Note: pCAG-T7pol is an eukaryotic expression vector capable of expressing T7 RNA polymerase within host cells. This enzyme drives the transcription of viral cDNA into viral RNA, providing crucial support for the rescue of recombinant Hazaranero virus.
基本信息
- 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℃
产品信息
质粒编号:V001259
质粒名称:pCAG-T7pol
规格:5 μg (冻干粉)
下载资源
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。
确保质粒的关键元件正确，但是我们并不能保证实验效果。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。
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 质粒 (编号: V001259)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JOURNAL Unpublished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"Unpublished"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