathway, amino acid transport pathway, ATP synthesis, cytoskeleton synthesis, NADPH / NADP + synthesis-related genes, NADH / NAD + synthesis-related genes, vitamin synthesis-related genes, one-carbon transport system-related genes, carbon dioxide fixation-related genes, DNA / RNA synthesis-related genes, amino acid synthesis-related genes, ribose synthesis-related genes, ribosome synthesis-related genes, ribosome assembly-related genes, aminoacyl-tRNA synthetase-related genes, and isozyme genes of the above genes.

[0019] According to an embodiment of the present invention, the elimination target of the plasmid elimination module is all microorganisms, animals, or plant cells that have stable free plasmids, where the free plasmids have a certain probability of not being distributed to daughter cells, and the elimination of free plasmids does not cause cell death or inability to proliferate.

[0020] To facilitate the understanding of the present invention by those skilled in the art, the main vocabulary explanations are as follows:

[0021] Gene circuit: A combination of DNA sequences that arranges specific gene elements such as promoters, terminators, protein translation regions, and specific protein binding regions in a unit point or multiple points according to certain rules to achieve specific functions.

[0022] Conveniently available: This module can be stored in the form of PCR products / plasmids, etc., and can be inserted into plasmids outside or inside cells through forms such as Gibson assembly, Golden Gate cloning, restriction enzyme digestion and ligation, and genome editing.

[0023] Programmable: The use of this module is not restricted by the plasmid copy number, and selective elimination can be achieved in cells containing multiple types of plasmids by combining different regulatory proteins.

[0024] Wide adaptability: This module can be used across species by optimizing / replacing relevant control elements with codons.

[0025] Strong stability: Even if there is a certain sequence deviation between the antisense RNA and the target mRNA, it does not affect the function of the module proposed in the present invention.

[0026] The required sequence is short: When using the native regulatory factors of cells, the total length of the plasmid elimination module sequence proposed by the present invention is usually less than 1 kbp, and even less than 100 bp. When using self - contained regulatory factors, the total length of the plasmid elimination module sequence proposed by the present invention usually does not exceed 3 kbp.

[0027] Constructing plasmids: mainly refers to the tool plasmids that carry the essential functional proteins for genome editing to assist in achieving the purpose of genome editing of genetic engineering cells. Such plasmids usually need to be eliminated after completing the genome editing of engineering cells to avoid introducing or causing additional mutations in the application of engineering cells.

[0028] Artificially constructed plasmids: mainly refer to plasmids that do not involve genome editing and are artificially constructed to express homologous / heterologous proteins or some functional gene circuits.

[0029] The backbone of the plasmid: the DNA sequence carrying the plasmid replicon and plasmid maintenance genes.

[0030] Free plasmids: linear or circular double - stranded DNA that can autonomously replicate in cells either independently or together with the genome, can be distributed to daughter cells along with cell proliferation, and can stably exist outside the genome.

[0031] Constitutive promoter: a specific DNA sequence that can be recognized by the E. coli σ factor and recruit RNA polymerase subunits for transcription downstream.

[0032] Illustrations are as follows:

[0033] E. coli - related sequences:

[0034] Full sequence of the interference region of the E. coli acnB gene:

[0035] ACTATGACAATGAGAGCGAGGAGAACCGTCGTGCTAGAAGAATACCGTAAGCACGT

[0036] AGCTGAGCGTGCCGCTGAGGGGATTGCGCCCAAACCCCTGGATGCAAACCAAATGGCC

[0037] GCACTTGTAGAGCTGCTGAAAAACCCGCCCGCGGGCGAAGAAGAATTCCTGTTAGATCT

[0038] GTTAACCAACCGTGTTCCCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCC

[0039] TGGCTGCTATCGCTAAAGGCGAAGCCAAATCCCCTCTGCTGACTCCGGAAAAAGCCATC

[0040] GAACTGCTGGGCACCATGCAGGGTGGTTACAACATTCATCCGCTGATCGACGCGCTGGA

[0041] TGATGCCAAACTGGCACCGATCGCTGCCAAAGCACTTTCTCACACACTGCTGATGTTCG

[0042] ATAACTTCTATGACGTAGAAGAGAAAGCGAAAGCAGGCAACGAATATGCGAAGCAGGT

[0043] AATGCAGTCCTGGGCGGATGCCGAATGGTTCCTGAATCGCCCGGCGCTGGCTGAAAAAC

[0044] TGACCGTTACCGTCTTCAAAGTCACTGGCGAAACCAACACCGATGACCTCTCTCCGGCA

[0045] CCGGATGCGTGGTCACGCCCGGATATCCCACTGCACGCGCTGGCGATGCTGAAAAACGC

[0046] CCGTGAAGGCATCGAGCCAGACCAGCCAGGTGTTGTTGGTCCGATCAAGCAAATCGAA

[0047] GCTCTGCAACAGAAAGGTTTCCCGCTGGCGTACGTCGGTGACGTTGTGGGTACGGGTTC

[0048] ATCGCGTAAATCCGCCACGAACTCCGTACTGTGGTTTATGGGTGATGATATTCCACATGT

[0049] GCCGAACAAACGCGGCGGTGGTTTGTGCCTCGGCGGTAAAATTGCACCAATCTTCTTTA

[0050] ACACAATGGAAGACGCAGGTGCACTGCCAATCGAAGTCGACGTCTCTAACCTGAACAT

[0051] GGGCGACGTAATTGACGTTTACCCGTACAAAGGTGAAGTGCGTAACCACGAAACCGGC

[0052] GAACTGCTGGCGACCTTCGAACTGAAAACCGACGTGCTGATTGATGAAGTGCGTGCTG

[0053] GCGGCCGTATTCCGCTGATTATTGGGCGTGGCCTGACCACCAAAGCGCGTGAAGCACTT

[0054] GGTCTGCCGCACAGTGATGTGTTCCGTCAGGCGAAAGATGTCGCTGAGAGCGATCGCG

[0055] GCTTCTCGCTGGCGCAAAAAATGGTAGGCCGTGCCTGTGGCGTGAAAGGCATTCGTCCG

[0056] GGCGCGTACTGCGAACCGAAAATGACTTCTGTAGGCTCTCAGGACACCACCGGCCCGAT

[0057] GACCCGTGATGAACTGAAAGACCTGGCGTGCCTGGGCTTCTCGGCTGACCTGGTGATGC

[0058] AGTCTTTCTGCCACACCGCGGCGTATCCGAAGCCAGTTGACGTGAACACGCACCACACG

[0059] CTGCCGGACTTCATTATGAACCGTGGCGGTGTGTCGCTGCGTCCGGGTGACGGCGTCAT

[0060] TCACTCCTGGCTGAACCGTATGCTGCTGCCGGATACCGTCGGTACCGGTGGTGACTCCCA

[0061] TACCCGTTTCCCGATCGGTATCTCTTTCCCGGCGGGTTCTGGTCTGGTGGCGTTTGCTGC

[0062] CGCAACTGGCGTAATGCCGCTGGATATGCCGGAATCCGTTCTGGTGCGCTTCAAAGGCA

[0063] AAATGCAGCCGGGCATCACCCTGCGCGATCTGGTACACGCGATCCCGCTGTATGCGATC

[0064] AAACAAGGTCTGCTGACCGTTGAGAAGAAAGGCAAGAAAAACATCTTCTCTGGCCGCA

[0065] TCCTGGAAATTGAAGGTCTGCCGGATCTGAAAGTTGAGCAGGCCTTTGAGCTAACCGAT

[0066] GCGTCCGCCGAGCGTTCTGCCGCTGGTTGTACCATCAAGCTGAACAAAGAACCGATCAT

[0067] CGAATACCTGAACTCTAACATCGTCCTGCTGAAGTGGATGATCGCGGAAGGTTACGGCG

[0068] ATCGTCGTACCCTGGAACGTCGTATTCAGGGCATGGAAAAATGGCTGGCGAATCCTGAG

[0069] CTGCTGGAAGCCGATGCAGATGCGGAATACGCGGCAGTGATCGACATCGATCTGGCGGA

[0070] TATTAAAGAGCCAATCCTCTGTGCACCGAACGACCCGGACGACGCACGTCCGCTGTCTG

[0071] CGGTACAGGGCGAGAAGATCGACGAAGTGTTTATCGGTTCCTGCATGACCAACATCGGT

[0072] CACTTCCGTGCTGCGGGTAAACTGCTGGATGCGCACAAAGGCCAGTTGCCGACGCGCCT

[0073] GTGGGTGGCACCGCCAACCCGTATGGATGCCGCGCAGTTGACCGAAGAAGGCTACTATA

[0074] GCGTCTTCGGTAAGAGCGGTGCGCGTATCGAGATCCCTGGCTGTTCCCTGTGTATGGGTA

[0075] ACCAGGCGCGTGTAGCAGACGGTGCGACGGTGGTTTCCACCTCTACCCGTAACTTCCCG

[0076] AACCGTCTGGGGACTGGCGCGAATGTCTTCCTGGCTTCTGCGGAACTGGCGGCTGTTGC

[0077] GGCGCTGATTGGCAAACTGCCGACGCCGGAAGAGTACCAGACCTACGTGGCGCAAGTA

[0078] GATAAAACTGCCGTTGATACTTATCGTTATCTGAACTTCAACCAGCTTTCTCAGTACACCGAAAAAGCCGATGGGGTGATTTTCCAGACTGCGGTTTAA(SEQ ID NO:1)

[0079] The complete sequence of pET28a plasmid:

[0080] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

[0081] CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAA

[0082] TATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGC

[0083] AGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG

[0084] GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT

[0085] ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGAT

[0086] TGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC

[0087] ATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA

[0088] CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAA

[0089] CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0090] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

[0091] CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC

[0092] AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

[0093] AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

[0094] GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

[0095] GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

[0096] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

[0097] AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0098] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

[0099] CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA

[0100] CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

[0101] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

[0102] ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0103] GAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATG

[0104] GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTAT

[0105] CGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC

[0106] TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG

[0107] CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA

[0108] GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCT

[0109] CGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGG

[0110] CGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG

[0111] TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT

[0112] GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0113] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCA

[0114] CAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA

[0115] CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTC

[0116] AGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCAT

[0117] TCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCAC

[0118] GATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAAC

[0119] GTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATAC

[0120] CGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATG

[0121] ACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAG

[0122] TGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT

[0123] CTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCG

[0124] TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0125] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCA

[0126] CCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG

[0127] CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACG

[0128] GCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACC

[0129] AACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG

[0130] CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0131] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTG

[0132] AGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC

[0133] TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTAC

[0134] CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAAT

[0135] AACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0136] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTAC

[0137] AGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0138] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGG

[0139] TGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA

[0140] ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG

[0141] CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGAC

[0142] ATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT

[0143] CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC

[0144] CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG

[0145] CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGG

[0146] GCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGA

[0147] TCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGT

[0148] GATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAAT

[0149] ACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT

[0150] TAACTTTAAGAAGGAGATATACCATGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC

[0151] TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:2)

[0152] Arabinose promoter sequence (arrow indicates the transcription start direction):

[0153] AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ IDNO:3)

[0154] T7 terminator sequence (arrow indicates the transcription termination direction):

[0155] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO:4)

[0156] Arabinose transcription factor expression cassette (including promoter, CDS region, terminator):

[0157]

[0158] Corynebacterium glutamicum - related sequences:

[0159] Full sequence of the CDS region of the acn gene in Corynebacterium glutamicum:

[0160] TTGGAGCTCACTGTGACTGAAAGCAAGAACTCCTTCAATGCTAAGAGCACCCTTGAAGT

[0161] TGGCGACAAGTCCTATGACTACTTCGCCCTCTCTGCAGTGCCTGGCATGGAGAAGCTGC

[0162] CGTACTCCCTCAAGGTTCTCGGAGAGAACCTTCTTCGTACCGAAGACGGCGCAAACATC

[0163] ACCAACGAGCACATTGAGGCTATCGCCAACTGGGATGCATCTTCCGATCCAAGCATCGA

[0164] AATCCAGTTCACCCCAGCCCGTGTTCTCATGCAGGACTTCACCGGTGTCCCTTGTGTAGT

[0165] TGACCTCGCAACCATGCGTGAGGCAGTTGCTGCACTCGGTGGCGACCCTAACGACGTCA

[0166] ACCCACTGAACCCAGCCGAGATGGTCATTGACCACTCCGTCATCGTGGAGGCTTTCGGC

[0167] CGCCCAGATGCACTGGCTAAGAACGTTGAGATCGAGTACGAGCGCAACGAGGAGCGTT

[0168] ACCAGTTCCTGCGTTGGGGTTCCGAGTCCTTCTCCAACTTCCGCGTTGTTCCTCCAGGA

[0169] ACCGGTATCGTCCACCAGGTCAACATTGAGTACTTGGCTCGCGTCGTCTTCGACAACGA

[0170] GGGCCTTGCATACCCAGATACCTGCATCGGTACCGACTCCCACACCACCATGGAAAACG

[0171] GCCTGGGCATCCTGGGCTGGGGCGTTGGTGGCATTGAGGCTGAAGCAGCAATGCTCGG

[0172] CCAGCCAGTGTCCATGCTGATCCCTCGCGTTGTTGGCTTCAAGTTGACCGGCGAGATCC

[0173] CAGTAGGCGTTACCGCAACTGACGTTGTGCTGACCATCACCGAAATGCTGCGCGACCAC

[0174] GGCGTCGTCCAGAAGTTCGTTGAGTTCTACGGCTCCGGTGTTAAGGCTGTTCCACTGGC

[0175] TAACCGTGCAACCATCGGCAACATGTCCCCAGAGTTCGGCTCCACCTGTGCGATGTTCC

[0176] CAATCGACGAGGAGACCACCAAGTACCTGCGCCTCACCGGCCGCCCAGAAGAGCAGGT

[0177] TGCACTGGTCGAGGCTTACGCCAAGGCGCAGGGCATGTGGCTCGACGAGGACACCGTT

[0178] GAAGCTGAGTACTCCGAGTACCTCGAGCTGGACCTGTCCACCGTTGTTCCTTCCATCGC

[0179] TGGCCCTAAGCGCCCACAGGACCGCATCCTTCTCTCCGAGGCAAAGGAGCAGTTCCGTA

[0180] AGGATCTGCCAACCTACACCGACGACGCTGTTTCCGTAGACACCTCCATCCCTGCAACC

[0181] CGCATGGTTAACGAAGGTGGCGGACAGCCTGAAGGCGGCGTCGAAGCTGACAACTACA

[0182] ACGCTTCCTGGGCTGGCTCCGGCGAGTCCTTGGCTACTGGCGCAGAAGGACGTCCTTCC

[0183] AAGCCAGTCACCGTTGCATCCCCACAGGGTGGCGAGTACACCATCGACCACGGCATGGT

[0184] TGCAATTGCATCCATCACCTCTTGCACCAACACCTCTAACCCATCCGTGATGATCGGCGC

[0185] TGGCCTGATCGCACGTAAGGCAGCAGAAAAGGGCCTCAAGTCCAAGCCTTGGGTTAAG

[0186] ACCATCTGTGCACCAGGTTCCCAGGTTGTCGACGGCTACTACCAGCGCGCAGACCTCTG

[0187] GAAGGACCTTGAGGCCATGGGCTTCTACCTCTCCGGCTTCGGCTGCACCACCTGTATTG

[0188] GTAACTCCGGCCCACTGCCAGAGGAAATCTCCGCTGCGATCAACGAGCACGACCTGAC

[0189] CGCAACCGCAGTTTTGTCCGGTAACCGTAACTTCGAGGGACGTATCTCCCCTGACGTTA

[0190] AGATGAACTACCTGGCATCCCCAATCATGGTCATTGCTTACGCAATCGCTGGCACCATGG

[0191] ACTTCGACTTCGAGAACGAAGCTCTTGGACAGGACCAGGACGGCAACGACGTCTTCCT

[0192] GAAGGACATCTGGCCTTCCACCGAGGAAATCGAAGACACCATCCAGCAGGCAATCTCC

[0193] CGTGAGCTTTACGAAGCTGACTACGCAGATGTCTTCAAGGGTGACAAGCAGTGGCAGG

[0194] AACTCGATGTTCCTACCGGTGACACCTTCGAGTGGGACGAGAACTCCACCTACATCCGC

[0195] AAGGCACCTTACTTCGACGGCATGCCTGTCGAGCCAGTGGCAGTCACCGACATCCAGGG

[0196] CGCACGCGTTCTGGCTAAGCTCGGCGACTCTGTCACCACCGACCACATCTCCCCTGCTT

[0197] CCTCCATTAAGCCAGGTACCCCTGCAGCTCAGTACTTGGATGAGCACGGTGTGGAACGC

[0198] CACGACTACAACTCCCTGGGTTCCAGGCGTGGTAACCACGAGGTCATGATGCGCGGCAC

[0199] CTTCGCCAACATCCGCCTCCAGAACCAGCTGGTTGACATCGCAGGTGGCTACACCCGCG

[0200] ACTTCACCCAGGAGGGTGCTCCACAGGCGTTCATCTACGACGCTTCCGTCAACTACAAG

[0201] GCTGCTGGCATTCCGCTGGTCGTCTTGGGCGGCAAGGAGTACGGCACCGGTTCTTCCCG

[0202] TGACTGGGCAGCTAAGGGCACTAACCTGCTCGGAATTCGCGCAGTTATCACCGAGTCCT

[0203] TCGAGCGTATTCACCGCTCCAACCTCATCGGTATGGGCGTTGTCCCACTGCAGTTCCCTG

[0204] CAGGCGAATCCCACGAGTCCCTGGGCCTTGACGGCACCGAGACCTTCGACATCACCGG

[0205] ACTGACCGCACTCAACGAGGGCGAGACTCCTAAGACTGTCAAGGTCACCGCAACCAAG

[0206] GAGAACGGCGACGTCGTCGAGTTCGACGCAGTTGTCCGCATCGACACCCCAGGTGAGG

[0207] CTGACTACTACCGCCACGGCGGCATCCTGCAGTACGTGCTGCGTCAGATGGCTGCTTCTTCTAAGTAA(SEQ ID NO:6)

[0208] Corynebacterium glutamicum pEC-XK99E plasmid sequence:

[0209] GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGG

[0210] CTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAA

[0211] GCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCC

[0212] ATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGC

[0213] GAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGC

[0214] CTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGG

[0215] AGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCA

[0216] TAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTT

[0217] TCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTA

[0218] ATTCCGCTAGATGACGTGCGGCTTCGACCTCCTGGGCGTGGCGCTTGTTGGCGCGCTCG

[0219] CGGCTGGCTGCGGCACGACACGCGTCTGAGCAGTATTTTGCGCGCCGTCCTCGTGGGTC

[0220] AGGCCGGGGTGGGATCAGGCCACCGCAGTAGGCGCAGCTGATGCGATCCTCCACTACTG

[0221] CGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCGGCCAGCTCTTCAAGGTCGGCC

[0222] ACAAGCGTTTCTAGGTCGCTCGCGGCACTTGCCCAGTCGCGTGATGCTGGCGCGTCTGT

[0223] CGTATCGAGGGCGCGGAAAAATCCGATCACCGTTTTTAAATCGACGGCGGCATCGAGTG

[0224] CGTCGGACTCCAGCGCGACATCGGAGAGATCCACCGCTGATGCTTCAGGCCAGTTTTGG

[0225] TACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTTAAAAATTCTAGCCCC

[0226] AATTCTGATAATTTCTTCCGGCACTCCTGCGAAAACCTGCGAGACTTCTTGCCCAGAAA

[0227] AAACGCCAAGCGCAGCGGTTACCGCACTTTTTTTCCAGGTGATTTCACCCTGACCAGCG

[0228] AAGCGGCACTTTAGTGCATGAGGTGTGCCCCTGGTTTCCCCTCTTTGGAGGGTTCAACC

[0229] CAAAAAAGCACACAAGCAAAAATGAAAATCATCATGAGCAAGTTGGTGCGAAGCAGCA

[0230] ACGCGCTAGCTCCAAAAAGGTCTCCAGGATCTCGAGGAGATTTTTGAGGGGGAGGGAG

[0231] TCGAGGAAGAGCCAGAGCAGAAGGCGGGGAACCGTTCTCTGCCGACAGCGTGAGCCC

[0232] CCCTTAAAAATCAGGCCGGGGAGGAACCGGGGAGGGATCAGAGCTAGGAGCGAGACA

[0233] CCCTAAAGGGGGGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGT

[0234] GGATAGCGGAGGGTGAGGGCAAGTGAGAGCCAGAGCAAGGACGGGACCCCTAAAGGG

[0235] GGGAACCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGACGGCCGCG

[0236] TTTAGCTTCCATTCCAAGTGCCTTTCTGACTTGTTGGATGCGCCTTTCACTGACACCTAG

[0237] TTCGCCTGCAAGCTCACGAGTCGAGGGATCAGCAACCGATTGAGAACGGGCATCCAGG

[0238] ATCGCAGTTTTGACGCGAAGTTCGAGCAACTCGCCTGTCATTTCTCGGCGTTTGTTTGCT

[0239] TCCGCTAATCGCTGTCGCGTCTCCTGCGCATACTTACTTTCTGGGTCAGCCCATCTGCGT

[0240] GCATTCGATGTAGCTGCGCCCCGTCGCCCCATCGTCGCTAGAGCTTTCCGCCCTCGGCTG

[0241] CTCTGCGTTTCCACCCGACGAGCAGGGACGACTGGCTGGCCTTTAGCCACGTAGCCGCG

[0242] CACACGACGCGCCATCGTCAGGCGATCACGCATGGCGGGAAGATCCGGCTCCCGGCCG

[0243] TCTGCACCGACCGCCTGGGCAACGTTGTACGCCACTTCATACGCGTCGATGATCTTGGCA

[0244] TCTTTTAGGCGCTCACCAGCAGCTTTGAGCTGGTATCCCACGGTCAACGCGTGGCGAAA

[0245] CGCGGTCTCGTCGCGCGCTCGCTCTGGATTTGTCCAGAGCACTCGCACGCCGTCGATCA

[0246] GGTCGCCGGACGCGTCCAGGGCGCTCGGCAGGCTCGCGTCCAAAATCGCTAGCGCCTT

[0247] GGCTTCTGCGGTGGCGCGTTGTGCCGCTTCAATGCGGGCGCGTCCGCTGGAAAAGTCCT

[0248] GCTCAATGTACTTTTTCGGCTTCTGTGATCCGGTCATCGTTCGAGCAATCTCCATTAGGTC

[0249] GGCCAGCCGATCCACACGATCATGCTGGCAGTGCCATTTATAGGCTGTCGGATCGTCTGA

[0250] GACGTGCAGCGGCCACCGGCTCAGCCTATGCGAAAAAGCCTGGTCAGCGCCGAAAACA

[0251] CGAGTCATTTCTTCCGTCGTTGCAGCCAGCAGGCGCATATTTGGGCTGGTTTTACCTGCT

[0252] GCGGCATACACCGGGTCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAGCGGATTCAC

[0253] GCCGATCCAAGCCGGCGCTTTTTCTAGGCGTGCCCATTTCTCTAAAATCGCGTAGACCTG

[0254] CGGGTTTACGTGCTCAATCTTCCCGCCGGCCTGGTGGCTGGGCACATCGATGTCAAGCA

[0255] CGATCACCGCGGCATGTTGCGCGTGCGTCAGCGCAACGTACTGGCACCGCGTCAGCGCT

[0256] TTTGAGCCAGCCCGGTAGAGCTTTGGTTGGGTTTCGCCGGTATCCGGGTTTTTAATCCAG

[0257] GCGCTCGCGAAATCTCTTGTCTTGCTGCCCTGGAAGCTTTCGCGTCCCAGGTGAGCGAG

[0258] CAGTTCGCGGCGATCTTCTGCCGTCCAGCCGCGTGAGCCGCAGCGCATAGCTTCGGGGT

[0259] GGGTGTCGAACAGATCGGCGGACAATTTCCACGCGCTAGCTGTGACTGTGTCCTGCGGA

[0260] TCGGCTAGAGTCATGTCTTGAGTGCTTTCTCCCAGCTGATGACTGGGGGTTAGCCGACG

[0261] CCCTGTGAGTTCCCGCTCACGGGGCGTTCAACTTTTTCAGGTATTTGTGCAGCTTATCGT

[0262] GTTTTCTTCGTAAATGAACGCTTAACTACCTTGTTAAACGTGGCAAATAGGCAGGATTGA

[0263] TGGGGATCTAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAG

[0264] CGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCT

[0265] ACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCA

[0266] GTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAA

[0267] TTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGG

[0268] CTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGA

[0269] TGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTG

[0270] GGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCG

[0271] CCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC

[0272] GGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG

[0273] GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTA

[0274] TTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGT

[0275] ATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATT

[0276] CGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTT

[0277] GTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCG

[0278] CCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGC

[0279] CTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG

[0280] GCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAG

[0281] AGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT

[0282] CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTT

[0283] CGCGGAATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC

[0284] CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTT

[0285] GCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA

[0286] ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA

[0287] GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

[0288] CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG

[0289] GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT

[0290] GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

[0291] GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC

[0292] GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT

[0293] CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGT

[0294] CAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGC

[0295] CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC

[0296] CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG

[0297] CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATC

[0298] TGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA

[0299] GTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACAC

[0300] CCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAG

[0301] ACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA

[0302] AACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTACGTT

[0303] GACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAG

[0304] TCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCG

[0305] GTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAA

[0306] ACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGG

[0307] CACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCC

[0308] CTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGC

[0309] CAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTG

[0310] CACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAG

[0311] GATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTG

[0312] ACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTG

[0313] GAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTC

[0314] TGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCA

[0315] GCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATG

[0316] CAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGC

[0317] GCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGG

[0318] TAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCACCATCA

[0319] AACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG

[0320] GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCA

[0321] CCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGC

[0322] TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA

[0323] GTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTT

[0324] CTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCA

[0325] TAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATA

[0326] ACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATG (SEQ ID NO: 7) Arabinose promoter sequence (arrow indicates the transcription start direction):

[0327] AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT

[0328] TCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACC

[0329] AAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACA

[0330] TTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT→(SEQ ID NO: 8) T7 terminator sequence (the arrow indicates the transcription termination direction):

[0331] →CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG(SEQ ID NO: 9) Arabinose transcription factor expression cassette (including promoter, CDS region, terminator):

[0332] GGATTTCTGTTCATGGGGGTAATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACC

[0333] TGCATCGATTTATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCA

[0334] CGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTG

[0335] ATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAA

[0336] GCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACT

[0337] GCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCT

[0338] GGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTAC

[0339] CCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAA

[0340] TTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTT

[0341] AATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGA

[0342] ACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGA

[0343] CGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATG

[0344] AATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCT

[0345] GATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCA

[0346] GCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCC

[0347] ACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATA

[0348] CTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGC

[0349] CGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAA

[0350] AGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCT(SEQ ID NO:10)

[0351] Saccharomyces cerevisiae related sequence:

[0352] Full sequence of the CDS region of the Saccharomyces cerevisiae ACO1 / GLU1 gene:

[0353] ATGCTGTCTGCACGTTCTGCCATCAAGAGACCCATTGTTCGTGGTCTTGCGACAGTCTCC

[0354] AACTTGACTAGAGATTCAAAAGTCAACCAAAACTTATTAGAAGATCATTCTTTTATTAAC

[0355] TACAAGCAGAATGTGGAAACGCTGGATATCGTAAGAAAAAGATTAAACAGGCCATTTAC

[0356] CTACGCGGAAAAGATTTTGTACGGTCACTTGGATGACCCTCATGGTCAAGATATTCAGAG

[0357] AGGTGTTTCATACCTAAAATTAAGACCAGATCGTGTTGCCTGTCAAGATGCTACTGCTCA

[0358] AATGGCTATTTTACAATTTATGTCCGCTGGTTTACCACAGGTTGCTAAGCCAGTCACTGTC

[0359] CACTGTGACCATTTGATTCAAGCACAAGTTGGTGGTGAAAAAGATTTGAAGAGAGCTAT

[0360] AGATCTAAACAAGGAAGTTTATGATTTCTTGGCCTCTGCCACTGCGAAATATAACATGGG

[0361] TTTCTGGAAGCCAGGTTCCGGTATCATTCACCAAATTGTTCTGGAAAACTACGCTTTCCC

[0362] AGGTGCTTTGATCATTGGTACTGACTCCCATACACCAAATGCTGGTGGTTTAGGTCAATT

[0363] GGCTATTGGTGTTGGTGGTGCTGATGCCGTTGATGTTATGGCAGGTCGTCCATGGGAATT

[0364] GAAGGCTCCAAAGATCTTAGGTGTTAAGTTGACTGGTAAGATGAACGGTTGGACTTCTC

[0365] CAAAGGATATTATTTTGAAATTGGCTGGTATCACAACTGTCAAAGGTGGTACTGGTAAAA

[0366] TTGTTGAATATTTCGGTGATGGTGTTGACACCTTCTCCGCTACTGGTATGGGTACCATTTG

[0367] TAATATGGGTGCTGAAATCGGTGCTACCACATCTGTTTTCCCATTCAACAAATCTATGATT

[0368] GAATATTTGGAAGCAACTGGTCGTGGTAAGATCGCTGACTTTGCTAAATTATACCACAAG

[0369] GACCTATTATCTGCTGATAAGGATGCTGAATACGATGAGGTCGTCGAAATTGACTTGAAC

[0370] ACTCTGGAACCATACATCAATGGGCCATTTACCCCCGATTTGGCTACTCCAGTTTCTAAG

[0371] ATGAAGGAAGTTGCTGTTGCTAATAACTGGCCATTGGATGTCAGAGTCGGTTTGATCGGT

[0372] TCTTGTACCAATTCCTCTTATGAAGATATGTCTCGTTCAGCATCCATTGTCAAGGATGCTG

[0373] CTGCTCATGGTTTGAAATCCAAGACCATTTTCACTGTTACTCCAGGTTCTGAACAAATCA

[0374] GAGCCACTATTGAACGTGATGGCCAATTAGAAACCTTCAAAGAATTTGGTGGTATCGTTT

[0375] TGGCAAACGCCTGTGGCCCATGTATTGGTCAATGGGATCGTAGAGATATCAAGAAAGGT

[0376] GACAAGAATACTATCGTTTCCTCTTACAACAGAAATTTCACTTCTAGAAATGATGGTAAC

[0377] CCACAAACTCATGCTTTTGTTGCATCTCCAGAATTAGTAACTGCGTTCGCCATTGCGGGT

[0378] GATTTGAGATTCAACCCTCTAACAGACAAATTAAAGGACAAGGATGGTAATGAGTTCAT

[0379] GTTGAAACCACCACATGGTGATGGTTTGCCTCAAAGAGGTTATGATGCTGGTGAGAACA

[0380] CTTACCAAGCTCCACCTGCAGACCGTAGCACCGTTGAAGTTAAAGTTTCTCCAACTTCA

[0381] GACCGTCTACAACTGTTGAAACCATTCAAACCTTGGGATGGTAAGGATGCTAAAGACAT

[0382] GCCAATCTTGATTAAGGCCGTCGGTAAGACAACTACTGATCATATTTCTATGGCTGGTCCA

[0383] TGGTTGAAATACAGAGGTCATTTAGAAAACATTTCTAATAACTATATGATTGGTGCTATTA

[0384] ATGCTGAAAACAAGAAGGCTAACTGTGTTAAAAATGTATATACTGGTGAATACAAAGGT

[0385] GTTCCAGACACTGCTAGAGATTACAGAGACCAAGGTATCAAGTGGGTTGTTATTGGTGA

[0386] TGAAAACTTTGGTGAAGGTTCCTCTCGTGAACACGCTGCTTTGGAACCAAGATTCTTGG

[0387] GCGGTTTCGCTATCATCACAAAGTCTTTCGCTCGTATCCATGAAACTAACTTGAAAAAAC

[0388] AAGGTCTATTGCCATTGAACTTCAAGAACCCAGCTGACTATGACAAGATCAACCCTGAT

[0389] GACAGAATCGATATTCTGGGTCTAGCTGAATTGGCTCCAGGTAAGCCTGTAACAATGAG

[0390] AGTTCATCCAAAGAATGGTAAGCCATGGGATGCTGTGTTGACCCATACTTTCAACGATGA

[0391] GCAAATTGAATGGTTCAAATATGGTTCTGCCTTAAATAAAATTAAGGCCGATGAGAAGAAATAA(SEQ ID NO: 11)

[0392] Saccharomyces cerevisiae 2μm shuttle plasmid sequence:

[0393] CAACTTTGTATAGAAAAGTTGACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGA

[0394] AGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATG

[0395] TGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATG

[0396] AAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATT

[0397] AACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAG

[0398] CGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTT

[0399] AACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCA

[0400] ACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCAAGTTTGT

[0401] ACAAAAAAGCAGGCTGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTG

[0402] GTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCG

[0403] GCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC

[0404] CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGT

[0405] GCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCC

[0406] GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCC

[0407] GCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAT

[0408] CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGC

[0409] CACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT

[0410] CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

[0411] CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGC

[0412] CCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC

[0413] GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAACCCAGCTTTCTTGTAC

[0414] AAAGTGGTAGCGGCCGCTCGAGCATGCATCTAGAGGGCCGCATCATGTAATTAGTTATGT

[0415] CACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGA

[0416] CAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATT

[0417] TATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGA

[0418] AAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCC

[0419] TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC

[0420] GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC

[0421] TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA

[0422] TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT

[0423] TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG

[0424] TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT

[0425] GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG

[0426] AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG

[0427] CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG

[0428] GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC

[0429] ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG

[0430] GTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC

[0431] AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA

[0432] GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA

[0433] GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGG

[0434] GATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

[0435] AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA

[0436] TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC

[0437] CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT

[0438] ACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAA

[0439] GGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT

[0440] GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT

[0441] GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC

[0442] CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT

[0443] CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGC

[0444] AGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG

[0445] TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC

[0446] GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA

[0447] ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT

[0448] AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT

[0449] GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAAT

[0450] GTTGAATACTCATACTCTTCCTTTTTCAATGGGTAATAACTGATATAATTAAATTGAAGCTC

[0451] TAATTTGTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTC

[0452] AAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACG

[0453] AGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGC

[0454] TATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGC

[0455] GGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCT

[0456] CGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGA

[0457] TGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGC

[0458] TCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAG

[0459] CCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACA

[0460] GGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACA

[0461] ACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTG

[0462] CCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGG

[0463] GCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGT

[0464] GCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCC

[0465] ATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTT

[0466] AAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAA

[0467] GTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTC

[0468] AAGGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAAAGCTAGCT

[0469] TATCGATGATAAGCTGTCAAACATGAGAATTAATTCCACGGACTATAGACTATACCTAGTA

[0470] TACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTAC

[0471] CACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCT

[0472] TCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTT

[0473] CTACAATGGCTGCCATCATTATTATCCGATGTGACGCTGCAGCTTCTCAATGATATTCGAA

[0474] TACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTA

[0475] CTTGTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATA

[0476] GTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATT

[0477] TCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCG

[0478] GTTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTG

[0479] TGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAAT

[0480] CTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAG

[0481] AATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACA

[0482] AAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCA

[0483] ACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCT

[0484] AACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGAT

[0485] AACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCT

[0486] CTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGG

[0487] GTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCAT

[0488] ACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACG

[0489] GTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTC

[0490] GATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATA

[0491] AACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATG

[0492] GGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGT

[0493] TTGTGGAAGCGGTATTCGCAATGGGAAGCTCCACCCCGGTTGATAATCAGAAAAGCCCC

[0494] AAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAGCGTTAATATTTTGTTAAAAT

[0495] TCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAAT

[0496] CCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACA

[0497] AGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA

[0498] GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCC

[0499] GTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAA

[0500] GCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGG

[0501] CGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT(SEQ ID NO: 12)

[0502] Neomycin sulfate resistance gene sequence of Saccharomyces cerevisiae (including promoter, expression cassette,Terminator): GTGAGTTGAGTATACATGCATTTACTTATAATACAGTTTTTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTG(SEQ ID NO: 13),

[0503] E. coli replicon sequence of the shuttle vector:

[0504] TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT

[0505] GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG

[0506] CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA

[0507] AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC

[0508] TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG

[0509] TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA

[0510] CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG

[0511] TGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCA

[0512] GTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAG

[0513] CGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA(SEQ ID NO: 14)

[0514]

[0515] Sequence of the inducible promoter GAL1 of Saccharomyces cerevisiae:

[0516] ACGGATTAGAGCCGCCGAGCGACGAGCGAGCGGAGCGCAGCAGCGCCGAAGGAAGAC

[0517] TCTCCTCCGTGCGCTCCTCGTCTCGTCTTCACCGGTCGTCGTCCTGAAACGAGATGTGTG

[0518] CCTCGCGCCGCACTGCTCCGAACAATAATACAATAAAGATTCTACAATACTAGCTATGGTT

[0519] ATGGTTATGAAGAGAAAAATGGCAGTACCTGGCCCCAAACCTCAAATGAACGAATCAAT

[0520] TAACAACCATAGGATGATGATAATGCGATTAGTTAGCTTATTCTATTTCTGGGGTAATTAAT

[0521] CAGCGAAGCGATGATGATTTTGATCTATTACAGATATATAATGCAAAACTGCATAACCACT

[0522] TAACTAACTAATATAATACTTTCAACATTTTCGTGTTGATTACTTCTTATCAAATGTAATACAAAAATTGTAATATATACCTATACTACTACTACTTAACGTCAAGGAGACC(SEQ ID NO: 16) Sequence of the yeast terminator CYC1:

[0523] ATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCG

[0524] AAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTT

[0525] AGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACG

[0526] CATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCT(SEQ ID NO:17)

[0527] The beneficial effects of the present invention are as follows:

[0528] The present invention designs an antisense RNA-based and modular plasmid elimination technology. The components required for constructing the plasmid elimination module are simple and easy to obtain. The plasmid module has a high elimination efficiency, and the elimination target can be freely set according to needs, enabling the selective or synchronous elimination of multiple plasmids. Moreover, the present invention has a wide application range, and this technology can be applied in both eukaryotes and prokaryotes. Antisense RNAs related to genes in the central metabolic pathway can all be combined with antisense RNAs for plasmid elimination. Antisense RNAs related to genes in metabolic pathways other than the central metabolic pathway, such as those involved in amino acid, nucleic acid, lipid synthesis pathways, transcription and translation, DNA replication, cell wall, cell membrane, endoplasmic reticulum, etc., can also be applied to this technology. The elimination effect can also be enhanced by strategies such as increasing the types of antisense RNAs and extending the lengths of antisense RNAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0529] Figure 1 It is the interference result diagram of Example 4.

[0530] Figure 2 It is the interference result diagram of Example 5.

[0531] Figure 3 It is the interference result diagram of Example 6.

[0532] Figure 4 It is the schematic diagram of the principle of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0533] The following specifically introduces the present invention in conjunction with the accompanying drawings and specific embodiments.

[0534] The principle of plasmid elimination by antisense RNA is as follows: We place antisense RNAs that can specifically bind to the mRNAs of key metabolic genes, such as genes related to the TCA cycle, protein synthesis, DNA / RNA synthesis, amino acid synthesis, ribose synthesis, ribosome synthesis, and ribosome assembly, downstream of an inducible promoter, and control the transcription of antisense RNAs of key metabolic genes by adding / removing an inducer. When the antisense RNA is transcribed, the antisense RNA can bind to the mRNA of the key metabolic gene in the form of hydrogen bond pairing, thereby inhibiting the translation of the key metabolic gene, making the growth rate of cells expressing the antisense RNA significantly lower than that of cells not expressing the antisense RNA. Since plasmids have a spontaneous loss phenomenon, plasmid elimination can be achieved by subculturing to enrich plasmid-free cells. (The principle is as shown in Figure 4as shown

[0535] The inducible promoter controls the expression of the antisense RNA of the key metabolic gene on the plasmid to be eliminated through an inducer, thereby achieving controllable cell growth inhibition, controllable plasmid-free cell enrichment, and plasmid elimination.

[0536] Instrumentation equipment:

[0537] 1. PCR instrument

[0538] 2. Electrophoresis instrument

[0539] 3. Electrophoresis tank

[0540] 4. Ultra-low temperature freezer

[0541] 5. Biochemical incubator

[0542] 6. Autoclave

[0543] 7. Water bath

[0544] 8. Medical freezer

[0545] 9. Tabletop centrifuge

[0546] 10. Pipettor

[0547] 11. Oven

[0548] 12. Electroporator and its supporting 1mm electroporation cuvette Reagents:

[0549]

[0550]

[0551] Biomaterials:

[0552] E. coli-related plasmids and primers:

[0553] Primers:

[0554] EP001: CGGGTGGGCCTTTCTTCGAATGAATCACCGATACGCGAGCGAACG (SEQ ID NO: 18)

[0555] EP002: CGCAACTCTCTACTGTTTCTCCATGGAATTGTGAGCGGATAACAATTCC (SEQ ID NO: 19)

[0556] EP003: CGAAGAAAGGCCCACCCGTGAAGGTGAGCCTTATGACAACTTGACGGCTACATC (SEQ ID NO: 20)

[0557] EP004: GGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGG (SEQ ID NO: 21) EP005: GATAGCAGCCAGGAAGCCTGCTTTGACATAGGCGGCTTCATCGACGCCTGGGGGGAATTGTTATCCGCTCACAATTCC (SEQ ID NO: 22)

[0558] EP006:

[0559] GCAGGCTTCCTGGCTGCTATCGCTAAAGGCGAAGCCAAATCCCCCTAGCATAACCCCTT G GGGCCTC (SEQ ID NO: 23)

[0560] EP007: CATGTTGTTGCTCAGGTCGCAGACG (SEQ ID NO: 24)

[0561] EP008: GGAAGGGAAGAAAGCGAAAGGAGC (SEQ ID NO: 25)

[0562] Plasmids and primers related to Corynebacterium glutamicum:

[0563] Primers:

[0564] GP001: CCATGAACAGAAATCCCGTAAATGCATGCCGCTTCGCCTTCG (SEQ ID NO: 26) GP002:

[0565] CGCAACTCTCTACTGTTTCTCCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGG (SEQ ID NO: 27)

[0566] GP003: GGATTTCTGTTCATGGGGGTAATG (SEQ ID NO: 28)

[0567] GP004: ATGGAGAAACAGTAGAGAGTTGCG (SEQ ID NO: 29)

[0568] GP005: CGGCCGAAAGCCTCCACGATGACGGAGTGGTCAATGACCATCTCGGCTGGGCAA ATAAAACGAAAGGCTCAGTCGAAAGACTGGG (SEQ ID NO: 30)

[0569] GP006: CGTGGAGGCTTTCGGCCGCCCAGATGCACTGGCATGGAGAAACAGTAGAGAGTTGCG (SEQ ID NO: 31)

[0570] GP007: GCTTACAGACAAGCTGTGACCGTCTC (SEQ ID NO: 32)

[0571] GP008: CCGTTGCTTCGCAACGTTCAAATCC (SEQ ID NO: 33)

[0572] Plasmids and primers related to Saccharomyces cerevisiae:

[0573] Primers:

[0574] SP001: TGATGCCGTTGATGTTATGGCAGGTCGTCCATGGGAATTGAAGGCTCCGGTTTTTTCTCCTTGACGTTAAAGTATAGAGG (SEQ ID NO: 34)

[0575] SP002: CCATAACATCAACGGCATCAGCACCACCAACACCAATAGCCAATTGACATCATGTAATTAGTTATGTCACGCTTACATTC (SEQ ID NO: 35)

[0576] SP003: GATTTAGAGCTTGACGGGGAAAGCC (SEQ ID NO: 36)

[0577] SP004: CTCACATGTTCTTTCCTGCGTTATCC (SEQ ID NO: 37)

[0578] Example 1 Construction of the plasmid elimination module of the arabinose promoter in Escherichia coli

[0579] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0580] 1. Design primers EP001 and EP002 to amplify the plasmid template pET28a plasmid according to the following system and PCR program, and design primers EP003 and EP004 to amplify the plasmid template pkd46 plasmid according to the same system and PCR program.

[0581] System:

[0582]

[0583]

[0584] PCR procedure:

[0585]

[0586] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products EP001-EP002 and EP003-EP004 according to the purification kit instructions.

[0587] 3. Connect the two purified products EP001-EP002 / EP003-EP004 according to the operation procedure shown in the ready-to-use seamless cloning kit instructions.

[0588] 4. Transform according to the following steps After overnight culture of 5α Chemically Competent Cell:

[0589] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (pipette gently or flick the tube wall several times), and let stand on ice for 5 min.

[0590] 2) Heat shock in a 42 °C water bath for 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0591] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and resuscitate at 37 °C and 200 rpm for 40 min.

[0592] 4) Take an appropriate volume of the resuscitation solution and spread it evenly on an LB solid medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator overnight.

[0593] (2) Plasmid extraction experiment:

[0594] Select available single colonies on the transformation plate in (1) and inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin for overnight activation, and complete plasmid extraction according to the instructions of the Tiangen plasmid miniprep kit.

[0595] (3) Second high-fidelity enzyme amplification and plasmid construction experiment:

[0596] 1. Amplify the plasmid obtained in (2) (the plasmid concentration needs to be diluted to less than 1 ng / μL) with primers EP005 and EP006 according to the following system and PCR procedure.

[0597] System:

[0598]

[0599]

[0600] PCR procedure:

[0601]

[0602] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR products EP005 - EP006 according to the purification kit instructions.

[0603] 3. Connect the two purified fragments EP001 - EP002 / EP003 - EP004 according to the operation procedure shown in the ready-to-use seamless cloning kit instructions.

[0604] 4. Transform according to the following steps After overnight culture of 5α Chemically Competent Cell:

[0605] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (pipette gently or flick the tube wall several times), and let stand on ice for 5 min.

[0606] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0607] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C and 200 rpm for 40 min.

[0608] 4) Take an appropriate volume of the recovered solution and spread it evenly on a medium containing 50 μg / mL kanamycin antibiotic, and incubate it upside down in a 37 °C incubator overnight.

[0609] (4) Sequencing verification experiment for the construction result:

[0610] Take the single colonies obtained from the spread plate and send them for sequencing with primers EP007 and EP008 to verify the construction result.

[0611] Plasmid construction result:

[0612] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCT

[0613] ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT

[0614] TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCAC

[0615] TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

[0616] TATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA

[0617] TTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA

[0618] ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC

[0619] GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA

[0620] AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC

[0621] AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAA

[0622] CCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

[0623] CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAA

[0624] TATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGC

[0625] AGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG

[0626] GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT

[0627] ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGAT

[0628] TGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC

[0629] ATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA

[0630] CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAA

[0631] CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0632] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

[0633] CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC

[0634] AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

[0635] AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

[0636] GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

[0637] GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC

[0638] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG

[0639] AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA

[0640] CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

[0641] CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA

[0642] CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT

[0643] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

[0644] ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0645] GAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATG

[0646] GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTAT

[0647] CGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC

[0648] TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG

[0649] CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA

[0650] GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCT

[0651] CGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGG

[0652] CGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGG

[0653] TAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACAT

[0654] GCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACC

[0655] AGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCA

[0656] CAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGA

[0657] CTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTC

[0658] AGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCAT

[0659] TCGAAGAAAGGCCCACCCGTGAAGGTGAGCCTTATGACAACTTGACGGCTACATCATTC

[0660] ACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAAT

[0661] ACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAG

[0662] GCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAG

[0663] CTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACA

[0664] AGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTG

[0665] ATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATC

[0666] GCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAG

[0667] CGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTG

[0668] GTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCC

[0669] ATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCC

[0670] TCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGG

[0671] TCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATT

[0672] GAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGC

[0673] CTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGG

[0674] GATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATT

[0675] GTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAA

[0676] ACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAA

[0677] AAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCAC

[0678] GGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGAC

[0679] GCTTTTTATCGCAACTCTCTACTGTTTCTCCATGGAATTGTGAGCGGATAACAATTCCCCC

[0680] CAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGC

[0681] GAAGCCAAATCCCCCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT(SEQ ID NO:38)

[0682] Among them, the antisense RNA sequence is:

[0683] CCCCAGGCGTCGATGAAGCCGCCTATGTCAAAGCAGGCTTCCTGGCTGCTATCGCTAAAGGCGAAGCCAAATCCCC. (SEQ ID NO:39)

[0684] Example 2 Construction of the Arabinose Promoter Plasmid Elimination Module of Corynebacterium glutamicum

[0685] (1) High-fidelity enzyme amplification and plasmid construction experiments:

[0686] 1. Design primers GP001 and GP002 and amplify the plasmid template pEC-XK99E plasmid according to the following system and PCR program; design primers GP003 and GP004 and amplify the codon-optimized arabinose induction system according to the following system and PCR program.

[0687] System:

[0688]

[0689] PCR program:

[0690]

[0691] 2. Assemble the SanPrep Column PCR Product Purification Kit using a ready-to-use seamless cloning kit, and purify the PCR products GP001 - GP002 / GP003 - GP004 according to the purification kit instructions.

[0692] 3. Ligate the purified products GP001 - GP002 / GP003 - GP004 according to the operation procedure shown in the ready-to-use seamless cloning kit instructions.

[0693] 4. Transform according to the following steps 5α Chemically Competent Cell and incubate overnight:

[0694] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (pipette gently or flick the tube wall several times), and let it stand on ice for 5 min.

[0695] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let it stand for 2 min.

[0696] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C, 200 rpm for 40 min.

[0697] 4) Take an appropriate volume of the recovered solution and spread it evenly on a medium containing 50 μg / mL kanamycin, and incubate it upside down in a 37 °C incubator

[0698] overnight.

[0699] (2) Plasmid extraction experiment:

[0700] Select available single colonies on the transformation plate in (1) and inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin for overnight activation, and complete plasmid extraction according to the Tiangen Plasmid Mini Kit instructions.

[0701] (3) Second-round high-fidelity enzyme amplification and plasmid construction experiment:

[0702] 1. Design primers GP005 and GP006 and amplify the plasmid extracted in (2) according to the following system and PCR program.

[0703] System:

[0704]

[0705] PCR program:

[0706]

[0707] 2. Assemble the SanPrep Column PCR Product Purification Kit using a ready-to-use seamless cloning kit, and purify the PCR products GP005-GP006 according to the instructions of the purification kit.

[0708] 3. Connect the purified products GP005-GP006 according to the operation procedure shown in the instructions of the ready-to-use seamless cloning kit.

[0709] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[0710] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (pipette gently or flick the tube wall several times), and let stand on ice for 5 min.

[0711] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0712] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C and 200 rpm for 40 min.

[0713] 4) Take an appropriate volume of the recovered solution and spread it evenly on a medium containing 50 μg / mL kanamycin antibiotic, and incubate it upside down in a 37 °C incubator overnight.

[0714] overnight.

[0715] (4) Sequencing verification experiment for the construction result

[0716] Take the single colonies obtained from the spread plate and send them for sequencing verification of the construction result using the GP007 / GP008 primers.

[0717] Plasmid construction result:

[0718]

[0719] AAAGGTCTCCAGGATCTCGAGGAGATTTTTGAGGGGGAGGGAGTCGAGGAAGAGCCAG

[0720] AGCAGAAGGCGGGGAACCGTTCTCTGCCGACAGCGTGAGCCCCCCTTAAAAATCAGGC

[0721] CGGGGAGGAACCGGGGAGGGATCAGAGCTAGGAGCGAGACACCCTAAAGGGGGGGAA

[0722] CCGTTTTCTGCTGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGATAGCGGAGGGTGA

[0723] GGGCAAGTGAGAGCCAGAGCAAGGACGGGACCCCTAAAGGGGGGAACCGTTTTCTGC

[0724] TGACGGTGTTTCGTTTATTAGTTTTCAGCCCGTGGACGGCCGCGTTTAGCTTCCATTCCA

[0725] AGTGCCTTTCTGACTTGTTGGATGCGCCTTTCACTGACACCTAGTTCGCCTGCAAGCTCA

[0726] CGAGTCGAGGGATCAGCAACCGATTGAGAACGGGCATCCAGGATCGCAGTTTTGACGC

[0727] GAAGTTCGAGCAACTCGCCTGTCATTTCTCGGCGTTTGTTTGCTTCCGCTAATCGCTGTC

[0728] GCGTCTCCTGCGCATACTTACTTTCTGGGTCAGCCCATCTGCGTGCATTCGATGTAGCTG

[0729] CGCCCCGTCGCCCCATCGTCGCTAGAGCTTTCCGCCCTCGGCTGCTCTGCGTTTCCACCC

[0730] GACGAGCAGGGACGACTGGCTGGCCTTTAGCCACGTAGCCGCGCACACGACGCGCCAT

[0731] CGTCAGGCGATCACGCATGGCGGGAAGATCCGGCTCCCGGCCGTCTGCACCGACCGCCT

[0732] GGGCAACGTTGTACGCCACTTCATACGCGTCGATGATCTTGGCATCTTTTAGGCGCTCAC

[0733] CAGCAGCTTTGAGCTGGTATCCCACGGTCAACGCGTGGCGAAACGCGGTCTCGTCGCGC

[0734] GCTCGCTCTGGATTTGTCCAGAGCACTCGCACGCCGTCGATCAGGTCGCCGGACGCGTC

[0735] CAGGGCGCTCGGCAGGCTCGCGTCCAAAATCGCTAGCGCCTTGGCTTCTGCGGTGGCGC

[0736] GTTGTGCCGCTTCAATGCGGGCGCGTCCGCTGGAAAAGTCCTGCTCAATGTACTTTTTCG

[0737] GCTTCTGTGATCCGGTCATCGTTCGAGCAATCTCCATTAGGTCGGCCAGCCGATCCACAC

[0738] GATCATGCTGGCAGTGCCATTTATAGGCTGTCGGATCGTCTGAGACGTGCAGCGGCCAC

[0739] CGGCTCAGCCTATGCGAAAAAGCCTGGTCAGCGCCGAAAACACGAGTCATTTCTTCCGT

[0740] CGTTGCAGCCAGCAGGCGCATATTTGGGCTGGTTTTACCTGCTGCGGCATACACCGGGTC

[0741] AATGAGCCAGATGAGCTGGCATTTCCCGCTCAGCGGATTCACGCCGATCCAAGCCGGCG

[0742] CTTTTTCTAGGCGTGCCCATTTCTCTAAAATCGCGTAGACCTGCGGGTTTACGTGCTCAA

[0743] TCTTCCCGCCGGCCTGGTGGCTGGGCACATCGATGTCAAGCACGATCACCGCGGCATGT

[0744] TGCGCGTGCGTCAGCGCAACGTACTGGCACCGCGTCAGCGCTTTTGAGCCAGCCCGGTA

[0745] GAGCTTTGGTTGGGTTTCGCCGGTATCCGGGTTTTTAATCCAGGCGCTCGCGAAATCTCT

[0746] TGTCTTGCTGCCCTGGAAGCTTTCGCGTCCCAGGTGAGCGAGCAGTTCGCGGCGATCTT

[0747] CTGCCGTCCAGCCGCGTGAGCCGCAGCGCATAGCTTCGGGGTGGGTGTCGAACAGATC

[0748] GGCGGACAATTTCCACGCGCTAGCTGTGACTGTGTCCTGCGGATCGGCTAGAGTCATGT

[0749] CTTGAGTGCTTTCTCCCAGCTGATGACTGGGGGTTAGCCGACGCCCTGTGAGTTCCCGC

[0750] TCACGGGGCGTTCAACTTTTTCAGGTATTTGTGCAGCTTATCGTGTTTTCTTCGTAAATGA

[0751] ACGCTTAACTACCTTGTTAAACGTGGCAAATAGGCAGGATTGATGGGGATCTAGCTTCAC

[0752] GCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAG

[0753] CCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACA

[0754] AGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGAT

[0755] AGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCC

[0756] CTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGA

[0757] TCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATG

[0758] ATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGG

[0759] CTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAG

[0760] CGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTC

[0761] CAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTG

[0762] TGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGG

[0763] GCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGC

[0764] AATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAAC

[0765] ATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTG

[0766] GACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGA

[0767] TGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGG

[0768] TGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGC

[0769] TATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

[0770] GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATC

[0771] GCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCGGAATCATGACCAAAAT

[0772] CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT

[0773] CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC

[0774] TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT

[0775] GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA

[0776] CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT

[0777] GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTAC

[0778] CGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA

[0779] GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

[0780] CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA

[0781] GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT

[0782] TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT

[0783] GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT

[0784] CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT

[0785] GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGA

[0786] AGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG

[0787] CATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTC

[0788] CGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGAC

[0789] GCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC

[0790] CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAG

[0791] ATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTACGGGATTTCTGTTCATGGGGGT

[0792] AATGATACCGATGGCGCACATTTCCCCGAAAAGTGCCACCTGCATCGATTTATTATGACA

[0793] ACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTG

[0794] GCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTG

[0795] CGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGAT

[0796] ACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTG

[0797] ACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCT

[0798] GTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGG

[0799] ATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTAT

[0800] CGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACA

[0801] GGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAAT

[0802] ATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTG

[0803] GTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGA

[0804] ACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCT

[0805] GACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAA

[0806] AAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAA

[0807] CGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCC

[0808] ATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTA

[0809] CTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAG

[0810] CGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAA

[0811] GTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT(SEQ ID NO:40)

[0812] The antisense RNA sequence therein:

[0813] GCCAGTGCATCTGGGCGGCCGAAAGCCTCCACGATGACGGAGTGGTCAATGACCAT CTCGGCTGGG(SEQ ID NO:41)

[0814] Example 3 Construction of the inducible GAL1 promoter plasmid elimination module in Saccharomyces cerevisiae

[0815] (1) High-fidelity enzyme amplification and plasmid construction experiment:

[0816] 1. Design primers SP001-SP002 and amplify the plasmid template Saccharomyces cerevisiae 2μ plasmid according to the following system and PCR program; amplify the codon-optimized LacI protein expression cassette with primers SP002-SP003.

[0817] System:

[0818]

[0819] PCR program:

[0820]

[0821] 2. Use a ready-to-use seamless cloning kit to assemble the SanPrep column PCR product purification kit, and purify the PCR product SP001-SP002 according to the purification kit instructions.

[0822] 3. Connect the purified products SP001-SP002 respectively according to the operation process shown in the ready-to-use seamless cloning kit instructions; self-ligate the purified product SP009-SP010 with the ready-to-use seamless cloning kit.

[0823] 4. Transform according to the following steps 5α Chemically Competent Cell and culture overnight:

[0824] 1) Take 100 μL of competent cells melted on ice, add the ligation product in "3", mix gently (gently pipette or flick the tube wall several times), and let stand on ice for 5 min.

[0825] 2) Heat shock in a 42 °C water bath for 45 - 60 s, quickly transfer to an ice bath, and let stand for 2 min.

[0826] 3) Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube, mix well, and recover at 37 °C and 200 rpm for 40 min.

[0827] 4) Take an appropriate volume of the recovered solution and evenly spread it on an LB solid medium containing 100 μg / mL amp antibiotic, and incubate it upside down in a 37 °C incubator overnight.

[0828] Incubate overnight.

[0829] (2) Sequencing verification experiment for the construction result:

[0830] Take the single colonies obtained from the two coated plates and use the SP003 / SP004 primers to send them for sequencing to verify the construction result, and obtain plasmids with correct sequencing.

[0831] Plasmid construction result:

[0832]

[0833] AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA

[0834] GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT

[0835] TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT

[0836] TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG

[0837] CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC

[0838] TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGT

[0839] AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTC

[0840] TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG

[0841] GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA

[0842] GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAA

[0843] CTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCC

[0844] AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC

[0845] GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC

[0846] CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGT

[0847] TGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC

[0848] ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG

[0849] TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAG

[0850] CAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGA

[0851] TCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAG

[0852] CATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCA

[0853] AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATG

[0854] GGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGTTGAGTATACATGCATTTA

[0855] CTTATAATACAGTTTTTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGC

[0856] GAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAA

[0857] GCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCA

[0858] GCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAG

[0859] CAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCT

[0860] GGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGA

[0861] CAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCG

[0862] AATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGAT

[0863] ACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCA

[0864] ATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAAC

[0865] GCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCAC

[0866] CGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACA

[0867] CGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCC

[0868] ACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGATTTATCTTCGT

[0869] TTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTT

[0870] TCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTT

[0871] CTGTTCGGAGATTACCGAATCAAAAAAATTTCAAGGAAACCGAAATCAAAAAAAAGAA

[0872] TAAAAAAAAAATGATGAATTGAAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAG

[0873] AATTAATTCCACGGACTATAGACTATACCTAGTATACTCCGTCTACTGTACGATACACTTC

[0874] CGCTCAGGTCCTTGTCCTTTAACGAGGCCTTACCACTCTTTTGTTACTCTATTGATCCAGC

[0875] TCAGCAAAGGCAGTGTGATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACC

[0876] GAGAAAGAGACTAGAAATGCAAAAGGCACTTCTACAATGGCTGCCATCATTATTATCCGA

[0877] TGTGACGCTGCAGCTTCTCAATGATATTCGAATACGCTTTGAGGAGATACAGCCTAATAT

[0878] CCGACAAACTGTTTTACAGATTTACGATCGTACTTGTTACCCATCATTGAATTTTGAACAT

[0879] CCGAACCTGGGAGTTTTCCCTGAAACAGATAGTATATTTGAACCTGTATAATAATATATAG

[0880] TCTAGCGCTTTACGGAAGACAATGTATGTATTTCGGTTCCTGGAGAAACTATTGCATCTAT

[0881] TGCATAGGTAATCTTGCACGTCGCATCCCCGGTTCATTTTCTGCGTTTCCATCTTGCACTT

[0882] CAATAGCATATCTTTGTTAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAAC

[0883] GCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATG

[0884] CAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAA

[0885] ATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAAC

[0886] AGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCT

[0887] ACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCT

[0888] CCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGT

[0889] TAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTC

[0890] CCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCC

[0891] GATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGA

[0892] TGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGT

[0893] ATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAA

[0894] TTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAG

[0895] ATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGA

[0896] TATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATGGGAAGC

[0897] TCCACCCCGGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATAT

[0898] TTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCAT

[0899] TTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA

[0900] TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCC

[0901] AACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCAC

[0902] CCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGG

[0903] AGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGG

[0904] AAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGC

[0905] GTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATCGATCCACTAGT(SEQID NO:42)

[0906] The antisense RNA sequence therein:

[0907] GGAGCCTTCAATTCCCATGGACGACCTGCCATAACATCAACGGCATCAGCACCACCAACACCAATAGCCAATTGAC(SEQ ID NO:43)

[0908] (3) Plasmid extraction experiment:

[0909] Select available single colonies on the transformation plate in (1) according to the sequencing alignment results in (2), inoculate them into 5 mL of LB liquid medium containing 100 μg / mL amp antibiotic for overnight activation, and complete plasmid extraction according to the instructions of the Tiangen plasmid miniprep kit.

[0910] Example 4 Plasmid elimination using the Escherichia coli arabinose promoter plasmid elimination module

[0911] 1. Prepare competent cells from the Escherichia coli to be transformed according to the procedure described in the super competent cell preparation kit.

[0912] 2. Pipette 100 μL of competent cells thawed on ice into the plasmid elimination module constructed in Example 1, and gently mix (pipette gently or flick the tube wall several times), then let it stand on ice for 5 min.

[0913] 3. Heat shock in a 42 °C water bath for 60 s, then quickly transfer to an ice bath and let it stand for 2 min (do not shake the sample during the standing on ice process, otherwise the transformation efficiency will be reduced).

[0914] 4. Add 700 μL of sterile liquid medium without antibiotics (SOB or LB) to the centrifuge tube and mix well.

[0915] 5. Take an appropriate volume of the resuscitation solution and evenly spread it on a culture medium containing 50 μg / mL kanamycin antibiotic, and incubate it upside down in a 37 °C incubator overnight.

[0916] 6. Add the Escherichia coli constructed in "6" or the frozen bacteria / stab culture of Escherichia coli containing the module constructed in "5" to an LB liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, and culture at 37 °C and 220 rpm.

[0917] 7. Add an inducer of 1% arabinose at a final concentration to the LB medium in "6".

[0918] 8. After overnight induction of Escherichia coli (12 h), streak it on an antibiotic-free LB solid medium plate.

[0919] 9. Take the single colonies obtained in "8" and spot them on both antibiotic-free and antibiotic-containing solid medium plates respectively (each single colony needs to be spotted on the plates twice to ensure the distinction of phenotypic differences), and select the colonies that do not grow on the antibiotic-containing plate but grow on the antibiotic-free plate for colony PCR to verify whether the plasmid has been eliminated.

[0920] The specific results are as Figure 1 shown. After plasmid elimination, most of the bacteria lost their resistance to antibiotics and were no longer able to grow in the antibiotic-containing plate, indicating that the relevant resistance genes had been lost and the plasmid had been successfully eliminated. Inoculating the corresponding strain with successful elimination can obtain the strain with successfully eliminated plasmid.

[0921] Example 5 Plasmid Elimination Using the Arabinose Promoter Plasmid Elimination Module of Corynebacterium glutamicum

[0922] 1. Activate the Corynebacterium glutamicum ATCC13032 strain overnight in 5 ml of brain heart infusion liquid medium.

[0923] 2. Inoculate all the activated Corynebacterium glutamicum bacterial liquid into 45 mL of Epo liquid medium to make the OD600 reach 0.2, and grow at 200 rpm and 30 °C until the OD600 reaches 0.9.

[0924] 3. Transfer the bacterial solution to a sterile 50 mL centrifuge tube, centrifuge to remove the supernatant, wash the bacterial cells 4 times with ice-cold 10% sterilized glycerol, aliquot 100 μL per tube into sterile 1.5 mL centrifuge tubes, and store at -70°C or use immediately.

[0925] 4. For electroporation, thaw the aliquoted competent cells on ice and add 5 μL of the plasmid obtained in Example 2.

[0926] 5. Transfer the mixture to a cold electroporation cuvette (0.1 cm), and perform pulsed electroporation at 1.8 kV, 200 Ω, 25 μF, and 5 ms. Immediately after electroporation, add 1 mL of LBHIS medium to the cuvette, mix well, and transfer to a 1.5 mL centrifuge tube.

[0927] 6. Incubate at 30°C for 1 hour.

[0928] 7. Finally, spread on a brain heart infusion agar plate containing 50 μg / mL kanamycin.

[0929] 8. Add the frozen stock / puncture stock of Corynebacterium glutamicum constructed in "7" or Corynebacterium glutamicum containing the construction module in "7" to a non-antibiotic brain heart infusion liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, and incubate at 30°C and 220 rpm.

[0930] 9. Add an arabinose inducer with a final concentration of 1% to the medium described in "8".

[0931] 10. After overnight induction of Corynebacterium glutamicum (18 h), streak on a non-antibiotic LB solid medium plate.

[0932] 11. Pick the single colonies obtained in "10" and spot plate on non-antibiotic and antibiotic-containing solid medium plates respectively (each single colony needs to be spot plated twice to ensure the distinction of phenotypic differences).

[0933] 12. The specific results are as Figure 2 shown. After plasmid curing, most of the cultured bacteria lost their resistance to antibiotics and could no longer grow on the antibiotic-containing plates, indicating that the relevant resistance genes had been lost and the plasmid had been successfully cured. Inoculating the corresponding strain with successful plasmid curing can obtain the strain with successfully cured plasmid.

[0934] Example 6 Plasmid curing using the plasmid curing module with the GAL1 promoter of Saccharomyces cerevisiae

[0935] First, prepare the following reagents:

[0936] 10×TE buffer

[0937] 100 mM LiAC buffer

[0938] 1 M DTT buffer

[0939] ddH2O

[0940] 1M sorbitol solution

[0941] Preparation of competent cells of Saccharomyces cerevisiae:

[0942] 1. Pre-cool the reagents.

[0943] 2. Pipette 30 μL from the glycerol tube of Saccharomyces cerevisiae and inoculate it into 10 mL of liquid YPD medium. Culture it in a constant temperature shaker at 30 °C for 24 h, and then pipette 100 μL from it and inoculate it into 100 mL of liquid YPD medium until the cell concentration OD600 grows to 1.3 - 1.5.

[0944] 3. Divide the bacterial solution into three pre-cooled 50 mL centrifuge tubes, centrifuge at 4 °C and 5000 r×min-1 for 5 min, discard the supernatant, and collect the cells.

[0945] 4. Resuspend the cells by pipetting with 4 mL of pre-cooled ddH2O. Combine the three tubes into one tube, and add 2 mL of pre-cooled 10×TE buffer, 2 mL of 100 mM LiAC buffer, and 0.5 mL of 1M DTT buffer. Pipette and mix well, and place the centrifuge tube in a constant temperature water bath shaker at 30 °C and 50 r×min-1 for 45 min.

[0946] 5. Add 13.5 mL of pre-cooled ddH2O to a 50 mL centrifuge tube, centrifuge at 4 °C and 5000 r×min-1 for 5 min, discard the supernatant, and collect the cells

[0947] 6. Add 25 mL of pre-cooled ddH2O to a 50 mL centrifuge tube and pipette to resuspend the cells. Centrifuge at 5000 r×min-1 for 5 min, discard the supernatant, and collect the cells. Then wash the cells twice with 1M sorbitol (25 mL for the first time and 20 mL for the second time)

[0948] 7. Add 1 mL of pre-cooled sorbitol to the centrifuge tube to resuspend the cells, and dispense them into 1.5 mL EP tubes, 80 μL per tube, for immediate use.

[0949] 8. During electroporation, place the dispensed competent cells on ice and add 5 μL of the plasmid obtained in Example 3.

[0950] 9. Transfer the mixture to a cold electroporation cuvette (0.2 cm), and perform pulsed electroporation at 1.5 kV, 200 Ω, 25 μF, and 5 ms. Immediately after electroporation, add 1 mL of antibiotic-free YPD medium to the cuvette, mix well, and transfer it to a 1.5 mL centrifuge tube. Incubate it in a shaker at 30 °C and 200 rpm for 1 - 3 h.

[0951] 10. Centrifugally collect the bacterial cells. Take 150 μl of the bacterial solution and spread it on a resistant plate, and culture at 30 °C for 2 - 3 days.

[0952] 11. Add the cryopreserved bacteria / puncture bacteria of Saccharomyces cerevisiae constructed in "10" or Saccharomyces cerevisiae containing the construction module in "10" into a non - antibiotic YPD liquid medium with a volume not exceeding 1 / 5 of the conical flask volume, and culture at 30 °C, 220 rpm.

[0953] 12. When the OD600 of the bacterial solution in "11" reaches 2, add a galactose inducer with a final concentration of 2%.

[0954] 13. After inducing Saccharomyces cerevisiae for 48 h, streak it on a non - antibiotic YPD solid medium plate containing 5% galactose.

[0955] Take the single colonies obtained in "13" and spot them on non - antibiotic and antibiotic (neomycin sulfate 500 μg / mL) YPD solid medium plates respectively (each single colony needs to be spotted twice to ensure the distinction of phenotypic differences). After culturing for 2 - 3 days, select the colonies that do not grow on the antibiotic plate but grow on the non - antibiotic plate for colony PCR to verify whether the plasmid has been eliminated.

[0956] The specific results are as Figure 3 shown. After plasmid elimination, most of the bacteria lost their resistance to antibiotics and could no longer grow in the antibiotic - containing plate, indicating that the relevant resistance genes have been lost and the plasmid has been successfully eliminated. Inoculating the corresponding strains with successful elimination can obtain the strains with successfully eliminated plasmids.

[0957] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. An inducible promoter-based plasmid elimination module, characterized in that, It includes two types of independent expression regions. One type of region is the inducer transcription region, which consists of a constitutive promoter carried by the transcription factor fragment, including its RNA polymerase binding site, transcription start site, ribosome binding site, inducer transcription factor translation region, and terminator. The other type of region is the antisense RNA expression region, which consists of an inducer promoter corresponding to the inducer transcription factor, antisense RNA, and terminator. The antisense RNA is the antisense RNA of the key metabolic gene mRNA, and it is an RNA fragment that can bind to the key metabolic gene mRNA to hinder the translation of the key metabolic gene mRNA.

2. The plasmid elimination module according to claim 1, wherein The inducer transcription factor is a protein that can induce the transcription of genes downstream of the binding site after binding to a specific compound; or a protein that can induce the transcription of downstream genes before binding to a specific compound and cannot induce the transcription of genes downstream of the binding site after binding to a specific compound.

3. The plasmid elimination module according to claim 1, characterized in that The constitutive promoter carried by the transcription factor fragment is the constitutive promoter carried by the arabinose transcription factor.

4. The plasmid elimination module according to claim 1, characterized in that The antisense RNA expression region is carried on the plasmid to be eliminated.

5. The plasmid elimination module according to claim 4, wherein The plasmid to be eliminated is a circular or linear DNA that can replicate independently outside the genome and is stably free in the cell, or can replicate with the genome and be passed on to daughter cells.

6. The plasmid elimination module according to claim 5, wherein The plasmid to be eliminated includes any one of pet28A, PEC-XK99E, and yeast 2μ plasmid.

7. The plasmid elimination module according to claim 1, wherein The gene interfered by the antisense RNA is the key metabolic gene.

8. The plasmid elimination module according to claim 7, characterized in that, The metabolic key genes described above are genes related to the TCA cycle, genes related to protein synthesis, genes related to aerobic respiration, genes related to anaerobic respiration, genes related to lipid synthesis, genes related to cell wall synthesis, genes related to sugar transport pathways, genes related to amino acid transport pathways, genes related to ATP synthesis, genes related to cytoskeleton synthesis, genes related to NADPH / NADP + synthesis, genes related to NADH / NAD + synthesis, genes related to vitamin synthesis, genes related to one-carbon transport systems, genes related to carbon dioxide fixation, genes related to DNA / RNA synthesis, genes related to amino acid synthesis, genes related to ribose synthesis, genes related to ribosome synthesis, genes related to ribosome assembly, genes related to aminoacyl-tRNA synthetase, and isozyme genes of the above genes.

9. The plasmid elimination module according to any one of claims 1 to 8, characterized in that, The elimination target of the plasmid elimination module is all microorganisms, animals, or plant cells that have stably free plasmids, there is a certain probability that the free plasmids will not be distributed to daughter cells, and the cells will not die or be unable to proliferate after the free plasmids are eliminated.

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