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 pCAG-T7pol is an expression vector designed specifically for the T7 RNA polymerase gene.

载体名称:
pCAG-T7pol
载体抗性:
Ampicillin
载体长度:
7457 bp
载体类型:
Mammalian Expression
复制子:
ori
载体来源:
Ian Wickersham Lab
拷贝数:
High Copy
启动子:
CAG
5'引物:
CTCTAGAGCCTCTGCTAACc
3'引物:
CAGAAGTCAGATGCTCAAG
感受态:
DH10B
培养温度:
37℃

pCAG-T7pol 载体图谱

pCAG-T7pol7457 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200CMV enhancerchicken beta-actin promoterchimeric intronpCAG-FT7 RNA polymeraseBglob-pA-Rbeta-globin poly(A) signalM13 revlac operatorlac promoterCAP binding siteSV40 promoterSV40 poly(A) signalL4440oriAmpRAmpR 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。

pCAG-T7pol 载体序列

LOCUS       Exported                7457 bp DNA     circular SYN 31-AUG-2024
DEFINITION  Exported.
ACCESSION   V001259
VERSION     .
KEYWORDS    pCAG-T7pol
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7457)
  TITLE     Genetic Neuroengineering Group Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7457)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7457)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7457)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     CDS             1742..4390
                     /codon_start=1
                     /label=T7 RNA polymerase
                     /note="T7 RNA polymerase from Escherichia phage T7.
                     Accession#: P00573"
                     /translation="MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYE
                     MGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQ
                     FLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFK
                     KNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIE
                     STGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGI
                     TGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANV
                     ITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLE
                     FMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYY
                     WLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFE
                     YAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAK
                     KVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVM
                     TLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVE
                     AMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLG
                     QFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHD
                     SFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLN
                     LRDILESDFAFA"
     primer_bind     complement(4479..4498)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4544..4599
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4598..4617)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(4960..4976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4984..5000)
                     /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)."
     promoter        complement(5008..5038)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5053..5074)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5132..5328
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     polyA_signal    5334..5468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5536..5553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5707..6295)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6469..7326)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7327..7431)
                     /label=AmpR promoter