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。
The pEarleyGate 100 is a Gateway-compatible plant transformation vector with no encoded tag.
- 载体名称:
- pEarleyGate 100
- 载体抗性:
- Chloramphenicol
- 载体长度:
- 11649 bp
- 载体类型:
- Plant Vectors
- 复制子:
- ori
- 载体来源:
- Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS.
- 拷贝数:
- High copy number
- 启动子:
- MAS
- 感受态:
- DB3.1
- 培养温度:
- 37℃
pEarleyGate 100 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。
pEarleyGate 100 载体序列
LOCUS 40924_16460 11649 bp DNA circular SYN 01-JAN-1980 DEFINITION Gateway(R)-compatible plant transformation vector with no encoded tag. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11649) AUTHORS Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS. TITLE Gateway-compatible vectors for plant functional genomics and proteomics. JOURNAL Plant J. 2006;45:616-29. PUBMED 16441352 REFERENCE 2 (bases 1 to 11649) AUTHORS Pikaard Lab TITLE Direct Submission REFERENCE 3 (bases 1 to 11649) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Plant J."; date: "2006"; volume: "45"; pages: "616-29" COMMENT SGRef: number: 2; type: "Journal Article" COMMENT The sequence was corrected by inserting a G at position 7757. FEATURES Location/Qualifiers source 1..11649 /mol_type="other DNA" /organism="synthetic DNA construct" misc_feature 1..25 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" terminator complement(111..363) /label=MAS terminator /note="mannopine synthase terminator" CDS complement(376..924) /codon_start=1 /label=BlpR /note="phosphinothricin acetyltransferase" /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF WQLDFSLPVPPRPVLPVTEI" promoter complement(930..1310) /label=MAS promoter /note="mannopine synthase promoter (Velten et al., 1984)" promoter 2306..2651 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" protein_bind 2663..2782 /label=attR1 /note="recombination site for the Gateway(R) LR reaction" promoter 2812..2842 /label=lac UV5 promoter /note="E. coli lac promoter with an 'up' mutation" CDS 2896..3552 /codon_start=1 /label=CmR /note="chloramphenicol acetyltransferase" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" CDS 3897..4199 /codon_start=1 /label=ccdB /note="CcdB, a bacterial toxin that poisons DNA gyrase" /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" protein_bind complement(4243..4367) /label=attR2 /note="recombination site for the Gateway(R) LR reaction" terminator 4420..5127 /label=OCS terminator /note="octopine synthase terminator" misc_feature 5371..5395 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" CDS 6695..7321 /codon_start=1 /label=pVS1 StaA /note="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" CDS 7753..8823 /codon_start=1 /label=pVS1 RepA /note="replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /translation="VSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI LVMRYRNLIEGEASAGS" rep_origin 8892..9086 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" misc_feature 9430..9570 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(9756..10344) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(10434..11225) /codon_start=1 /label=KanR /note="aminoglycoside phosphotransferase" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"