Note:
基本信息
- 载体名称:
- pOSIP-KT
- 载体抗性:
- Kanamycin
- 载体长度:
- 6944 bp
- 载体类型:
- Cloning Vectors
- 复制子:
- R6K γ ori
- 载体来源:
- St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE.
- 拷贝数:
- High copy number
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。
pOSIP-KT 质粒 (编号: V012432)序列
LOCUS 40924_33922 6944 bp DNA circular SYN 18-DEC-2018
DEFINITION Chromosomal integration vector pOSIP-KT, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6944)
AUTHORS St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE.
TITLE One-step cloning and chromosomal integration of DNA
JOURNAL ACS Synth Biol 2 (9), 537-541 (2013)
PUBMED 24050148
REFERENCE 2 (bases 1 to 6944)
AUTHORS St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE.
TITLE Direct Submission
JOURNAL Submitted (12-MAY-2013) Molecular and Biomedical Science, The
University of Adelaide, Molecular Life Sciences Building, Adelaide,
South Australia 5005, Australia
REFERENCE 3 (bases 1 to 6944)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6944)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol"; date: "2013"; volume: "2"; issue: "9"; pages: "537-541"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(12-MAY-2013) Molecular and Biomedical Science, The University of
Adelaide, Molecular Life Sciences Building, Adelaide, South
Australia 5005, Australia"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6944
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 142..444
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
rep_origin 477..1065
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 1125..1145
/label=BioBrick suffix
/note="universal suffix for all parts"
terminator 1200..1227
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator complement(1276..1307)
/label=tonB terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"
protein_bind 1384..1417
/label=FRT (minimal)
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
CDS complement(1427..2137)
/label=lambda repressor (ts)
/note="temperature-sensitive variant of the phage lambda
repressor"
RBS 2226..2234
/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 2299..3438
/label=phage phi-21 integrase
/note="integrase from phage phi-21"
CDS 3676..4488
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin 4613..5001
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
protein_bind 5012..5059
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
misc_feature 5026..5059
/note="FRT; Flp recombinase target site"
protein_bind 5139..5638
/label=phage phi-21 attP
/note="attachment site of phage phi-21"
terminator complement(5659..5905)
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
terminator 6225..6311
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 6406..6492
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 6587..6673
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator complement(6771..6814)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"