Note:
基本信息
- 载体名称:
- pSEVA238
- 载体抗性:
- Kanamycin
- 载体长度:
- 5081 bp
- 载体类型:
- Cloning vector
- 复制子:
- pBBR1 oriV
- 载体来源:
- Silva-Rocha R, Martinez-Garcia E, Calles B, Chavarria M, Arce-Rodriguez A, de Las Heras A, Paez-Espino AD, Durante-Rodriguez G, Kim J, Nikel PI, Platero R, de Lorenzo V.
- 启动子:
- Pm
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。
pSEVA238 质粒 (编号: V003344)序列
LOCUS 40924_39363 5081 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pSEVA238, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5081)
AUTHORS Silva-Rocha R, Martinez-Garcia E, Calles B, Chavarria M,
Arce-Rodriguez A, de Las Heras A, Paez-Espino AD, Durante-Rodriguez
G, Kim J, Nikel PI, Platero R, de Lorenzo V.
TITLE The Standard European Vector Architecture (SEVA): a coherent
platform for the analysis and deployment of complex prokaryotic
phenotypes
JOURNAL Nucleic Acids Res. 41 (DATABASE ISSUE), D666-D675 (2013)
PUBMED 23180763
REFERENCE 2 (bases 1 to 5081)
AUTHORS Silva-Rocha R, Martinez-Garcia E, de Lorenzo V.
TITLE Direct Submission
JOURNAL Submitted (01-APR-2013) Systems Biology Program, CNB-CSIC, Darwin 3,
Madrid, Madrid 28029, Spain
REFERENCE 3 (bases 1 to 5081)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5081)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res. 41 (DATABASE ISSUE), D666-D675 (2013)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(01-APR-2013) Systems Biology Program, CNB-CSIC, Darwin 3, Madrid,
Madrid 28029, Spain"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..5081
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(22..984)
/codon_start=1
/label=XylS
/note="XylS regulator encoded by the Pseudomonas putida TOL
plasmid pWWO"
/translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
promoter 1884..1964
/label=Pm promoter
/note="The bacterial Pm promoter is activated by XylS in
the presence of benzoate or m-toluate (Marques et al.,
1999)."
misc_feature 2014..2070
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(2079..2102)
/label=F24
/note="F24"
terminator 2113..2207
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS 2339..3151
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
oriT 3321..3429
/label=oriT
/note="incP origin of transfer"
CDS complement(3443..4102)
/codon_start=1
/label=pBBR1 Rep
/note="replication protein for the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica"
/translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
rep_origin complement(4103..4874)
/direction=LEFT
/label=pBBR1 oriV
/note="replication origin of the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica; requires the pBBR1
Rep protein for replication"
terminator complement(4989..5075)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"