质粒编号
质粒名称
规格
价格
V004775
pME6032
5 μg
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。
- 载体名称:
- pME6032
- 载体抗性:
- Tetracycline
- 载体长度:
- 9816 bp
- 载体类型:
- Shuttle vector
- 复制子:
- p15A ori
- 载体来源:
- Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U, O'Gara F, Haas D.
pME6032 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。
pME6032 载体序列
LOCUS 40924_30560 9816 bp DNA circular SYN 18-DEC-2018
DEFINITION Shuttle vector pME6032, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9816)
AUTHORS Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U,
O'Gara F, Haas D.
TITLE Small, stable shuttle vectors based on the minimal pVS1 replicon for
use in gram-negative, plant-associated bacteria
JOURNAL Mol. Plant Microbe Interact. 13 (2), 232-237 (2000)
PUBMED 10659714
REFERENCE 2 (bases 1 to 9816)
AUTHORS Heeb S, Blumer C, Haas D.
TITLE Regulatory RNA as mediator in GacA/RsmA-dependent global control of
exoproduct formation in Pseudomonas fluorescens CHA0
JOURNAL J. Bacteriol. 184 (4), 1046-1056 (2002)
PUBMED 11807065
REFERENCE 3 (bases 1 to 9816)
AUTHORS Heeb S.
TITLE Direct Submission
JOURNAL Submitted (19-MAY-2006) Institute of Infection, Immunity and
Inflammation, University of Nottingham, Centre for Biomolecular
Sciences, Nottingham NG7 2RD, United Kingdom
REFERENCE 4 (bases 1 to 9816)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 9816)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Plant
Microbe Interact."; date: "2000"; volume: "13"; issue: "2"; pages:
"232-237"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "J.
Bacteriol."; date: "2002"; volume: "184"; issue: "4"; pages:
"1046-1056"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(19-MAY-2006) Institute of Infection, Immunity and Inflammation,
University of Nottingham, Centre for Biomolecular Sciences,
Nottingham NG7 2RD, United Kingdom"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9816
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 195..881
/codon_start=1
/product="pVS1 resolvase"
/label=pVS1 resolvase
/note="ORF1; not required for stable maintenance"
/protein_id="ABG33931.1"
/translation="MNKSAAAGLLGYARVSTDDQDLTNQRAELHAAGCTKLFSEKITGT
RRDRPELARMLDHLRPGDVVTVTRLDRLARSTRDLLDIAERIQEAGAGLRSLAEPWADT
TTPAGRMVLTVFAGIAEFERSLIIDRTRSGREAAKARGVKFGPRPTLTPAQIAHARELI
DQEGRTVKEAAALLGVHRSTLYRALERSEEVTPTEARRRGAFREDALTEADALAAAENE
RQEEQA"
CDS 878..1093
/codon_start=1
/product="hypothetical protein"
/label=hypothetical protein
/note="ORF2; not required for stable maintenance"
/protein_id="ABG33932.1"
/translation="MKPHQDGQDEPFFITEEIEAEMIAAGYVFEPPAHVSTVRLHEILA
GLSDAKLAAWPASLAAEETERRRLKR"
CDS 1180..1806
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
CDS 1830..2045
/codon_start=1
/product="ParG"
/label=ParG
/note="pVS1 partitioning protein"
/protein_id="ABG33934.1"
/translation="MSKSTNTLSAGRPSARSSKAATLASLADTPAMKRVNFQLPAEDHT
KLKMYAVRQGKTITELLSEYIAQLPE"
CDS 2238..3308
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
rep_origin 3377..3571
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
rep_origin 4281..4826
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin."
oriT 5068..5089
/label=p15A origin of transfer
/note="p15A origin of transfer"
regulatory 5148..5414
/label=T4 transcription terminator
/note="T4 transcription terminator"
/regulatory_class="terminator"
misc_feature 5402..5421
/note="sequencing oligonucleotide P6032:
5'-CCCTCACTGATCCGCTAGTC-3'"
misc_feature 5416..5473
/label=multiple cloning site
/note="multiple cloning site"
regulatory complement(5478..5481)
/regulatory_class="ribosome_binding_site"
protein_bind complement(5490..5506)
/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)."
misc_feature complement(5509)
/label=start of transcription
/note="start of transcription"
promoter complement(5514..5542)
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
promoter 5776..5853
/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 5977..6792
/codon_start=1
/product="LacIQ repressor"
/label=LacIQ repressor
/note="tac promoter repressor"
/protein_id="ABG33936.1"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSR
ADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPA
LFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY
LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLR
VGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTTWTACCNSLRARR"
CDS complement(7824..8471)
/label=TetR
/note="tetracycline resistance regulatory protein"
CDS 8577..9773
/label=TcR
/note="tetracycline efflux protein"