pP02218-nat-cel8A 载体 (V004191)

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:
pP02218-nat-cel8A
载体抗性:
Ampicillin
载体长度:
6904 bp
载体类型:
Shuttle vector
复制子:
ori
载体来源:
Heinze S, Zimmermann K, Ludwig C, Heinzlmeir S, Schwarz WH, Zverlov VV, Liebl W, Kornberger P.

pP02218-nat-cel8A 载体图谱

pP02218-nat-cel8A6904 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900oriAmpRAmpR promoterTraJoriTKanRrepBP02218 promoter from P. polymyxa DSM292Endoglucanase A6xHis

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。

pP02218-nat-cel8A 载体序列

LOCUS       V004191                 6904 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004191
VERSION     V004191
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6904)
  AUTHORS   Heinze S, Zimmermann K, Ludwig C, Heinzlmeir S, Schwarz WH, Zverlov
            VV, Liebl W, Kornberger P.
  TITLE     Efficient secretory heterologous enzyme production in the novel host
            Paenibacillus polymyxa by usage of its promoter sequences
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6904)
  AUTHORS   Heinze S, Zimmermann K, Ludwig C, Heinzlmeir S, Schwarz WH, Zverlov
            VV, Liebl W, Kornberger P.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-MAY-2018) Department for Microbiology, Technical
            University of Munich, Emil-Ramann-Str. 4, Freising, Bavaria 85354,
            Germany
REFERENCE   3  (bases 1 to 6904)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6904)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (09-MAY-2018) Department for Microbiology, Technical University of
            Munich, Emil-Ramann-Str. 4, Freising, Bavaria 85354, Germany"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(79..667)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(841..1698)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(1699..1803)
                     /label="AmpR promoter"
     CDS             complement(1832..2116)
                     /codon_start=1
                     /gene="traJ"
                     /product="TraJ"
                     /label="traJ"
                     /protein_id="AXF54530.1"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGQQKAHYVNHHPNQVFWGRGAVKH"
     gene            complement(1832..2116)
                     /gene="traJ"
                     /label="traJ"
     oriT            complement(2149..2258)
                     /direction=LEFT
                     /label="oriT"
                     /note="incP origin of transfer"
     CDS             complement(2515..3276)
                     /codon_start=1
                     /gene="kanR"
                     /product="KanR"
                     /label="kanR"
                     /note="Kanamycin Resistance"
                     /protein_id="AXF54531.1"
                     /translation="MNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTD
                     GPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFF
                     SILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTTFLP
                     SLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSDSEK
                     LLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     gene            complement(2515..3276)
                     /gene="kanR"
                     /label="kanR"
     CDS             complement(3448..4449)
                     /label="repB"
                     /note="RepB replication protein"
     regulatory      4866..5277
                     /label="P02218 promoter from P. polymyxa DSM292"
                     /note="P02218 promoter from P. polymyxa DSM292"
                     /regulatory_class="promoter"
     CDS             5308..6717
                     /gene="celA"
                     /label="Endoglucanase A"
                     /note="Endoglucanase A from Acetivibrio thermocellus
                     (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 /
                     NCIMB 10682 / NRRL B-4536 / VPI 7372). Accession#: A3DC29"
     CDS             6724..6741
                     /label="6xHis"
                     /note="6xHis affinity tag"