Note: pHSP-MCS-mGFP (GenBank KP893621; Saidi, Moody & Coates) is a plant heat-shock reporter vector. The soybean Gmhsp17.5 promoter drives stress-inducible expression of genes cloned into the MCS as C-terminal fusions to mGFP5, a folding-enhanced GFP. A 35S terminator, T7/T3 promoters, f1 origin and AmpR complete the pUC/pBluescript-derived backbone for plant stress-response studies.
基本信息
- 载体名称:
- pHSP-MCS-mGFP
- 载体抗性:
- Ampicillin
- 载体长度:
- 4344 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 载体来源:
- Saidi Y, Moody LA, Coates JC.
产品信息
质粒编号:V005485
质粒名称:pHSP-MCS-mGFP
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，供学术研究使用。
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
- Fujita Y, Tohda H, Giga-Hama Y, Takegawa K. Heat shock-inducible expression vectors for use in Schizosaccharomyces pombe. FEMS Yeast Research. 2006;6(6):883-887. doi: 10.1111/j.1567-1364.2006.00093.x
- Hampton RY, Koning A, Wright R, Rine J. In vivo examination of membrane protein localization and degradation with green fluorescent protein. Proceedings of the National Academy of Sciences. 1996;93(2):828-833. doi: 10.1073/pnas.93.2.828.
pHSP-MCS-mGFP 质粒 (编号: V005485)序列
LOCUS 40924_25116 4344 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pHSP-MCS-mGFP, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4344)
AUTHORS Saidi Y, Moody LA, Coates JC.
TITLE Direct Submission
JOURNAL Submitted (07-MAR-2015) Department of Biosciences, University of
Birmingham, Edgbaston, Birmingham B15 2TT, UK
REFERENCE 2 (bases 1 to 4344)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4344)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Submitted
(07-MAR-2015) Department of Biosciences, University of Birmingham,
Edgbaston, Birmingham B15 2TT, UK"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4344
/mol_type="other DNA"
/organism="synthetic DNA construct"
regulatory 21..521
/label=Gmhsp17.5
/note="Gmhsp17.5"
/regulatory_class="promoter"
misc_feature 522..557
/label=MCS
/note="MCS"
CDS 558..1271
/label=mgfp5
/note="GFP with folding enhancement mutations"
regulatory 1281..1475
/label=35S Ter
/note="35S Ter"
/regulatory_class="terminator"
promoter complement(1490..1508)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1518..1534)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin complement(1675..2130)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2157..2261
/label=AmpR promoter
CDS 2262..3119
/label=AmpR
/note="beta-lactamase"
rep_origin 3293..3881
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 4169..4190
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4205..4235
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4243..4259
/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 4267..4283
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 4304..4322
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"