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
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ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7457) TITLE Genetic Neuroengineering Group Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 7457) TITLE Direct Submission REFERENCE 3 (bases 1 to 7457) TITLE Direct Submission REFERENCE 4 (bases 1 to 7457) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Unpublished" COMMENT SGRef: number: 2; type: "Journal Article" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..7457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