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。
The complexity of the transformation operation was avoided by the development of pCold TF DNA, a fusion cold shock expression vector with the E. coli chaperone protein Trigger Factor (TF) as a soluble tag.TF has a molecular weight of 48 kDa, and Trigger Factor is a prokaryotic ribosome-binding chaperone protein that promotes co-translation and folding of nascent peptide chains.TF is derived from E. coli and has a high efficiency of expression compared to other biologically-derived tags.TF has a high efficiency of expression compared to other biologically-derived tags. Folding.TF is derived from E. coli and is expressed in E. coli with high efficiency and solubility compared to tags from other biological sources. This vector inserts a 5' non-coding region (5'UTR), TEE (Translation Enhancement Element) sequence, His-Tag sequence, TF sequence, and Multiple Cloning Site (MCS) downstream of the cspA promoter. A lac operator was also inserted downstream of the cspA promoter to tightly regulate protein expression. And cut-off sites for HRV 3C Protease, Thrombin and Factor Xa are inserted between the TF sequence and MCS, which are used to eliminate tags of fusion proteins. pCold TF DNA applies the promoter of E. coli, and like the other pCold DNAs, most of E. coli can be used as an expression host. Using pCold TF DNA, soluble expression of difficult-to-express genes can be achieved through the soluble tagging function of TF and the molecular chaperone effect.
- 载体名称:
- pCold TF
- 载体抗性:
- Ampicillin
- 载体长度:
- 5769 bp
- 载体类型:
- Expression vector
- 复制子:
- ori
- 载体来源:
- Shodai T, Takakura H.
pCold TF 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。
pCold TF 载体序列
LOCUS 40924_12715 5769 bp DNA circular SYN 17-DEC-2018
DEFINITION Expression vector pColdTF DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5769)
AUTHORS Shodai T, Takakura H.
TITLE Expression vector pColdTF
JOURNAL Published Only in Database (2005)
REFERENCE 2 (bases 1 to 5769)
AUTHORS Shodai T, Takakura H.
TITLE Direct Submission
JOURNAL Submitted (17-MAY-2005) Toshihiro Shodai, TAKARA BIO INC.;
Seta3-4-1, OTSU, SHIGA 520-2193, Japan
(E-mail:shiyoudait@takara-bio.co.jp, Tel:81-77-543-7229,
Fax:81-77-543-7238)
REFERENCE 3 (bases 1 to 5769)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5769)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Published
Only in Database (2005)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(17-MAY-2005) Toshihiro Shodai, TAKARA BIO INC."; volume: "
Seta3-4-1, OTSU, SHIGA 520-2193, Japan
(E-mail:shiyoudait@takara-bio.co.jp, Tel:81-77-543-7229, Fax";
pages: "81-77-543-7238"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5769
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 15..81
/label=cspA promoter
/note="promoter of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
protein_bind 84..100
/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)."
5'UTR 122..255
/label=cspA 5'UTR
/note="5'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
CDS 256..270
/label=TEE
/note="translation enhancing element for E. coli (Qing et
al., 2004)"
CDS 271..288
/label=6xHis
/note="6xHis affinity tag"
CDS 289..1584
/label=Trigger Factor
/note="ribosome-associated chaperone from E. coli (Hoffmann
et al., 2010)"
CDS 1594..1617
/label=HRV 3C site
/note="recognition and cleavage site for human rhinovirus
3C and PreScission proteases"
CDS 1627..1644
/label=thrombin site
/note="thrombin recognition and cleavage site"
CDS 1651..1662
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
misc_feature 1663..1722
/gene="tig"
/note="MCS = Multiple cloning sites contain the follow
restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI,
HindIII, SalI, PstI, XbaI"
3'UTR 1730..1874
/label=cspA 3'UTR
/note="3'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
rep_origin complement(2073..2528)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2628..2732
/label=AmpR promoter
CDS 2733..3590
/label=AmpR
/note="beta-lactamase"
rep_origin 3764..4352
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind complement(4490..4511)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(4527..5606)
/label=lacI
/note="lac repressor"
promoter complement(5607..5684)
/label=lacI promoter