我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
所有的产品都严格仅供科学研究使用，不能应用于临床试验，包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确，但是我们并不能保证实验结果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
The pK18mobsacB vector contains the sacB element. The SacB gene, from Bacillus subtilis and related to sugar metabolism, converts sucrose to levan.
In Escherichia coli lacking genes to process levan, expressing SacB leads to the accumulation of levan and causes bacterial death. Thus, by simply adding 10% sucrose to the medium, E. coli can be screened.
In the CRISPR Cas9 system for gene knockout and targeted insertion in microorganisms, including E. coli, the sacB gene in the Cas9 plasmid helps eliminate the plasmid post-recombination. When sucrose is present, the protein encoded by SacB converts sucrose to fructose, and fructose accumulation is toxic to E. coli. Strains that can lose the plasmid survive under this pressure, while those unable to do so die.
- 载体名称:
- pK18mobsacB
- 载体抗性:
- Kanamycin
- 载体长度:
- 5719 bp
- 载体类型:
- Corynebacterium glutamicum knockout vectors
- 复制子:
- ori
- 载体来源:
- Kvitko BH, Collmer A.
- 启动子:
- sacB
- 克隆方法:
- Enzyme digestion and ligation
- 感受态:
- JM108
- 培养温度:
- 37℃
pK18mobsacB 载体图谱
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态， 要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
pK18mobsacB 载体序列
LOCUS Exported 5719 bp DNA circular SYN 29-AUG-2024
DEFINITION Cloning vector pK18mobsacB, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5719)
AUTHORS Kvitko BH, Collmer A.
TITLE Construction of Pseudomonas syringae pv. tomato DC3000 mutant and
polymutant strains
JOURNAL Methods Mol. Biol. 712, 109-128 (2011)
PUBMED 21359804
REFERENCE 2 (bases 1 to 5719)
AUTHORS Kvitko BH, Collmer A.
TITLE Direct Submission
JOURNAL Submitted (04-NOV-2008) Plant Pathology and Plant-Microbe Biology,
Cornell University, 334 Plant Science, Ithaca, NY 14853, USA
REFERENCE 3 (bases 1 to 5719)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5719)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 5719)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Methods
Mol. Biol. 712, 109-128 (2011)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(04-NOV-2008) Plant Pathology and Plant-Microbe Biology, Cornell
University, 334 Plant Science, Ithaca, NY 14853, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5719
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 346..1137
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter 1186..1631
/label=sacB promoter
/note="sacB promoter and control region"
CDS 1632..3050
/codon_start=1
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
oriT complement(3407..3516)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
promoter complement(3751..3841)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
promoter complement(4035..4125)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
rep_origin 4485..5073
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 5361..5382
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5397..5427
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5435..5451
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 5459..5475
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 5484..5540
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(5544..5560)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"