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pCold TF 质粒 (编号: V008303)

Note: pCold TF is a cold shock protein expression vector. It utilizes the cspA promoter for induction at low temperatures and fuses the target protein to a Trigger Factor (TF) chaperone to enhance solubility and correct folding in E. coli. It includes tags for purification and protease sites for tag removal.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.

pCold TF5769 bp60012001800240030003600420048005400cspA promoterlac operator5'UTRTEE6xHisTrigger FactorHRV 3C sitethrombin siteFactor Xa sitetig3'UTRf1 oriAmpR promoterAmpRoriCAP binding sitelacIlacI promoter
复制序列 NovoBuilder中查看图谱

基本信息

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.
培养温度:
37℃

产品信息

质粒编号:V008303
质粒名称:pCold TF
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，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。

参考文献

  • Sui B, Wang X, Zhao T, Zhen J, Ren H, Liu W, Zhang X, Zhang C. Design, Screening, and Characterization of Engineered Phage Endolysins with Extracellular Antibacterial Activity against Gram-Negative Bacteria. Appl Environ Microbiol. 2023 Jul 26;89(7):e0058123. doi:10.1128/aem.00581-23. Epub 2023 Jun 20. PMID:37338346; PMCID: PMC10370328.

pCold TF 质粒 (编号: V008303)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