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:
- pPKm-232
- 载体抗性:
- Ampicillin
- 载体长度:
- 9204 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 载体来源:
- Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
- 启动子:
- EF-1α
pPKm-232 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。
pPKm-232 载体序列
LOCUS V004064 9204 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V004064
VERSION V004064
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 9204)
AUTHORS Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao
SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
TITLE Biosynthesis of Orthogonal Molecules Using Ferredoxin and
Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB
Optogenetics
JOURNAL ACS Synth Biol (2018) In press
PUBMED 29301067
REFERENCE 2 (bases 1 to 9204)
AUTHORS Kyriakakis P.
TITLE Direct Submission
JOURNAL Submitted (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive,
PFBH room 251 MC0412, La Jolla, CA 92093, United States of America
REFERENCE 3 (bases 1 to 9204)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 9204)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol (2018) In press"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, PFBH room 251
MC0412, La Jolla, CA 92093, United States of America"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9204
/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"
CDS 1264..1338
/label="COX8 presequence"
/note="mitochondrial presequence of human cytochrome c
oxidase subunit VIII (Rizutto et al., 1989)"
regulatory 1360..1369
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1369..1398
/label="Myc"
/note="Myc (human c-Myc proto-oncogene) epitope tag"
misc_feature 1399..2127
/label="tHO1"
/note="tHO1"
CDS 2137..2193
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 2194..2268
/codon_start=1
/product="mitochondrial presequence of human cytochrome c
oxidase subunit VIII (Rizutto et al., 1989)"
/label="COX8 presequence"
/translation="MSVLTPLLLRGLTGSARRLPVPRAK"
CDS 2296..2319
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
misc_feature 2326..3051
/label="tPCYA"
/note="tPCYA"
regulatory 2760..2769
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
promoter 3089..3418
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
CDS 3428..4024
/label="PuroR"
/note="puromycin N-acetyltransferase"
LTR 4320..4553
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 4710..4934
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
regulatory 4977..4986
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
promoter 5030..5134
/label="AmpR promoter"
CDS 5135..5992
/label="AmpR"
/note="beta-lactamase"
rep_origin 6166..6754
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 6913..7546
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 7593..7718
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 8211..8444
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 8629..8673
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 8822..8863
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 8960..9077
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 9204
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"