pCGL0609 载体 (V008539)

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:
pCGL0609
载体抗性:
Kanamycin
载体长度:
6398 bp
载体类型:
Shuttle vector
复制子:
ColA ori
载体来源:
Reyes O, Guyonvarch A, Bonamy C, Salti V, David F, Leblon G.

pCGL0609 载体图谱

pCGL06096398 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300SK primerKanRrepBl1p15A orif1 ori

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。

pCGL0609 载体序列

LOCUS       40924_10581        6398 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Shuttle vector pCGL0609, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6398)
  AUTHORS   Reyes O, Guyonvarch A, Bonamy C, Salti V, David F, Leblon G.
  TITLE     'Integron'-bearing vectors: a method suitable for stable chromosomal
            integration in highly restrictive corynebacteria
  JOURNAL   Gene 107 (1), 61-68 (1991)
  PUBMED    1660430
REFERENCE   2  (bases 1 to 6398)
  AUTHORS   Ankri S, Reyes O, Leblon G.
  TITLE     Electrotransformation of highly DNA-restrictive corynebacteria with 
            synthetic DNA
  JOURNAL   Plasmid 35 (1), 62-66 (1996)
  PUBMED    8693028
REFERENCE   3  (bases 1 to 6398)
  AUTHORS   Ben-Samoun K, Leblon G, Reyes O.
  TITLE     Positively regulated expression of the Escherichia coli araBAD 
            promoter in Corynebacterium glutamicum
  JOURNAL   FEMS Microbiol. Lett. 174 (1), 125-130 (1999)
  PUBMED    10234830
REFERENCE   4  (bases 1 to 6398)
  AUTHORS   Ben-Samoun K, Leblon G, Reyes O.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-SEP-1998) Laboratoire de Biologie Moleculaire des 
            Corynebacteries, Institut de Genetique et Microbiologie URA 2225, 
            Universite de Paris XI, Orsay F-91405, France
REFERENCE   5  (bases 1 to 6398)
  TITLE     Direct Submission
REFERENCE   6  (bases 1 to 6398)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene"; 
            date: "1991"; volume: "107"; issue: "1"; pages: "61-68"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Plasmid"; 
            date: "1996"; volume: "35"; issue: "1"; pages: "62-66"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "FEMS 
            Microbiol. Lett."; date: "1999"; volume: "174"; issue: "1"; pages: 
            "125-130"
COMMENT     SGRef: number: 4; type: "Journal Article"; journalName: "Submitted 
            (17-SEP-1998) Laboratoire de Biologie Moleculaire des 
            Corynebacteries, Institut de Genetique et Microbiologie URA 2225, 
            Universite de Paris XI, Orsay F-91405, France"
COMMENT     SGRef: number: 5; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..88
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             675..1466
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     gene            complement(2411..3799)
                     /gene="repBl1"
                     /label=repBl1
     CDS             complement(2495..3700)
                     /codon_start=1
                     /gene="repBl1"
                     /product="replicase"
                     /label=repBl1
                     /note="minimally known pBl1 region able to promote
                     replication"
                     /protein_id="AAD08692.1"
                     /translation="MYKITNSKALAGCHRWRRDEAVAVSWSSNGASQFEGLQNSHSRWG
                     SPLAELEVMGERRIELAIATKNHLAAGGALMMFVGTVRHNRSQSFAQVEAGIKTAYSSM
                     VKTSQWKKERARYGVEHTYSDYEVTDSWANGWHLHRNMLLFLDRPLSDDELKAFEDSMF
                     SRWSAGVVKAGMDAPLREHGVKLDQVSTWGGDAAKMATYLAKGMSQELTGSATKTASKG
                     SYTPFQMLDMLADQSDAGEDMDAVLVARWREYEVGSKNLRSSWSRGAKRALGIDYIDAD
                     VRREMEEELYKLAGLEAPERVESTRVAVALVKPDDWKLIQSDFAVRQYVLDCVDKAKDV
                     AAAQRVANEVLASLGVDSTPCMIVMDDVDLDAVLPTHGDATKRDLNAAVFAGNEQTILR
                     TH"
     rep_origin      complement(5287..5821)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     rep_origin      5889..6317
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"