scAAV-CMV-GFP 载体 (V001596)

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

scAAV-CMV-GFP5899 bp60012001800240030003600420048005400AAV2 terminal repeatbGH poly(A) signalSV40 poly(A) signalEGFPSV40 intronCMV promoterCMV enhancerAAV2 terminal repeatM13 oriAmpR promoterAmpRori

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"