基本信息
- 载体名称:
- pYT3
- 载体抗性:
- Ampicillin
- 载体长度:
- 7596 bp
- 载体类型:
- Shuttle vector
- 复制子:
- ori
- 宿主:
- Yeast
- 载体来源:
- Tan X, Waterham HR, Veenhuis M, Cregg JM.
- 启动子:
- LEU2
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。
pYT3 质粒 (编号: V001867)序列
LOCUS 40924_48278 7596 bp DNA circular SYN 18-DEC-2018
DEFINITION Shuttle vector pYT3, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7596)
AUTHORS Tan X, Waterham HR, Veenhuis M, Cregg JM.
TITLE The Hansenula polymorpha PER8 gene encodes a novel peroxisomal
integral membrane protein involved in proliferation
JOURNAL J. Cell Biol. 128 (3), 307-319 (1995)
PUBMED 7844145
REFERENCE 2 (bases 1 to 7596)
AUTHORS Baerends RJS., van Dijk R, Cregg JM, Veenhuis M.
TITLE Direct Submission
JOURNAL Submitted (08-FEB-2001) Eukaryotic Microbiology, Groningen
Biomolecular Sciences and Biotechnology Institute, Kerklaan 30,
Haren, Gn 9751 NN, the Netherlands
REFERENCE 3 (bases 1 to 7596)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7596)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Cell
Biol."; date: "1995"; volume: "128"; issue: "3"; pages: "307-319"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(08-FEB-2001) Eukaryotic Microbiology, Groningen Biomolecular
Sciences and Biotechnology Institute, Kerklaan 30, Haren, Gn 9751
NN, the Netherlands"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7596
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 28..2724
/note="derived from Hansenula polymorpha; confers
autonomous replication activity (HARS) in Hansenula
polymorpha"
RBS 1139..1147
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS complement(3218..4309)
/label=LEU2
/note="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
promoter complement(4310..4717)
/label=LEU2 promoter
primer_bind complement(4989..5005)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(5013..5029)
/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)."
promoter complement(5037..5067)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5082..5103)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5391..5979)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6153..7010)
/label=AmpR
/note="beta-lactamase"
promoter complement(7011..7115)
/label=AmpR promoter