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:
- scAAV-CMV-GFP
- 载体抗性:
- Ampicillin
- 载体长度:
- 5899 bp
- 载体类型:
- Insertion vector
- 复制子:
- ori
- 载体来源:
- Rosas LE, Grieves JL, Zaraspe K, La Perle KM, Fu H, McCarty DM.
- 启动子:
- CMV
scAAV-CMV-GFP 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。
scAAV-CMV-GFP 载体序列
LOCUS 40924_48768 5899 bp DNA circular SYN 18-DEC-2018 DEFINITION Insertion vector scAAV-CMV-GFP, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5899) AUTHORS Rosas LE, Grieves JL, Zaraspe K, La Perle KM, Fu H, McCarty DM. TITLE Patterns of scAAV Vector Insertion Associated With Oncogenic Events in a Mouse Model for Genotoxicity JOURNAL Mol. Ther. (2012) In press PUBMED 22990674 REFERENCE 2 (bases 1 to 5899) AUTHORS Rosas LE, Grieves JL, Zaraspe K, La Perle KMD., Fu H, McCarty DM. TITLE Direct Submission JOURNAL Submitted (01-AUG-2012) Pediatrics/Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, 700 Children's Dr, Columbus, OH 43205, USA REFERENCE 3 (bases 1 to 5899) TITLE Direct Submission REFERENCE 4 (bases 1 to 5899) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Ther. (2012) In press" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (01-AUG-2012) Pediatrics/Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, 700 Children's Dr, Columbus, OH 43205, USA" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..5899 /mol_type="other DNA" /organism="synthetic DNA construct" repeat_region 1..112 /label=AAV2 terminal repeat /note="AAV2 terminal repeat" polyA_signal complement(202..313) /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 405..539 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" CDS complement(555..1271) /codon_start=1 /label=EGFP /note="enhanced GFP" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" intron complement(1348..1444) /label=SV40 intron /note="modified SV40 intron with splice donor and acceptor sites" promoter complement(1582..1785) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" enhancer complement(1786..2069) /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" repeat_region 2136..2272 /label=AAV2 terminal repeat /note="AAV2 terminal repeat" rep_origin 2683..3196 /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 3942..4046 /label=AmpR promoter CDS 4047..4904 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 5078..5666 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"