质粒编号
质粒名称
规格
价格
V005932
pT3-myr-AKT-HA
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学术研究使用，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。
Note:
- 载体名称:
- pT3-myr-AKT-HA
- 载体抗性:
- Ampicillin
- 载体长度:
- 7091 bp
- 载体类型:
- Mammalian Expression
- 复制子:
- ori
- 启动子:
- EF-1α
- 克隆方法:
- Gateway Cloning
- 5'引物:
- EF-1a-Fwd
- 3'引物:
- pT3EF1a-R 5'-TGACACCTACTCAGACAATG
pT3-myr-AKT-HA 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。
pT3-myr-AKT-HA 载体序列
LOCUS 40924_42264 7091 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7091)
AUTHORS Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, Destefanis G,
Delogu S, Zimmermann A, Ericsson J, Brozzetti S, Staniscia T, Chen
X, Dombrowski F, Evert M
TITLE Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling,
promotes development of human hepatocellular carcinoma.
JOURNAL Gastroenterology. 2011 Mar;140(3):1071-83. Epub 2010 Dec 11.
PUBMED 21147110
REFERENCE 2 (bases 1 to 7091)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7091)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Gastroenterology."; date: "2011-03"; volume: "140(3)"; pages:
"1071-83. Epub 2010 Dec 11"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7091
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..1179
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
promoter 1196..1214
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1293..1317
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 1338..1358
/codon_start=1
/label=myr
/note="N-myristoylation signal from Src kinase (Pellman et
al., 1985; Kaplan et al., 1988)"
/translation="MGSSKSK"
CDS 1371..2810
/codon_start=1
/label=Akt1
/note="human serine/threonine protein kinase activated by
growth factors"
/translation="MNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDV
DQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQ
TVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK
VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR
LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM
LDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDA
KEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM
ECVDSERRPHFPQFSYSASGTA"
CDS 2811..2837
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
protein_bind complement(2857..2881)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
CDS 2934..2975
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
polyA_signal 3019..3243
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 3313..3346
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
primer_bind 3402..3422
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
primer_bind complement(3804..3820)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4029..4048)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4148..4170
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4208..4226)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4294..4398
/label=AmpR promoter
CDS 4399..5256
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5430..6018
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6172..6189
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 6306..6327
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6342..6372
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6380..6396
/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 6404..6420
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6846..6865)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
protein_bind complement(6918..6951)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
promoter 7091
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"