CN116555259B - Nucleic acid molecules and their use as promoters - Google Patents
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 21
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 21
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 239000002773 nucleotide Substances 0.000 claims abstract description 14
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 14
- 210000003527 eukaryotic cell Anatomy 0.000 claims abstract description 6
- 239000013604 expression vector Substances 0.000 claims description 10
- 238000013518 transcription Methods 0.000 claims description 10
- 230000035897 transcription Effects 0.000 claims description 10
- 239000013603 viral vector Substances 0.000 claims description 10
- 230000002103 transcriptional effect Effects 0.000 claims description 3
- 102000034287 fluorescent proteins Human genes 0.000 claims description 2
- 108091006047 fluorescent proteins Proteins 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 7
- 210000004027 cell Anatomy 0.000 description 52
- 241000701022 Cytomegalovirus Species 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- 239000013598 vector Substances 0.000 description 18
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 16
- 238000001890 transfection Methods 0.000 description 14
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 12
- 239000005090 green fluorescent protein Substances 0.000 description 12
- 108010054624 red fluorescent protein Proteins 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 239000013607 AAV vector Substances 0.000 description 3
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 102100030801 Elongation factor 1-alpha 1 Human genes 0.000 description 2
- 101000920078 Homo sapiens Elongation factor 1-alpha 1 Proteins 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108700026226 TATA Box Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000013326 plasmid cotransfection Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
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Abstract
The present invention relates to the field of bioengineering, and in particular to nucleic acid molecules and their use as promoters. The present invention provides a nucleic acid molecule having: nucleotide sequences shown in any of SEQ ID NO. 1-SEQ ID NO. 4; or a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown; or a nucleotide sequence having at least 85% sequence homology with said nucleotide sequence. Compared with the existing common short-version promoter, the promoter provided by the invention can improve the expression of the target gene in eukaryotic cells, and has stronger application value under certain scenes with higher requirement on the expression quantity of the target gene but limitation on the length of the promoter.
Description
Technical Field
The present invention relates to the field of bioengineering, and in particular to nucleic acid molecules and their use as promoters.
Background
The Promoter (Promoter) is a DNA sequence located upstream of the 5' end of the structural gene, which activates RNA polymerase to bind precisely to the template DNA and has specificity for transcription initiation. The general structure of a promoter includes a core promoter element, which in turn includes a transcription initiation point and a TATA box, which are the primary sites for RNA polymerase to bind and initiate transcription, and an upstream regulatory element; upstream regulatory elements are then able to alter the efficiency of transcription by binding to the corresponding trans-acting factors. The transcriptional capacity of different promoters varies due to the elements and positions; promoters will generally be classified into weak promoters and strong promoters, depending on the degree to which they regulate transcription levels. For example, the CMV promoter is a strong promoter found in human Cytomegalovirus (CMV) and can be widely and highly expressed in most eukaryotic cells.
Viral vector systems in gene transfer vector systems can achieve higher efficiency delivery, but there are often disadvantages of limited vector capacity. Among viral vectors in common use are lentiviral vectors, adenoviral vectors, AAV vectors, etc., wherein AAV vectors are considered one of the most promising gene transfer vectors for their advantages of safety and high infection efficiency, and are widely used in gene therapy and vaccine research worldwide.
The genomic capacity of AAV is about 4.7kb, and if the length of the gene to be delivered is substantially up to this length limit, it will be difficult to deliver using AAV vectors, one solution being to use short length promoters and ployA elements, as much as possible to make room for the loading of the gene.
Therefore, a number of nucleotide sequence segments with certain functions and activities and relatively short overall length, including truncated versions of CMV promoters, such as CMVd1, have been obtained later by artificial engineering; and translation elongation factor fragments such as EFS promoter, etc. Although the promoter fragments of these versions are sufficiently short in length, their promoter transcriptional activity is much lower than that of the commonly used promoters; there is a problem that the expression level of the target gene is insufficient. In general, there still exists a blank region in which both the expression amount is required to be high and the length of the promoter is required to be compressed to some extent.
Disclosure of Invention
In view of this, the present invention provides nucleic acid molecules and their use as promoters. Compared with the existing common short-version promoter, the promoter provided by the invention can improve the expression of the target gene in eukaryotic cells, and has stronger application value under certain scenes with higher requirement on the expression quantity of the target gene but limitation on the length of the promoter.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a nucleic acid molecule having:
(1) Nucleotide sequences shown in any of SEQ ID NO. 1-SEQ ID NO. 4; or (b)
(2) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (1); or (b)
(3) A nucleotide sequence having at least 85% sequence homology with the nucleotide sequence of (1) or (2).
In some embodiments of the invention, the sequence of SEQ ID NO. 1 is: GGTCTTTCATT ATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC.
In some embodiments of the invention, the sequence of SEQ ID NO. 2 is: CCCCCAAGGA CCTGAAATGACCCTGCGCCCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCAAAACAAACTCCCATTGACTCACTCGGCGCGCCAGTCCTCCG.
In some embodiments of the invention, the sequence of SEQ ID NO. 3 is: ACTCCGCCCA GTTCCGCCCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGCCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCACTTTCCCAGTTTACCCTCCCCTCGCAGCCCCGGTTTGACTCACGGCCGGCGCTCCGGGGAGCACGAGGCGAAGGTATCGAAAGCAGCGAGACAGGCGCGAAGGTGCCACCAGATTCGCACGCGGCGGCCCCAGCGCCCAGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTT.
In some embodiments of the invention, the sequence of SEQ ID NO. 4 is: TCTGATGGTTC TCTAGAAACTGCTGAGGGCGGGACCGCATCTGGCCTAAAGACGACGTACTCCAAAAGCTCGAGAGAGCCGGGGCGGTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTG.
In some embodiments of the invention, the size of the nucleic acid molecule described above is no greater than 400bp.
The invention also provides application of the nucleic acid molecule as a promoter.
The invention also provides a transcription element comprising the nucleic acid molecule and a gene of interest.
In some embodiments of the invention, the transcription element further comprises a fluorescent protein.
The invention also provides an expression vector comprising the above nucleic acid molecule or the above transcription element.
In some embodiments of the invention, the expression vector described above includes a viral vector or a non-viral vector.
The invention also provides a host, transformed or transfected with the above expression vector.
In some embodiments of the invention, the host described above comprises a eukaryotic cell.
The invention also provides the application of the nucleic acid molecule, the transcription element, the expression vector or the host in a eukaryotic expression system.
The present invention provides a nucleic acid molecule having:
(1) Nucleotide sequences shown in any of SEQ ID NO. 1-SEQ ID NO. 4; or (b)
(2) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (1); or (b)
(3) A nucleotide sequence having at least 85% sequence homology with the nucleotide sequence of (1) or (2).
The invention obtains four recombinant promoters with shorter length by artificial transformation of CMV promoter, the length of which is basically similar to EFS promoter, but the expression intensity is obviously higher than EFS and CMVd1 promoters. The four recombinant promoters can be widely applied to eukaryotic expression systems, ensure that the target genes are expressed strongly, save more target gene loading space, and are suitable for being widely applied to various viral vectors and non-viral vector systems.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 illustrates a carrier frame;
FIG. 2 is a 24-hour fluorescence observation chart of effect example 1; wherein: grey is a cell picture photographed under the light field of an inverted fluorescent microscope, and is used for observing the growth density, state and the like of cells, white light rings (halation) are arranged at the edges of the cells, and the background is grey black; the green is an inverted fluorescence microscope green channel shooting picture, and is used for observing the expression condition of EGFP green fluorescent protein on a recombinant promoter carrier; the red is an inverted fluorescence microscope red channel shooting picture, and is used for observing the expression condition of mCherry red fluorescent protein on a cotransfection control carrier;
FIG. 3 is a view showing a 48-hour fluorescence observation of effect example 1; wherein: grey is a cell picture taken under the bright field of an inverted fluorescence microscope; the green color is the image taken by the inverted fluorescence microscope green channel; the red color is the red channel of the inverted fluorescence microscope to shoot pictures;
FIG. 4 is a bar graph showing the 24-hour fluorescence positive rate of effect example 1; wherein, green is the ratio of cells expressing green fluorescent protein EGFP in all detected cells; red is the ratio of cells expressing red fluorescent protein mCherry to total cells tested;
FIG. 5 is a bar graph showing the 24-hour average fluorescence intensity of effect example 1; wherein, green is the average fluorescence intensity of green fluorescent protein EGFP; red is the average fluorescence intensity of red fluorescent protein mCherry;
FIG. 6 is a bar graph showing the 48-hour fluorescence positive rate of effect example 1; wherein, green is the ratio of cells expressing green fluorescent protein EGFP in all detected cells; red is the ratio of cells expressing red fluorescent protein mCherry to total cells tested;
FIG. 7 is a bar graph showing the average fluorescence intensity at 48 hours in effect example 1; wherein, green is the average fluorescence intensity of green fluorescent protein EGFP; red is the average fluorescence intensity of red fluorescent protein mCherry;
FIG. 8 is a view showing a 48-hour fluorescence observation of effect example 2; wherein: grey is a cell picture taken under the bright field of an inverted fluorescence microscope; the green color is the image taken by the inverted fluorescence microscope green channel; the red color is the red channel of the inverted fluorescence microscope to shoot pictures;
FIG. 9 is a bar graph showing the 48h fluorescence positive rate of effect example 2; wherein, green is the ratio of cells expressing green fluorescent protein EGFP in all detected cells; red is the ratio of cells expressing red fluorescent protein mCherry to total cells tested;
FIG. 10 is a bar graph showing the average fluorescence intensity at 48 hours in effect example 2; wherein, green is the average fluorescence intensity of green fluorescent protein EGFP; red is the average fluorescence intensity of red fluorescent protein mCherry;
FIG. 11 is a view showing a 48-hour fluorescence observation of effect example 3; wherein: grey is a cell picture taken under the bright field of an inverted fluorescence microscope; the green color is the image taken by the inverted fluorescence microscope green channel; the red color is the red channel of the inverted fluorescence microscope to shoot pictures;
FIG. 12 is a bar graph showing the 48h fluorescence positive rate of effect example 3; wherein, green is the ratio of cells expressing green fluorescent protein EGFP in all detected cells; red is the ratio of cells expressing red fluorescent protein mCherry to total cells tested;
FIG. 13 is a bar graph showing the average fluorescence intensity at 48 hours in effect example 3; wherein, green is the average fluorescence intensity of green fluorescent protein EGFP; red is the average fluorescence intensity of red fluorescent protein mCherry.
Detailed Description
The invention discloses a nucleic acid molecule and application thereof as a promoter.
It should be understood that the expression "one or more of … …" individually includes each of the objects recited after the expression and various combinations of two or more of the recited objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.
The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.
It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.
The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.
In the present invention example 1 and effect examples 1 to 3, all the raw materials and reagents used were commercially available.
The invention is further illustrated by the following examples:
example 1
(1) Multiple recombinant promoters were obtained and experimentally verified for differences in expression intensity from the CMVd1 and EFS promoters.
(2) The upstream is constructed as each recombinant promoter, the downstream is an expression vector of a green fluorescent protein (EGFP) gene, the vector frame is shown in figure 1, and the links are https:// www.vectorbuilder.cn/vector/VB 900138-0425yph. The EFS promoter was replaced by the recombinant promoter by a conventional ligation method.
(3) Each expression vector containing the recombinant promoter is evenly mixed with a vector pAAV [ Exp ] -EF1A > mCherry of WPRE (https:// en. Vectoruilder. Com/vector/VB211103-1345tgg. Html) plasmid for driving and expressing red fluorescent protein by EF1A, and then transferred into 293T, hela and BHK21 eukaryotic cells through a Trans Hi transfection reagent. It can be observed whether the transfection efficiency of each experimental group is substantially uniform by comparing the positive rate of red fluorescence with the expression intensity.
(4) After 24 and 48 hours of transfection, the fluorescent microscope is used for observation and recording, and the expression strength of each recombinant promoter and the control promoter is counted by flow analysis of the corresponding fluorescent expression condition of each recombinant promoter.
Effect example 1
20ng of the recombinant promoter vector obtained in example 1 +60ng of pAAV [ exp ] -EF1A > mCherry:WPRE vector were transfected into 293T cells.
As shown in FIGS. 2 and 3, the cell state and growth density after transfection of plasmids 24h and 48h were normal; the expression conditions of red fluorescence in each group are consistent, so that the transfection expression conditions of the control vector are not obvious, and the cell state differences of each group are not obvious; the green fluorescence brightness of the expression of the four recombinant promoters is obviously stronger than that of the CMV d1 and EFS promoters under the same experimental conditions, which shows that the expression strength of the four recombinant promoters is better than that of the CMV d1 and EFS promoters.
Table 124h stream statistics
Statistical analysis was performed on the difference in fluorescence signal of cells 24h after transfection by flow, and specific results are shown in table 1, fig. 4 and fig. 5. The positive rate of red fluorescent cells and the average fluorescence intensity in each group are consistent, the positive rate of green fluorescent cells of the four recombinant promoters is slightly higher than that of CMV d1 and EFS promoters, and the corresponding average fluorescence intensity is obviously higher than that of CMV d1 and EFS promoters.
Table 248h stream statistics
Statistical analysis was performed on the difference in fluorescence signal of cells after 48h of transfection by flow-through, and specific results are shown in table 2, fig. 6 and fig. 7. The positive rate and average fluorescence intensity of red fluorescent cells in each group are consistent, the positive rate of green fluorescent cells of the four recombinant promoters are higher than those of CMV d1 and EFS promoters, and the corresponding average fluorescence intensity is obviously higher than those of CMV d1 and EFS promoters and consistent with the trend of transfection for 24 hours.
Effect example 2
100ng of the recombinant promoter vector obtained in example 1+50 ng of pAAV [ exp ] -EF1A > mCherry:WPRE vector were transfected into HeLa cells.
As shown in FIG. 8, the cell state and growth density were normal after 48h of plasmid transfection; the expression conditions of red fluorescence in each group are consistent, so that the transfection expression conditions of the control vector are not obvious, and the cell state differences of each group are not obvious; the green fluorescence brightness of the expression of the four recombinant promoters is obviously stronger than that of the CMV d1 and EFS promoters under the same experimental conditions, which shows that the expression strength of the four recombinant promoters is better than that of the CMV d1 and EFS promoters.
Table 348h stream statistics
Statistical analysis was performed on the difference in fluorescence signal of cells after 48h of transfection by flow-through, and specific results are shown in table 3, fig. 9 and fig. 10. The positive rate and average fluorescence intensity of red fluorescent cells in each group are consistent, the positive rate of green fluorescent cells of the four recombinant promoters are higher than those of CMV d1 and EFS promoters, and the corresponding average fluorescence intensity is obviously higher than those of CMV d1 and EFS promoters.
Effect example 3
100ng of the recombinant promoter vector +50ng EF1A>mCherry vector obtained in example 1 transfected BHK21 cells.
As shown in FIG. 11, the cell state and growth density were normal after 48h of plasmid transfection; the expression conditions of red fluorescence in each group are consistent, so that the transfection expression conditions of the control vector are not obvious, and the cell state differences of each group are not obvious; the green fluorescence brightness of the expression of the four recombinant promoters is obviously stronger than that of the CMV d1 and EFS promoters under the same experimental conditions, which shows that the expression strength of the four recombinant promoters is better than that of the CMV d1 and EFS promoters.
Table 448h flow statistics
Statistical analysis was performed on the difference in fluorescence signal of cells after 48h of transfection by flow-through, and specific results are shown in table 4, fig. 12 and fig. 13. The positive rate and average fluorescence intensity of red fluorescent cells in each group are consistent, the positive rate of green fluorescent cells of the four recombinant promoters are higher than those of CMV d1 and EFS promoters, and the corresponding average fluorescence intensity is obviously higher than those of CMV d1 and EFS promoters.
In summary, the different recombinant promoters obtained in example 1 showed higher expression intensity in different cells than CMVd1 and EFS, and can drive the target gene to be expressed better.
The same plasmid cotransfection with the same quality is used as an internal reference to observe whether the transfection and expression states of cells are relatively consistent, and the red fluorescence positive rate and the mean value difference in different groups fluctuate within a smaller range, so that experimental errors are eliminated. The cell pictures of different groups are all photographed under the same condition, the expression intensities of the four recombinant promoters, CMVd1 and EFS obtained in the embodiment 1 can be observed directly through the green fluorescence brightness of the pictures, and then the different fluorescence brightness of the cells are quantitatively analyzed through a flow cytometer, so that more accurate data can be obtained for comparison. In 293T, hela cells and BHK21 cells, the four recombinant promoters obtained in example 1 showed higher expression levels and higher positive rates of green fluorescent protein than CMVd1 and EFS, and were particularly significant in 293T and Hela cells. Four recombinant promoters have the ability to drive green fluorescent protein expression in more cells and this driving effect is stronger than CMVd1, EFS.
In addition, the four recombinant promoters obtained in example 1 are similar in length to EFS, but have higher expression strength than EFS in three common cell lines, and are suitable for being widely applied to eukaryotic expression systems, so that the target genes can be ensured to have stronger expression, and meanwhile, more target gene loading space can be saved, and the recombinant promoters are suitable for being widely applied to various viral vectors and non-viral vector systems.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A nucleic acid molecule characterized by the sequence: the nucleotide sequence is shown as SEQ ID NO. 1-SEQ ID NO. 4.
2. The nucleic acid molecule of claim 1, wherein the size of the nucleic acid molecule is no greater than 400bp.
3. Use of a nucleic acid molecule according to claim 1 or 2 as a promoter.
4. A transcription element comprising the nucleic acid molecule of claim 1 or 2 and a gene of interest.
5. The transcriptional element of claim 4, further comprising a gene encoding a fluorescent protein.
6. Expression vector, characterized in that it comprises a nucleic acid molecule according to claim 1 or 2 or a transcription element according to claim 4 or 5.
7. The expression vector of claim 6, comprising a viral vector or a non-viral vector.
8. A host transformed or transfected with the expression vector of claim 6 or 7.
9. The host of claim 8, comprising a eukaryotic cell.
10. Use of a nucleic acid molecule according to claim 1 or 2, a transcription element according to claim 4 or 5, an expression vector according to claim 6 or 7 or a host according to claim 8 or 9 in a eukaryotic expression system.
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| CN118667810A (en) * | 2024-05-06 | 2024-09-20 | 云舟生物科技(广州)股份有限公司 | 5' UTR element, expression vector and application thereof |
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| CN112553196A (en) * | 2020-11-06 | 2021-03-26 | 中山大学 | Nucleic acid sequences and their use as promoters |
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| WO2024217518A1 (en) | 2024-10-24 |
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