pCBcth4 载体 (V008689)

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:
pCBcth4
载体抗性:
Ampicillin
载体长度:
5905 bp
载体类型:
Cloning vector
复制子:
p15A ori
载体来源:
Zheng T, Cui J, Bae HR, Lynd LR, Olson DG.

pCBcth4 载体图谱

pCBcth45905 bp600120018002400300036004200480054002638AdhAChloramphenicol acetyltransferaseAmpRAmpR promoterp15A orirepB

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。

pCBcth4 载体序列

LOCUS       V008689                 5905 bp    DNA     circular SYN 17-DEC-2018
DEFINITION  Exported.
ACCESSION   V008689
VERSION     V008689
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 5905)
  AUTHORS   Zheng T, Cui J, Bae HR, Lynd LR, Olson DG.
  TITLE     Expression of adhA from different organisms in Clostridium
            thermocellum
  JOURNAL   Biotechnol Biofuels 10, 251 (2017)
   PUBMED   29213311
REFERENCE   2  (bases 1 to 5905)
  AUTHORS   Zheng T.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-SEP-2017) Biology, Dartmouth College, 14 Engineering
            Drive, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 5905)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5905)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName: "Biotechnol
            Biofuels 10, 251 (2017)"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (28-SEP-2017) Biology, Dartmouth College, 14 Engineering Drive,
            Hanover, NH 03755, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1..209
                     /label="2638"
                     /note="2638"
                     /regulatory_class="promoter"
     CDS             210..1430
                     /codon_start=1
                     /gene="adhA"
                     /product="AdhA"
                     /label="adhA"
                     /note="derived from Thermococcus sp. strain AN1"
                     /protein_id="AUE21777.1"
                     /translation="MLWESQIPINQIFELRCRTIDYFGVGAINKFYDIAKDLKENRGIS
                     RVILVTGKSSYKKCGAWDVVKPALEENGIEYVHYDKVGPNPTVDMVDEAAQMGKEFGAQ
                     AVIGIGGGSPIDTAKSVAILLEYTDKTARELYKLQFSPRKAKPIIAINTTHGTGTEVNR
                     FAVASILEEEYKPAIAYDCIYPLYSIDDPTLMTKLPADQTRYVTIDALNHVNEAATTKV
                     ASPYSILLAKETARLIFDYLPEALIHPDNLQAKYYLLYASAIAGISFDNGLLHFTHALE
                     HPLSAVKPDLPHGLGLAILLPAVIKHIYPATARILAEIYRPLVPEVKGVPGEAELVAKK
                     VEEWLFAIGITQKLTDVGFSEDDVKRLTELAMTTPSLNLLLSLAPVEATKETIEAIYRD
                     SLYPLSK"
     gene            210..1430
                     /gene="adhA"
                     /label="adhA"
     CDS             1560..2207
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     CDS             complement(2291..3148)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(3149..3240)
                     /label="AmpR promoter"
     rep_origin      3840..4385
                     /direction=RIGHT
                     /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."
     CDS             4856..5857
                     /label="repB"
                     /note="RepB replication protein"