质粒编号
质粒名称
规格
价格
V003722
pRK442
5 μg (冻干粉)
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:
- pRK442
- 载体抗性:
- Tetracycline
- 载体长度:
- 9816 bp
- 载体类型:
- Cloning vector
- 复制子:
- oriV
- 载体来源:
- Scott HN, Laible PD, Hanson DK.
pRK442 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。
pRK442 载体序列
LOCUS 40924_37143 9816 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pRK442, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9816)
AUTHORS Scott HN, Laible PD, Hanson DK.
TITLE Sequences of versatile broad-host-range vectors of the RK2 family
JOURNAL Plasmid 50 (1), 74-79 (2003)
PUBMED 12826060
REFERENCE 2 (bases 1 to 9816)
AUTHORS Scott HN, Laible PD, Hanson DK.
TITLE Direct Submission
JOURNAL Submitted (18-DEC-2002) Biosciences Division, Argonne National
Laboratory, 9700 South Cass Ave., Argonne, IL 60439, USA
REFERENCE 3 (bases 1 to 9816)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 9816)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid";
date: "2003"; volume: "50"; issue: "1"; pages: "74-79"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(18-DEC-2002) Biosciences Division, Argonne National Laboratory,
9700 South Cass Ave., Argonne, IL 60439, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9816
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(152..863)
/direction=LEFT
/label=oriV
/note="incP origin of replication"
primer_bind 1373..1389
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1438..1454)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(1462..1478)
/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(1486..1516)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(1531..1552)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(1787..2155)
/codon_start=1
/label=traJ
/note="oriT-recognizing protein"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT complement(2188..2297)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
CDS complement(2981..3628)
/codon_start=1
/label=TetR
/note="tetracycline resistance regulatory protein"
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
CDS 3734..4930
/codon_start=1
/label=TcR
/note="tetracycline efflux protein"
/translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
"
CDS complement(6209..7354)
/codon_start=1
/label=trfA
/note="trans-acting replication protein that binds to and
activates oriV"
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(8081..9175)
/codon_start=1
/gene="incC"
/product="plasmid maintenance protein"
/label=incC
/protein_id="AAO47421.1"
/translation="MGVIHEETAYRKPVPGGDPGAGSGAADHRDSAGRLSRWEATGDVR
NVAGTDQGRSVASGASRVGRVRGQELARGVRAGNGGSAGTSGVHRPEVGSGRQEKTGNQ
TMKTLVTANQKGGVGKTSTLVHLAFDFFERGLRVAVIDLDPQGNASYTLKDFATGLHAS
KLFGAVPAGGWTETAPAAGDGQAARLALIESNPVLANAERLSLDDARELFGANIKALAN
QGFDVCLIDTAPTLGVGLAAALFAADYVLSPIELEAYSIQGIKKMVTTIANVRQKNAKL
QFLGMVPSKVDARNPRHARHQAELLAAYPKMMIPATVGLRSSIADALASGVPVWKIKKT
AARKASKEVRALADYVFTKMEISQ"
gene complement(8081..9175)
/gene="incC"
/label=incC
CDS complement(8857..9162)
/codon_start=1
/gene="korA"
/product="regulation of gene expression"
/label=korA
/protein_id="AAO47422.1"
/translation="MKKRLTESQFQEAIQGLEVGQQTIEIARGVLVDGKPQATFATSLG
LTRGAVSQAVHRVWAAFEDKNLPEGYARVTAVLPEHQAYIVRKWEADAKKKQETKR"
gene complement(8857..9162)
/gene="korA"
/label=korA