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。

The plasmid pKD13 contains the R6Kγ origin of replication (ori). This particular ori requires the λpir protein for initiation of replication.
For this reason, pKD13 cannot replicate in conventional bacterial strains such as DH5α. DH5α lacks the pir+ gene that is essential for expressing the λpir protein required for pKD13 replication.
However, pKD13 can proliferate in bacteria that contain the pir+ gene, such as S17-1λpir or DH5αλpir. These strains are engineered to express the λpir protein, which enables the replication of pKD13.
It provides specificity in genetic manipulation by only replicating in strains with the pir+ gene. This allows for precise engineering tasks and controlled experiments with reduced background noise.

载体名称:
pKD13
载体抗性:
Ampicillin, Kanamycin
载体长度:
3470 bp
载体类型:
Knockout Vectors
复制子:
R6K γ ori
克隆方法:
Enzyme digestion and ligation

pKD13 载体图谱

pKD133470 bp6001200180024003000FRTNeoR/KanRFRT (minimal)lambda tL3 terminatorR6K gamma oriAmpRAmpR promoterrrnB T1 terminatorrrnB T2 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。

pKD13 载体序列

LOCUS       Exported                3470 bp DNA     circular SYN 31-AUG-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3470)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3470)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3470)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2069..2105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(51..98)
                     /label=FLP recombinase from the Saccharomyces
                     cerevisi
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(114..905)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     protein_bind    complement(1273..1306)
                     /label=FRT (minimal)
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     terminator      1386..1632
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     rep_origin      complement(1693..2113)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(2186..3043)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(3044..3148)
                     /label=AmpR promoter
     terminator      3238..3324
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3416..3443
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"