基本信息
- 载体名称:
- ERM-APEX2
- 载体抗性:
- Ampicillin
- 载体长度:
- 8694 bp
- 载体类型:
- Mammalian Expression, Bacterial Expression, Lentiv
- 复制子:
- ori
- 启动子:
- hPGK
- 5'引物:
- CMV-forward
产品信息
质粒编号:V012475
质粒名称:ERM-APEX2
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，供学术研究使用。
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。
ERM-APEX2 质粒 (编号: V012475)序列
LOCUS 40924_785 8694 bp DNA circular SYN 13-MAY-2021
DEFINITION Soybean APEX2 anchored to cytosolic face of ER membrane with
N-terminal targeting sequence of residues 1-27 of ER-resident P450
oxidase 2C1..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8694)
AUTHORS Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK,
Ting AY
TITLE Directed evolution of APEX2 for electron microscopy and proximity
labeling.
JOURNAL Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
PUBMED 25419960
REFERENCE 2 (bases 1 to 8694)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8694)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8694
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 105..408
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 409..612
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 635..659
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 836..1582
/codon_start=1
/label=APEX2
/note="soybean ascorbate peroxidase, engineered to serve as
a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
YAADEDAFFADYAEAHQKLSELGFADA"
CDS 1583..1624
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
protein_bind complement(1632..1656)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
CDS 1658..1699
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
misc_feature 1741..2329
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 2401..2634
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 2712..2846
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 2873..3008
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(3029..3047)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3057..3073)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3215..3670
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3696..3800
/label=AmpR promoter
CDS 3801..4658
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4832..5420
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5574..5591
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 5708..5729
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5744..5774
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5782..5798
/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)."
primer_bind 5806..5822
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 5843..5861
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 5889..6115
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 6116..6296
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6343..6468
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 6961..7194
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7379..7423
/codon_start=1
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
/translation="KNEQELLELDKWASL"
promoter 7598..8098
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 8110..8505
/codon_start=1
/label=BSD
/note="blasticidin S deaminase"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 8565..8682
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"