质粒编号
质粒名称
规格
价格
V001326
pBOB-EF1-FastFUCCI-Puro
5 μg
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。
pBOB - EF1 - FastFUCCI - Puro is a third-generation lentiviral vector that encodes the FUCCI reporter gene for cell cycle analysis and puro for selection.
- 载体名称:
- pBOB-EF1-FastFUCCI-Puro
- 载体抗性:
- Ampicillin
- 载体长度:
- 12352 bp
- 载体类型:
- Mammalian Expression, Lentiviral
- 复制子:
- ori
- 筛选标记:
- Puromycin
- 拷贝数:
- High Copy
- 启动子:
- EF-1α
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- ggcattctgcacgcttca
- 感受态:
- stbl3
pBOB-EF1-FastFUCCI-Puro 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。
pBOB-EF1-FastFUCCI-Puro 载体序列
LOCUS V001326 12352 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001326
VERSION V001326
KEYWORDS pBOB-EF1-FastFUCCI-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12352)
AUTHORS Koh SB, Mascalchi P, Rodriguez E, Lin Y, Jodrell DI, Richards FM,
Lyons SK
TITLE Quantitative FastFUCCI assay defines cell cycle dynamics at
single-cell level.
JOURNAL J Cell Sci. 2016 Nov 25. pii: jcs.195164.
PUBMED 27888217
REFERENCE 2 (bases 1 to 12352)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12352)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J Cell Sci.
2016 Nov 25. pii: jcs.195164."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12352
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 172..551
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 552..754
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 769..949
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 996..1121
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1618..1851
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2036..2080
/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 2229..2270
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2332..2448
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2465..3643
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3664..4314
/label="mKO2"
/note="monomeric Kusabira-Orange 2 fluorescent protein"
CDS 4369..4641
/label="Cdt1 (30-120)"
/note="degron consisting of residues 30-120 of human Cdt1
(Zielke and Edgar, 2015)"
CDS 4648..4701
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 4708..5385
/codon_start=1
/product="monomeric Azami-Green fluorescent protein
(Karasawa et al., 2003)"
/label="mAzami-Green"
/translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
SQAK"
CDS 5416..5745
/label="Gem1 (1-110)"
/note="degron consisting of residues 1-110 of human Geminin
(Zielke and Edgar, 2015)"
promoter 5788..6287
/label="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 6331..6927
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 7092..7680
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 8091..8271
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 8303..8527
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 8573..9001
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 9015..9344
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 9392..9439
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 9458..9829
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 9962..10083
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(9999..10018)
/label="SV40pA-R"
/note="SV40 polyA, reverse primer"
primer_bind 10053..10072
/label="EBV-rev"
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(10132..10148)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10132..10148)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(10145..10167)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 10156..10172
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(10180..10210)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(10225..10246)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(10363..10380)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(10534..11122)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11296..12153)
/label="AmpR"
/note="beta-lactamase"
promoter complement(12154..12258)
/label="AmpR promoter"
primer_bind complement(12333..12352)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"