Improved promoter and application thereof

A promoter and target technology, applied in the field of genetic engineering, can solve the problem that the cloning vector cannot be cloned, and achieve the effect of avoiding false positive cloning

Active Publication Date: 2018-06-05
GENEWIZ INC SZ
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved in the present invention is to overcome the cloning vectors prepared in the prior art tha...

Method used

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  • Improved promoter and application thereof
  • Improved promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Codon optimized lacZα gene

[0082] Codon-optimized lacZα gene, comprising the following steps:

[0083] The lacZα gene (SEQ ID NO.39) of pUC57 was codon optimized using codon optimization software (Codon optimization software, developed by Suzhou Jinweizhi Biotechnology Co., Ltd.), and the optimized lacZα gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. , the nucleotide sequence is shown in SEQ ID No.39, specifically as follows:

[0084] lacZα基因(SEQ ID NO.39):ATGACCATGCTCGAGCCAAGCTTGCATGCAGGCCTCTGCAGTCGACGGGCCCGGGATCCGATATCTAGATGCATTCGCGAGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG;

[0085] 优化后的lacZα基因(SEQ ID NO.40):ATGACCATGCTGGAACCGAGCCTGCATGCAGGTCTGTGCAGCCGTCGTGCACGCGATCCGATTAGCCGCTGCATTCGCGAAGTGCCGAGCAGCAATAGCCTGGCCG...

Embodiment 2

[0086] Embodiment 2: Construction of high copy cloning vector

[0087] The construction method of high-copy cloning vector comprises the following specific steps:

[0088] 1) adopt the optimized lacZα gene in Example 1 to replace the lacZα gene of pUC57 (kanamycin resistance), specifically as follows:

[0089] (1) Using the pUC57 plasmid with kanamycin resistance as a template and using SEQ ID NO.41-42 as primers to carry out PCR amplification reaction, the specific sequence is as follows:

[0090] SEQ ID NO.41 (forward primer): ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC;

[0091] SEQ ID NO.42 (reverse primer): AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGACACCCGCCAACAC;

[0092] The PCR reaction system is shown in Table 1 below:

[0093] Table 1

[0094]

[0095]

[0096] Wherein, one group is the negative control of sample with water;

[0097] The reaction conditions are shown in Table 2 below:

[0098] Table 2

[0099]

[0100] (2) The PCR reaction ...

Embodiment 3 3

[0146] Example 3 Construction of Three Kinds of Mutant pUC57-lacZ-Mu-2 Plasmids

[0147] The method for pUC57-lacZ-Mu-2 plasmid construction comprises the steps:

[0148] (1) Use the plasmid pUC57-lacZ-Mu-2 constructed in Example 2 as a template, and use F1-del+R1-del, F2-del+R2-del, F3-del+R3-del as primers to perform PCR Amplification reaction, the nucleotide sequences of the primers F1-del, R1-del, F2-del, R2-del, F3-del, R3-del are shown in SEQ ID NO.49-SEQ ID NO.54, details as follows:

[0149] SEQ ID NO.49 (F1-del): ATACGAGCCGGAGAATCAAGTGTAAAGCCTGGGGTGCCTAAT;

[0150] SEQ ID NO. 50 (R1-del): GCTTTACACTTGATTCTCCGGCTCGTATGTTGTGTGGAATTG;

[0151] SEQ ID NO.51 (F2-del): TACGAGCCGGAGATTCAAGTGTAAAGCCTGGGGTGCCTAATG;

[0152] SEQ ID NO.52 (R2-del): GGCTTTACACTTGAATCTCCGGCTCGTATGTTGTGTGGAATTG;

[0153] SEQ ID NO.53 (F3-del): ATACGAGCCGGAGATCAAGTGTAAAGCCTGGGGTGCCTAATG;

[0154] SEQ ID NO. 54 (R4-del): GGCTTTACACTTGATCTCCGGCTCGTATGTTGTGTGGAATTG;

[0155] The PCR reaction sys...

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Abstract

The invention belongs to the field of gene engineering, and relates to an improved promoter and application thereof. The improved promoter is used for mutating nucleotide sequences from a -35 area toa -10 area in a promoter region into recognition sites of endonuclease. The improved promoter can be used for solving the problem that the cloning cannot be achieved, which is caused as a transcription or translation product of an exogenous gene, which is promoted by a strong promoter existing in a vector adopting blue and white screening, possibly has toxicity to a host, can be used for avoidingthe defect that the vector loses 1bp to 2bp at an enzyme digestion site to cause the frame shift mutation of a lacZalpha gene to generate false positive cloning, and can be used for eliminating the false negative phenomenon that a plate all has locus ceruleus, which is caused as an extraneous DNA (Deoxyribonucleic Acid) fragment is smaller and moreover a reading frame of the lacZalpha gene is notchanged by the insertion of extraneous DNA.

Description

technical field [0001] The invention belongs to the field of genetic engineering, relates to an improved promoter and its application, in particular to an improved promoter, a cloning vector with the improved promoter, a host cell with the cloning vector and its application . Background technique [0002] The invention of PCR technology is a major breakthrough in the fields of molecular biology and genetic engineering. After the birth of PCR technology, the technology of cloning PCR products into vectors (usually plasmids) has also been developed. Common and relatively simple cloning methods include TA cloning and blunt-end ligation. The PCR product amplified by Taq enzyme contains dAMP tail, and under the action of T4 ligase, it can be ligated with the carrier (T carrier) containing T-terminus, which is TA clone. High-fidelity DNA polymerases usually contain 3'-5' exonuclease activity, and the PCR products amplified by them are blunt ends. Ligation of these fragments wit...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70C12N15/66C12N1/21C12P21/00C12R1/19
CPCC12N9/2471C12N15/66C12N15/70C12P21/00C12Y302/01023C12N2800/22C12N15/72C12N2830/55
Inventor 薛高旭齐甜铭冯爱华谢正立贾延凯吴昕孙中平廖国娟
Owner GENEWIZ INC SZ
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