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。
Bacterial Resistance(s)Chloramphenicol, 25 μg/mL. It could be used as CRISPR/Cas based plant genome editing and gene regulation; used as template for making expression cassette with multiple gRNA target sites
- 载体名称:
- pCBC-DT2T3
- 载体抗性:
- Chloramphenicol
- 载体长度:
- 3528 bp
- 载体类型:
- Plant Expression, CRISPR
- 载体来源:
- Qi-Jun Chen
- 拷贝数:
- High copy number
- 启动子:
- U6
- 感受态:
- DH5alpha
- 培养温度:
- 37℃
pCBC-DT2T3 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。
pCBC-DT2T3 载体序列
LOCUS Exported 3528 bp DNA circular SYN 29-JUN-2024 DEFINITION CRISPR/Cas based plant genome editing and gene regulation; used as template for making expression cassette with multiple gRNA target sites. ACCESSION . VERSION . KEYWORDS pCBC-DT2T3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3528) AUTHORS Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ TITLE A CRISPR/Cas9 toolkit for multiplex genome editing in plants. JOURNAL BMC Plant Biol. 2014 Nov 29;14(1):327. PUBMED 25432517 REFERENCE 2 (bases 1 to 3528) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "BMC Plant Biol."; date: "2014-11-29"; volume: "14(1)"; pages: "327" FEATURES Location/Qualifiers source 1..3528 /mol_type="other DNA" /organism="synthetic DNA construct" misc_RNA 1..76 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(609..627) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(610..627) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(645..661) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(645..661) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(658..680) /label=M13/pUC Reverse /note="In lacZ gene" promoter complement(700..747) /label=EM7 promoter /note="synthetic bacterial promoter " primer_bind complement(758..781) /label=pENTR-F /note="pENTR vectors, forward primer" terminator 789..816 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 908..994 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" rep_origin complement(1159..1747) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1239..1258) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1972..2631) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2546..2565 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS complement(2980..3282) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(3082..3101) /label=ccdB-fwd /note="ccdB gene, forward primer" primer_bind 3405..3427 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3419..3436 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3420..3436 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3443..3462 /label=T7 /note="T7 promoter, forward primer" promoter 3443..3461 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase"