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

pRK41510690 bp5001000150020002500300035004000450050005500600065007000750080008500900095001000010500oriVMCSlac promoterCAP binding sitetraJoriTTetRTcRtrfAoriV

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"