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:
- p451-LII-F3-4
- 载体抗性:
- Streptomycin
- 载体长度:
- 6882 bp
- 载体类型:
- Cloning vector
- 复制子:
- RSF1010 oriV
- 载体来源:
- Chiasson D, Gimenez-Oya V, Bircheneder M, Bachmaier S
- 启动子:
- lac UV5
p451-LII-F3-4 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。
p451-LII-F3-4 载体序列
LOCUS 62056_2180 6882 bp DNA circular SYN 29-JUL-2019
DEFINITION Cloning vector p451 LII F3-4, complete sequence.
ACCESSION MK495774
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6882)
AUTHORS Chiasson D, Gimenez-Oya V, Bircheneder M, Bachmaier S, Studtrucker
T, Ryan J, Sollweck K, Leonhardt H, Boshart M, Dietrich P, Parniske
M.
TITLE A unified multi-kingdom Golden Gate cloning platform
JOURNAL Sci Rep 9 (1), 10131 (2019)
PUBMED 31300661
REFERENCE 2 (bases 1 to 6882)
AUTHORS Chiasson D.
TITLE Direct Submission
JOURNAL Submitted (06-FEB-2019) Biology, LMU Munich, Grosshaderner Str. 2-4,
Martinsried 82152, Germany
REFERENCE 3 (bases 1 to 6882)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep";
date: "2019"; volume: "9"; issue: "1"; pages: "10131"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(06-FEB-2019) Biology, LMU Munich, Grosshaderner Str. 2-4,
Martinsried 82152, Germany"
FEATURES Location/Qualifiers
source 1..6882
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(240..1208)
/codon_start=1
/label=RSF1010 RepB
/note="replication protein B of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
promoter complement(1232..1262)
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
rep_origin 1318..1712
/label=RSF1010 oriV
/note="replication origin of the broad-host-range plasmid
RSF1010; requires the RSF1010 RepA/B/C proteins for
replication (Scholz et al., 1989)"
terminator complement(1750..1836)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
promoter 2053..2083
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 2137..2793
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 3138..3440
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
terminator 3545..3639
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS 3875..4663
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
oriT 4812..4920
/label=oriT
/note="incP origin of transfer"
CDS complement(4934..5782)
/codon_start=1
/label=RSF1010 RepC
/note="replication protein C of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/translation="VVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
TAECHVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
AG"
CDS complement(5772..6608)
/codon_start=1
/label=RSF1010 RepA
/note="replication protein A of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"