Note:
基本信息
- 载体名称:
- PSM1
- 载体抗性:
- Chloramphenicol
- 载体长度:
- 6321 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 载体来源:
- Rahnama M, Forester N, Ariyawansa S, Voisey CR, Johnson LJ, Johnson RD, Fleetwood DJ.
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。
PSM1 质粒 (编号: V003138)序列
LOCUS 40924_40702 6321 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector PSM1, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6321)
AUTHORS Rahnama M, Forester N, Ariyawansa S, Voisey CR, Johnson LJ, Johnson
RD, Fleetwood DJ.
TITLE Efficient targeted mutagenesis in Epichloe festucae using a split
marker system
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6321)
AUTHORS Rahnama M, Fleetwood D.
TITLE Direct Submission
JOURNAL Submitted (27-SEP-2016) School of Biological Sciences, University of
Auckland, 3A Symonds Street, Auckland, Auckland 1010, New Zealand
REFERENCE 3 (bases 1 to 6321)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6321)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(27-SEP-2016) School of Biological Sciences, University of Auckland,
3A Symonds Street, Auckland, Auckland 1010, New Zealand"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6321
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(268..295)
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator complement(387..473)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
CDS complement(513..1124)
/codon_start=1
/gene="hph"
/product="hygromycin B phosphotransferase"
/label=hph
/protein_id="APD72149.1"
/translation="MPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLT"
gene complement(513..1124)
/gene="hph"
/label=hph
intron complement(1133..1248)
/label=gpdA intron
/note="intron from the Aspergillus nidulans
glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al.,
1990)"
primer_bind 2096..2112
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2129..2360
/label=attP1
/note="recombination site for the Gateway(R) BP reaction
(pDONR(TM)221 version)"
CDS complement(2759..3061)
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
CDS complement(3409..4065)
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
promoter complement(4066..4168)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
protein_bind complement(4313..4544)
/label=attP2
/note="recombination site for the Gateway(R) BP reaction
(pDONR(TM)221 version)"
promoter complement(4563..4581)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4586..4602)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
CDS 4715..5521
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin 5671..6259
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"