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。
- 载体名称:
- pRK415
- 载体抗性:
- Tetracycline
- 载体长度:
- 10690 bp
- 载体类型:
- Broad host range expression vector
- 复制子:
- oriV
- 载体来源:
- Hulter N, Wackernagel W.
- 启动子:
- lac
- 感受态:
- DH10B
- 培养温度:
- 37℃
pRK415 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。
pRK415 载体序列
LOCUS 40924_37123 10690 bp DNA circular SYN 18-DEC-2018 DEFINITION Broad host range expression vector pRK415, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10690) AUTHORS Hulter N, Wackernagel W. TITLE Double illegitimate recombination events integrate DNA segments through two different mechanisms during natural transformation of Acinetobacter baylyi JOURNAL Mol. Microbiol. 67 (5), 984-995 (2008) PUBMED 18194157 REFERENCE 2 (bases 1 to 10690) AUTHORS Huelter N, Wackernagel W. TITLE Direct Submission JOURNAL Submitted (13-FEB-2007) Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, Carl-von-Ossietzkystrasse 9-11, Oldenburg 26111, Germany REFERENCE 3 (bases 1 to 10690) TITLE Direct Submission REFERENCE 4 (bases 1 to 10690) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Microbiol."; date: "2008"; volume: "67"; issue: "5"; pages: "984-995" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (13-FEB-2007) Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, Carl-von-Ossietzkystrasse 9-11, Oldenburg 26111, Germany" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..10690 /mol_type="other DNA" /organism="synthetic DNA construct" rep_origin complement(71..602) /direction=LEFT /label=oriV /note="incP origin of replication" misc_feature 1239..1295 /label=MCS /note="pUC18/19 multiple cloning site" promoter complement(1356..1386) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(1401..1422) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(1657..2025) /codon_start=1 /label=traJ /note="oriT-recognizing protein" /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE KQDELGKVMMGVVRPRAEP" oriT complement(2058..2167) /direction=LEFT /label=oriT /note="incP origin of transfer" CDS complement(2851..3498) /codon_start=1 /label=TetR /note="tetracycline resistance regulatory protein" /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD" CDS 3604..4800 /codon_start=1 /label=TcR /note="tetracycline efflux protein" /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR " CDS complement(6077..7222) /codon_start=1 /label=trfA /note="trans-acting replication protein that binds to and activates oriV" /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR" rep_origin complement(join(10071..10690,1..2)) /direction=LEFT /label=oriV /note="incP origin of replication"