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pEX18Tc 质粒 (编号: V006436)

Note: Broad-host-range gene replacement vector. pEX18Tc and its derivatives could be maintained and selected in E. coli using medium supplemented with 10 µg/ml tetracycline at 25–30°C for 36–48h.

pEX18Tc6369 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300SacBsacB promoteroriTrrnB T2 terminatorrrnB T1 terminatorEschericia coli 5S ribosomal RNAM13 fwdMCSM13 revlac operatorlac promoterCAP binding siteoriTcRAmpR promoter
复制序列 NovoBuilder中查看图谱

基本信息

载体名称:
pEX18Tc
载体抗性:
Tetracycline
载体长度:
6369 bp
载体类型:
Cloning vector
复制子:
ori
载体来源:
Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
启动子:
sacB
克隆方法:
restriction endonuclease,
5'引物:
M13F (-47) CGCCAGGGTTTTCCCAGTCACGAC
3'引物:
M13R (-48) AGCGGATAACAATTTCACACAGGA
感受态:
Stbl3
培养温度:
30℃

产品信息

质粒编号:V006436
质粒名称:pEX18Tc
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

  • Huang W, Wilks A. A rapid seamless method for gene knockout in Pseudomonas aeruginosa. BMC Microbiol. 2017 Sep 19;17(1):199. doi: 10.1186/s12866-017-1112-5. PMID: 28927382; PMCID: PMC5606073.

  • Wang Y, Zhang C, Gong T, Zuo Z, Zhao F, Fan X, Yang C, Song C. An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440. J Microbiol Methods. 2015 Jun;113:27-33. doi: 10.1016/j.mimet.2015.03.022. Epub 2015 Mar 28. PMID: 25828098.

pEX18Tc 质粒 (编号: V006436)序列

LOCUS       Exported                6369 bp DNA     circular SYN 02-SEP-2024
DEFINITION  Cloning vector pEX18Tc, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6369)
  AUTHORS   Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
  TITLE     A broad-host-range Flp-FRT recombination system for site-specific 
            excision of chromosomally-located DNA sequences: application for 
            isolation of unmarked Pseudomonas aeruginosa mutants
  JOURNAL   Gene 212 (1), 77-86 (1998)
  PUBMED    9661666
REFERENCE   2  (bases 1 to 6369)
  AUTHORS   Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-FEB-1998) Microbiology, Colorado State University, 
            Fort Collins, CO 80523, USA
REFERENCE   3  (bases 1 to 6369)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6369)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6369)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene"; 
            date: "1998"; volume: "212"; issue: "1"; pages: "77-86"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (10-FEB-1998) Microbiology, Colorado State University, Fort Collins,
            CO 80523, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2899..2921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(212..1630)
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        complement(1631..2076)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     oriT            complement(2450..2559)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(3002..3029)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(3121..3207)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     rRNA            complement(3208..3327)
                     /product="Eschericia coli 5S ribosomal RNA"
     primer_bind     3557..3573
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(3577..3633)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3643..3658)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3666..3682)
                     /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(3690..3720)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3735..3756)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4044..4632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4908..6095)
                     /codon_start=1
                     /label=TcR
                     /note="tetracycline efflux protein"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     promoter        complement(6170..6274)
                     /label=AmpR promoter