pGFP-B-RS 载体 (V010487)

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:
pGFP-B-RS
载体抗性:
Kanamycin
载体长度:
7388 bp
载体类型:
Viral Expression & Packaging Vectors
复制子:
ori
载体来源:
OriGene
拷贝数:
High copy number
启动子:
CMV

pGFP-B-RS 载体图谱

pGFP-B-RS7388 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200U6 promotershRNA siteSV40 promoterBSDLTRorihGH poly(A) signalM13 revTurboGFPT7 promoterCMV promoterCMV enhancerKanRLTRMMLV Psigag (truncated)

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。

pGFP-B-RS 载体序列

LOCUS       pGFP-B-RS.        7388 bp DNA     circular SYN 01-JAN-1980
DEFINITION  HuSH-29 retroviral vector with kanamycin and blasticidin resistance 
            markers and a TurboGFP gene, for expressing an shRNA from the U6 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pGFP-B-RS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7388)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7388)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     The shRNA cassette consists of a 29-bp gene-specific sequence, 
            followed by a 7-bp loop, followed by the reverse complement of the 
            29-bp sequence, followed by a TTTTTT terminator.
FEATURES             Location/Qualifiers
     source          1..7388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        90..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    334..385
                     /label=shRNA site
                     /note="shRNA site"
                     /note="shRNA insertion site"
     promoter        441..649
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     CDS             676..1071
                     /label=BSD
                     /note="blasticidin S deaminase"
     LTR             1153..1746
                     /label=LTR
                     /note="long terminal repeat from Moloney murine leukemia
                     virus"
     rep_origin      complement(2118..2706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(2893..3369)
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     3380..3396
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3490..4185)
                     /label=TurboGFP
                     /note="improved green fluorescent protein from Pontellina 
                     plumata (Evdokimov et al., 2006)"
     promoter        complement(4229..4247)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4273..4476)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(4477..4856)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             complement(5094..5900)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     LTR             6034..6503
                     /label=LTR
                     /note="long terminal repeat from Moloney murine leukemia
                     virus"
     misc_feature    6568..6767
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus
                     (MMLV)"
     CDS             6960..7376
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"