pGRB2.1 载体 (V005896)

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:
pGRB2.1
载体抗性:
Ampicillin
载体长度:
5299 bp
载体类型:
Cloning vector
复制子:
ori
宿主:
Yeast
载体来源:
Zordan RE, Ren Y, Pan SJ, Rotondo G, Penas Ade L, Iluore J, Cormack BP.
启动子:
URA3

pGRB2.1 载体图谱

pGRB2.15299 bp6001200180024003000360042004800Candida glabrata CEN/ARSAmpR promoterAmpRoriCAP binding sitelac promoterlac operatorM13 revT3 promoterSK primerKS primerregion downstream of HIS3 orf in Candida glabrata BG2T7 promoterM13 fwdf1 oriURA3URA3 promoter

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。

pGRB2.1 载体序列

LOCUS       40924_22553        5299 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pGRB2.1, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5299)
  AUTHORS   Zordan RE, Ren Y, Pan SJ, Rotondo G, Penas Ade L, Iluore J, Cormack 
            BP.
  TITLE     Expression Plasmids for Use in Candida glabrata
  JOURNAL   G3 (Bethesda) 3 (10), 1675-1686 (2013)
  PUBMED    23934995
REFERENCE   2  (bases 1 to 5299)
  AUTHORS   Zordan RE, Cormack BP.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-MAY-2013) Molecular Biology and Genetics, Johns 
            Hopkins School of Medicine, 725 N Wolfe St., Hunterian 609, 
            Baltimore, MD 21205, USA
REFERENCE   3  (bases 1 to 5299)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5299)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "G3 
            (Bethesda)"; date: "2013"; volume: "3"; issue: "10"; pages: 
            "1675-1686"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (14-MAY-2013) Molecular Biology and Genetics, Johns Hopkins School 
            of Medicine, 725 N Wolfe St., Hunterian 609, Baltimore, MD 21205, 
            USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(66..664)
                     /label=Candida glabrata CEN/ARS
                     /note="Candida glabrata CEN/ARS"
     promoter        666..737
                     /label=AmpR promoter
     CDS             738..1595
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1769..2357
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2645..2666
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2681..2711
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2719..2735
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2743..2759
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2780..2798
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2835..2851
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2885..2901)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      2904..3301
                     /note="region downstream of HIS3 orf in Candida glabrata
                     BG2"
                     /regulatory_class="terminator"
     promoter        complement(3316..3334)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3344..3360)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3501..3956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(4090..4890)
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(4891..5106)
                     /label=URA3 promoter