Note: E. coli/C. glutamicum shuttle expression vectors based on the medium copy number plasmid pGA1 and on Ptrc.
基本信息
- 载体名称:
- pEC-XK99E
- 载体抗性:
- Kanamycin
- 载体长度:
- 7017 bp
- 载体类型:
- Shuttle expression vector
- 复制子:
- ori
- 载体来源:
- Kirchner O, Tauch A.
- 感受态:
- DH10B
- 培养温度:
- 37℃
产品信息
质粒编号:V007659
质粒名称:pEC-XK99E
规格:5 μg (冻干粉)
下载资源
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。
确保质粒的关键元件正确，但是我们并不能保证实验效果。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。
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。
参考文献
- Kirchner O, Tauch A. Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J Biotechnol. 2003;104(1-3):287-299. doi:10.1016/s0168-1656(03)00148-2
- Yang C, Peng Z, Yang L, Du B, Guo C, Sui S, Wang J, Li J, Wang J, Li N. Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum. Front Bioeng Biotechnol. 2023 May 30;11:1194511. doi: 10.3389/fbioe.2023.1194511. PMID: 37324439; PMCID: PMC10268032.
pEC-XK99E 质粒 (编号: V007659)序列
LOCUS Exported 7017 bp DNA circular SYN 02-SEP-2024
DEFINITION Shuttle expression vector pEC-XK99E, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7017)
AUTHORS Kirchner O, Tauch A.
TITLE Tools for genetic engineering in the amino acid-producing bacterium
Corynebacterium glutamicum
JOURNAL J. Biotechnol. 104 (1-3), 287-299 (2003)
PUBMED 12948646
REFERENCE 2 (bases 1 to 7017)
AUTHORS Kirchner O, Tauch A.
TITLE Direct Submission
JOURNAL Submitted (15-JAN-2003) Department of Genetics, University of
Bielefeld, Universitaetsstrasse 25, Bielefeld D-33615, Germany
REFERENCE 3 (bases 1 to 7017)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7017)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 7017)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J.
Biotechnol. 104 (1-3), 287-299 (2003)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(15-JAN-2003) Department of Genetics, University of Bielefeld,
Universitaetsstrasse 25, Bielefeld D-33615, Germany"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7017
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 1..57
/label=MCS
/note="pUC18/19 multiple cloning site"
terminator 260..346
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 438..465
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
CDS complement(704..919)
/codon_start=1
/gene="per"
/product="Per"
/label=per
/note="positive effector of replication"
/protein_id="AAO65195.1"
/translation="MDLSDVALESDALDAAVDLKTVIGFFRALDTTDAPASRDWASAAS
DLETLVADLEELADELRARQRQEDAQ"
gene complement(704..919)
/gene="per"
/label=per
CDS complement(1562..3025)
/codon_start=1
/gene="repA"
/product="RepA"
/label=repA
/note="replication protein"
/protein_id="AAO65196.1"
/translation="MTLADPQDTVTASAWKFSADLFDTHPELALRSRGWTAEDRRELLA
HLGRESFQGSKTRDFASAWIKNPDTGETQPKLYRAGSKALTRCQYVALTHAQHAAVIVL
DIDVPSHQAGGKIEHVNPQVYAILEKWARLEKAPAWIGVNPLSGKCQLIWLIDPVYAAA
GKTSPNMRLLAATTEEMTRVFGADQAFSHRLSRWPLHVSDDPTAYKWHCQHDRVDRLAD
LMEIARTMTGSQKPKKYIEQDFSSGRARIEAAQRATAEAKALAILDASLPSALDASGDL
IDGVRVLWTNPERAARDETAFRHALTVGYQLKAAGERLKDAKIIDAYEVAYNVAQAVGA
DGREPDLPAMRDRLTMARRVRGYVAKGQPVVPARRVETQSSRGRKALATMGRRGAATSN
ARRWADPESKYAQETRQRLAEANKRREMTGELLELRVKTAILDARSQSVADPSTRELAG
ELGVSERRIQQVRKALGMEAKRGRPRAEN"
gene complement(1562..3025)
/gene="repA"
/label=repA
CDS 3555..4346
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
rep_origin 4449..5037
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(5223..5363)
/label=bom
/note="basis of mobility region from pBR322"
promoter 5549..5626
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 5627..6706
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter 6941..6970
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 6978..6994
/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)."