pAV012 载体 (V009391)

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:
pAV012
载体抗性:
Ampicillin
载体长度:
4577 bp
载体类型:
Cloning vector
复制子:
R6K γ ori
载体来源:
White KP, Victorsen A.
启动子:
mPGK

pAV012 载体载体图谱

pAV0124577 bp600120018002400300036004200R6K gamma oriAmpR promoterAmpRsGFP exon 1; 1st exon of superfolder green fluorescent proteinloxPPGK promoterEM7 promoterNeoR/KanRloxPSP6 promotertetracysteine tagStrep-Tag IITEV siteFLAGreverse recombineering primer binding siteoriT

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。

pAV012 载体载体序列

LOCUS       40924_5089        4577 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pAV012, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4577)
  AUTHORS   White KP, Victorsen A.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-JUL-2013) Human Genetics, The University of Chicago, 
            5801 South Ellis Avenue, Chicago, IL 60637, USA
REFERENCE   2  (bases 1 to 4577)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4577)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (15-JUL-2013) Human Genetics, The University of Chicago, 5801 South 
            Ellis Avenue, Chicago, IL 60637, USA"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..414
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     promoter        509..613
                     /label=AmpR promoter
     CDS             614..1471
                     /label=AmpR
                     /note="beta-lactamase"
     misc_feature    1583..1867
                     /note="sGFP exon 1; 1st exon of superfolder green
                     fluorescent protein"
     primer_bind     1583..1604
                     /label=forward recombineering primer binding site
                     /note="forward recombineering primer binding site"
     misc_feature    1941..1974
                     /label=loxP
                     /note="loxP"
     protein_bind    1941..1974
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        1989..2488
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     promoter        2505..2552
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             2571..3371
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
     protein_bind    complement(3507..3540)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3604..3622
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             4109..4126
                     /label=tetracysteine tag
                     /note="tetracysteine peptide that binds biarsenical
                     labeling reagents"
     CDS             4148..4171
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     CDS             4172..4192
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             4235..4258
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     primer_bind     complement(4265..4288)
                     /label=reverse recombineering primer binding site
                     /note="reverse recombineering primer binding site"
     oriT            complement(4334..4443)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"