Note:
基本信息
- 载体名称:
- pEGFP-2.attB
- 载体抗性:
- Ampicillin
- 载体长度:
- 8865 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 载体来源:
- Bischof J, Bjorklund M, Furger E, Schertel C, Taipale J, Basler K.
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。
pEGFP-2.attB 质粒 (编号: V007601)序列
LOCUS 40924_17129 8865 bp DNA circular SYN 17-DEC-2018
DEFINITION Cloning vector pEGFP-2.attB, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8865)
AUTHORS Bischof J, Bjorklund M, Furger E, Schertel C, Taipale J, Basler K.
TITLE A versatile platform for creating a comprehensive UAS-ORFeome
library in Drosophila
JOURNAL Development 140 (11), 2434-2442 (2013)
PUBMED 23637332
REFERENCE 2 (bases 1 to 8865)
AUTHORS Bischof J, Bjorklund M, Furger E, Schertel C, Taipale J, Basler K.
TITLE Direct Submission
JOURNAL Submitted (13-APR-2013) Institute of Molecular Life Sciences,
University of Zurich, Winterthurerstrasse 190, Zurich 8057,
Switzerland
REFERENCE 3 (bases 1 to 8865)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8865)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Development"; date: "2013"; volume: "140"; issue: "11"; pages:
"2434-2442"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(13-APR-2013) Institute of Molecular Life Sciences, University of
Zurich, Winterthurerstrasse 190, Zurich 8057, Switzerland"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..8865
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 1..4136
/label=white dominant eye marker
/note="white dominant eye marker"
CDS join(479..550,952..1225,1300..1954,2016..2331,2552..2683,
2754..3368)
/codon_start=1
/gene="white"
/product="Drosophila white gene eye color pigment"
/label=mini-white
/note="This ia a modified version of the white gene lacking
part of the first intron."
/translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
protein_bind 4143..4176
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_feature 4189..4258
/label=multiple cloning site
/note="multiple cloning site"
CDS 4264..4980
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
intron 5073..5138
/label=small t intron
/note="SV40 (simian virus 40) small t antigen intron"
CDS 5268..5288
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
polyA_signal 5560..5694
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
protein_bind 5723..5792
/label=attB
/note="attB site for the phi-C31 integrase (Groth et al.,
2000)"
promoter complement(6011..6029)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(6050..6066)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6074..6090)
/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(6098..6128)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6143..6164)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6452..7040)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7214..8071)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8072..8176)
/label=AmpR promoter
rep_origin complement(8202..8657)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 8799..8815
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 8822..8840
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
gene 8865
/label=mini-white
/note="This modified version of the white gene lacks part
of the first intron."