质粒编号
质粒名称
规格
价格
V018125
pcDNA3-EGFP
5 μg (冻干粉)
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 pcDNA3-EGFP is a mammalian expression plasmid containing the enhanced green fluorescent protein (EGFP) gene driven by the CMV promoter for high-level expression in mammalian cells. It carries ampicillin resistance for bacterial selection and neomycin/G418 resistance for stable selection in eukaryotic cells. This plasmid is widely used as a reporter construct and for creating EGFP fusion proteins.
- 载体名称:
- pcDNA3-EGFP
- 载体抗性:
- Ampicillin
- 载体长度:
- 6159 bp
- 载体类型:
- Mammalian Expression Vectors
- 载体来源:
- Doug Golenbock
- 拷贝数:
- High copy number
- 启动子:
- CMV
- 培养温度:
- 37℃
pcDNA3-EGFP 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。
pcDNA3-EGFP 载体序列
LOCUS Exported 6159 bp DNA circular SYN 14-APR-2025
DEFINITION Mammalian expression vector for cloning your gene fused to EGFP.
ACCESSION .
VERSION .
KEYWORDS pcDNA3-EGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6159)
TITLE Fluorescent Protein Plasmids
REFERENCE 2 (bases 1 to 6159)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6159
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 629..648
/label=T7
/note="T7 promoter, forward primer"
promoter 629..647
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 743..1462
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 743..1459
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(788..809)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1049..1068)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1396..1417
/label=EGFP-C
/note="EGFP, forward primer"
promoter complement(1478..1496)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(1479..1496)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(1516..1533)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1522..1746
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 1792..2220
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(1879..1898)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2089..2110
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2229..2249)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 2234..2563
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 2414..2549
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2476..2495
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 2630..3424
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(2684..2703)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3294..3313
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 3598..3719
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3635..3654)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3689..3708
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(3768..3784)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3768..3784)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3781..3803)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3792..3808
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(3816..3846)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3861..3882
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(3999..4016)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4170..4758)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4250..4269)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4929..5789)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 5552..5571
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5790..5894)
/gene="bla"
/label=AmpR promoter
primer_bind complement(5969..5988)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"