pSC101-PhaC-pct 载体 (V015353)

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:
pSC101-PhaC-pct
载体抗性:
Ampicillin
载体长度:
7358 bp
载体类型:
Cloning vector
复制子:
pSC101 ori
载体来源:
Guo L.
启动子:
tetR/tetAs

pSC101-PhaC-pct 载体图谱

pSC101-PhaC-pct7358 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200T3Te terminatorTetRtetR/tetA promotersphaCpcttonB terminatorlambda t0 terminatorpSC101 oriRep101AmpRhis operon terminator

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。

pSC101-PhaC-pct 载体序列

LOCUS       62056_19380        7358 bp DNA     circular SYN 16-MAY-2020
DEFINITION  Cloning vector pSC101-PhaC-pct, complete sequence.
ACCESSION   MN623110
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7358)
  AUTHORS   Guo L.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-OCT-2019) Jiangnan University, State Key Laboratory of
            Food Science and Technology, 1800 Lihu Avenue, Wuxi, Jiangsu 214122,
            China
REFERENCE   2  (bases 1 to 7358)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (28-OCT-2019) Jiangnan University, State Key Laboratory of Food 
            Science and Technology, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, 
            China"
FEATURES             Location/Qualifiers
     source          1..7358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(100..129)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(185..808)
                     /label=TetR
                     /note="tetracycline repressor TetR"
     promoter        824..879
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     gene            909..2588
                     /gene="phaC"
                     /label=phaC
     CDS             909..2588
                     /codon_start=1
                     /transl_table=11
                     /gene="phaC"
                     /product="polyhydroxyalkanoate synthase"
                     /label=phaC
                     /protein_id="QJR97783.1"
                     /translation="MSNKNSDDLNRQASENTLGLNPVIGLRGKDLLTSARMVLTQAIKQ
                     PIHSVKHVAHFGIELKNVMFGKSKLQPESDDRRFNDPAWSQNPLYKRYLQTYLAWRKEL
                     HDWIGNSKLSEQDINRAHFVITLMTEAMAPTNSAANPAAVKRFFETGGKSLLDGLTHLA
                     KDLVNNGGMPSQVDMGAFEVGKSLGTTEGAVVFRNDVLELIQYRPTTEQVHERPLLVVP
                     PQINKFYVFDLSPDKSLARFCLSNNQQTFIVSWRNPTKAQREWGLSTYIDALKEAVDVV
                     SAITGSKDINMLGACSGGITCTALLGHYAALGEKKVNALTLLVTVLDTTLDSQVALFVD
                     EKTLEAAKRHSYQAGVLEGRDMAKVFAWMRPNDLIWNYWVNNYLLGNEPPVFDILFWNN
                     DTTRLPAAFHGDLIEMFKNNPLVRANALEVSGTPIDLKQVTADIYSLAGTNDHITPWKS
                     CYKSAQLFGGKVEFVLSSSGHIKSILNPPGNPKSRYMTSTDMPATANEWQENSTKHTDS
                     WWLHWQAWQAERSGKLKKSPTSLGNKAYPSGEAAPGTYVHER"
     gene            2616..4169
                     /gene="pct"
                     /label=pct
     CDS             2616..4169
                     /codon_start=1
                     /transl_table=11
                     /gene="pct"
                     /product="propionyl-CoA transferase"
                     /label=pct
                     /protein_id="QJR97784.1"
                     /translation="MRKVEIITAEQAAQLVKDNDTITSIGFVSSAHPEALTKALEKRFL
                     DTNTPQNLTYIYAGSQDKRDGRAAEHLAHTGLLKRAIIGHWQTVPAIGKLAVENKIEAY
                     NFSQGTLVHWFRALAGHKLGVFTDIGLETFLDPRQLGGKLNDVTKEDLVKLIEVDGHEQ
                     LFYPTFPVNVAFLRGTYADESGNITMDEEIGPFESTSVAQAVHNCGGKVVVQVKDVVAH
                     GSLDPRMVKIPGIYVDYVVVAAPEDHQQTYDCEYDPSLSGEHRAPEGATDAALPMSAKK
                     IIGRRGALELTENAVVNLGVGAPEYVASVAGEEGIADTITLTVEGGAIGGVPQGGARFG
                     SSRNADAIIDHTYQFDFYDGGGLDIAYLGLAQCDGSGNINVSKFGTNVAGCGGFPNISQ
                     QTPNVYFCGTFTAGGLKIAVEDGKVKILQEGKAKKFIKAVDQITFNGSYAARNGKHVLY
                     ITERCVFELTKEGLKLIEVAPGIDIEKDILAHMDFKPIIDNPKLMDARLFQDGPMGLKK
                     "
     terminator      4217..4248
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription
                     terminator"
     terminator      4373..4467
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4959..5181
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5229..6176
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
     CDS             6468..7325
                     /label=AmpR
                     /note="beta-lactamase"
     terminator      7339..7358
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E.
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."