质粒编号
质粒名称
规格
价格
V012538
pBR322
5 μg (冻干粉)
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学术研究使用，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。
Note:pBR322 is a 4,361 bp E. coli cloning vector containing ampicillin and tetracycline resistance genes. It features a pMB1 origin for high-copy replication and allows recombinant screening via antibiotic resistance loss when DNA is inserted into specific restriction sites.
- 载体名称:
- pBR322
- 载体抗性:
- Ampicillin
- 载体长度:
- 4361 bp
- 载体类型:
- Cloning Vectors
- 复制子:
- ori
- 载体来源:
- New England Biolabs
- 拷贝数:
- High copy number
- 启动子:
- tet
pBR322 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。
pBR322 载体序列
LOCUS ampicillin_resis 4361 bp DNA circular SYN 17-DEC-2018
DEFINITION Cloning vector pBR322, complete sequence.
ACCESSION .
VERSION .
KEYWORDS ampicillin resistance; beta-lactamase; cloning vector; drug re.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4361)
AUTHORS Sutcliffe JG.
TITLE Nucleotide sequence of the ampicillin resistance gene of Escherichia
coli plasmid pBR322
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 75 (8), 3737-3741 (1978)
PUBMED 358200
REFERENCE 2 (bases 1892 to 1893)
AUTHORS Sutcliffe JG.
TITLE Complete nucleotide sequence of the Escherichia coli plasmid pBR322
JOURNAL Cold Spring Harb. Symp. Quant. Biol. 43 (Pt 1), 77-90 (1979)
PUBMED 383387
REFERENCE 3 (bases 1 to 4361)
AUTHORS Reed RR, Young RA, Steitz JA, Grindley ND, Guyer MS.
TITLE Transposition of the Escherichia coli insertion element gamma
generates a five-base-pair repeat
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 76 (10), 4882-4886 (1979)
PUBMED 388421
REFERENCE 4 (bases 1 to 4361)
AUTHORS Covarrubias L, Cervantes L, Covarrubias A, Soberon X, Vichido I,
Blanco A, Kupersztoch-Portnoy YM, Bolivar F.
TITLE Construction and characterization of new cloning vehicles. V.
Mobilization and coding properties of pBR322 and several deletion
derivatives including pBR327 and pBR328
JOURNAL Gene 13 (1), 25-35 (1981)
PUBMED 6263753
REFERENCE 5 (bases 2351 to 2414)
AUTHORS Marians KJ, Soeller W, Zipursky SL.
TITLE Maximal limits of the Escherichia coli replication factor Y effector
site sequences in pBR322 DNA
JOURNAL J. Biol. Chem. 257 (10), 5656-5662 (1982)
PUBMED 6279609
REFERENCE 6 (bases 1 to 4361)
AUTHORS Brosius J, Cate RL, Perlmutter AP.
TITLE Precise location of two promoters for the beta-lactamase gene of
pBR322. S1 mapping of ribonucleic acid isolated from Escherichia
coli or synthesized in vitro
JOURNAL J. Biol. Chem. 257 (15), 9205-9210 (1982)
PUBMED 6178738
REFERENCE 7 (bases 1 to 4361)
AUTHORS Van Dyke MW, Hertzberg RP, Dervan PB.
TITLE Map of distamycin, netropsin, and actinomycin binding sites on
heterogeneous DNA: DNA cleavage-inhibition patterns with
methidiumpropyl-EDTA.Fe(II)
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 79 (18), 5470-5474 (1982)
PUBMED 6291045
REFERENCE 8 (bases 1 to 4361)
AUTHORS Peden KW, Nathans D.
TITLE Local mutagenesis within deletion loops of DNA heteroduplexes
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 79 (23), 7214-7217 (1982)
PUBMED 6760191
REFERENCE 9 (bases 1 to 4361)
AUTHORS Peden KW.
TITLE Revised sequence of the tetracycline-resistance gene of pBR322
JOURNAL Gene 22 (2-3), 277-280 (1983)
PUBMED 6307828
REFERENCE 10 (bases 1 to 4361)
AUTHORS Watabe H, Iino T, Kaneko T, Shibata T, Ando T.
TITLE A new class of site-specific endodeoxyribonucleases. Endo.Sce I
isolated from a eukaryote, Saccharomyces cerevisiae
JOURNAL J. Biol. Chem. 258 (8), 4663-4665 (1983)
PUBMED 6300094
REFERENCE 11 (bases 1 to 4361)
AUTHORS Livneh Z.
TITLE Directed mutagenesis method for analysis of mutagen specificity:
application to ultraviolet-induced mutagenesis
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 80 (1), 237-241 (1983)
PUBMED 6337373
REFERENCE 12 (bases 1 to 4361)
AUTHORS Mascharak PK, Sugiura Y, Kuwahara J, Suzuki T, Lippard SJ.
TITLE Alteration and activation of sequence-specific cleavage of DNA by
bleomycin in the presence of the antitumor drug
cis-diamminedichloroplatinum(II)
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 80 (22), 6795-6798 (1983)
PUBMED 6196777
REFERENCE 13 (bases 1 to 4361)
AUTHORS Schultz PG, Dervan PB.
TITLE Sequence-specific double-strand cleavage of DNA by
penta-N-methylpyrrolecarboxamide-EDTA X Fe(II)
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 80 (22), 6834-6837 (1983)
PUBMED 6417654
REFERENCE 14 (bases 1 to 4361)
AUTHORS Sutcliffe JG.
JOURNAL Unpublished
REFERENCE 15 (bases 1 to 4361)
AUTHORS Fuller RS, Funnell BE, Kornberg A.
TITLE The dnaA protein complex with the E. coli chromosomal replication
origin (oriC) and other DNA sites
JOURNAL Cell 38 (3), 889-900 (1984)
PUBMED 6091903
REFERENCE 16 (bases 1 to 4361)
AUTHORS Lathe R, Kieny MP, Skory S, Lecocq JP.
TITLE Linker tailing: unphosphorylated linker oligonucleotides for joining
DNA termini
JOURNAL DNA 3 (2), 173-182 (1984)
PUBMED 6327214
REFERENCE 17 (bases 1 to 4361)
AUTHORS Heusterspreute M, Davison J.
TITLE Restriction site bank vectors. II. DNA sequence analysis of plasmid
pJRD158
JOURNAL DNA 3 (3), 259-268 (1984)
PUBMED 6086259
REFERENCE 18 (bases 1 to 4361)
AUTHORS Abarzua P, Soeller W, Marians KJ.
TITLE Mutational analysis of primosome assembly sites. I. Distinct classes
of mutants in the pBR322 Escherichia coli factor Y DNA effector
sequences
JOURNAL J. Biol. Chem. 259 (22), 14286-14292 (1984)
PUBMED 6209275
REFERENCE 19 (bases 1 to 4361)
AUTHORS Soeller W, Abarzua P, Marians KJ.
TITLE Mutational analysis of primosome assembly sites. II. Role of
secondary structure in the formation of active sites
JOURNAL J. Biol. Chem. 259 (22), 14293-14300 (1984)
PUBMED 6150042
REFERENCE 20 (bases 1 to 4361)
AUTHORS Van Dyke MM, Dervan PB.
TITLE Echinomycin binding sites on DNA
JOURNAL Science 225 (4667), 1122-1127 (1984)
PUBMED 6089341
REFERENCE 21 (bases 1 to 4361)
AUTHORS Pouwels PH, Enger-Valk BE, Brammar WJ.
JOURNAL (in) CLONING VECTORS. Elsevier Scientific Publishing, Amsterdam
(1985)
REFERENCE 22 (bases 1892 to 1893)
AUTHORS Watson N.
TITLE A new revision of the sequence of plasmid pBR322
JOURNAL Gene 70 (2), 399-403 (1988)
PUBMED 3063608
REFERENCE 23 (bases 1 to 4361)
AUTHORS Gilbert W.
TITLE Obtained from VecBase 3.0
JOURNAL Unpublished
REFERENCE 24 (bases 1 to 4361)
TITLE Direct Submission
REFERENCE 25 (bases 1 to 4361)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
Acad. Sci. U.S.A."; date: "1978"; volume: "75"; issue: "8"; pages:
"3737-3741"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Cold Spring
Harb. Symp. Quant. Biol. 43 (Pt 1), 77-90 (1979)"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Proc. Natl.
Acad. Sci. U.S.A."; date: "1979"; volume: "76"; issue: "10"; pages:
"4882-4886"
COMMENT SGRef: number: 4; type: "Journal Article"; journalName: "Gene";
date: "1981"; volume: "13"; issue: "1"; pages: "25-35"
COMMENT SGRef: number: 5; type: "Journal Article"; journalName: "J. Biol.
Chem."; date: "1982"; volume: "257"; issue: "10"; pages: "5656-5662"
COMMENT SGRef: number: 6; type: "Journal Article"; journalName: "J. Biol.
Chem."; date: "1982"; volume: "257"; issue: "15"; pages: "9205-9210"
COMMENT SGRef: number: 7; type: "Journal Article"; journalName: "Proc. Natl.
Acad. Sci. U.S.A."; date: "1982"; volume: "79"; issue: "18"; pages:
"5470-5474"
COMMENT SGRef: number: 8; type: "Journal Article"; journalName: "Proc. Natl.
Acad. Sci. U.S.A."; date: "1982"; volume: "79"; issue: "23"; pages:
"7214-7217"
COMMENT SGRef: number: 9; type: "Journal Article"; journalName: "Gene 22
(2-3), 277-280 (1983)"
COMMENT SGRef: number: 10; type: "Journal Article"; journalName: "J. Biol.
Chem."; date: "1983"; volume: "258"; issue: "8"; pages: "4663-4665"
COMMENT SGRef: number: 11; type: "Journal Article"; journalName: "Proc.
Natl. Acad. Sci. U.S.A."; date: "1983"; volume: "80"; issue: "1";
pages: "237-241"
COMMENT SGRef: number: 12; type: "Journal Article"; journalName: "Proc.
Natl. Acad. Sci. U.S.A."; date: "1983"; volume: "80"; issue: "22";
pages: "6795-6798"
COMMENT SGRef: number: 13; type: "Journal Article"; journalName: "Proc.
Natl. Acad. Sci. U.S.A."; date: "1983"; volume: "80"; issue: "22";
pages: "6834-6837"
COMMENT SGRef: number: 14; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 15; type: "Journal Article"; journalName: "Cell";
date: "1984"; volume: "38"; issue: "3"; pages: "889-900"
COMMENT SGRef: number: 16; type: "Journal Article"; journalName: "DNA";
date: "1984"; volume: "3"; issue: "2"; pages: "173-182"
COMMENT SGRef: number: 17; type: "Journal Article"; journalName: "DNA";
date: "1984"; volume: "3"; issue: "3"; pages: "259-268"
COMMENT SGRef: number: 18; type: "Journal Article"; journalName: "J. Biol.
Chem."; date: "1984"; volume: "259"; issue: "22"; pages:
"14286-14292"
COMMENT SGRef: number: 19; type: "Journal Article"; journalName: "J. Biol.
Chem."; date: "1984"; volume: "259"; issue: "22"; pages:
"14293-14300"
COMMENT SGRef: number: 20; type: "Journal Article"; journalName: "Science";
date: "1984"; volume: "225"; issue: "4667"; pages: "1122-1127"
COMMENT SGRef: number: 21; type: "Journal Article"; journalName: "(in)
CLONING VECTORS. Elsevier Scientific Publishing, Amsterdam (1985)"
COMMENT SGRef: number: 22; type: "Journal Article"; journalName: "Gene";
date: "1988"; volume: "70"; issue: "2"; pages: "399-403"
COMMENT SGRef: number: 23; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 24; type: "Journal Article"
COMMENT On or before Apr 4, 2002 this sequence version replaced L08654.1,
gi:58257.
The circular sequence is numbered such that 0 is the middle of the
unique EcoRI site and the count increases first through the tet
genes, the pMB1 material, and finally through the Tn3 region.
Plasmid pBR322 contains ampicillin and tetracycline resistance
genes. The ampicillin resistance gene (amp-r) is a penicillin
beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene.
P3 is the natural promoter, and P1 is artificially created by the
ligation of two different DNA fragments to create pBR322. P2 is in
the same region as P1, but it is on the opposite strand and
initiates transcription in the direction of the tetracycline
resistance gene.
Mutational studies in the primosome assembly sites indicate four
types of mutations: Class I having no effect on the activities
elicited by the DNA site and the bases involved are probably
spacers; Class II requiring higher Mg-2+ concentrations than the
wild-type to be fully activated as factor Y ATPase effectors; Class
III co-inactivating both the ATPase effector and DNA replication
template activity of the site, indicating that they probably
represent essential contact points between factor Y and the DNA;
Class IV having a replication template activity intermediate that of
class III and class II mutant DNAs.
Specific sites within or near the origins of replication are
recognized by dnaA protein. Without dnaA binding to the origin of
replication chromosomal replication is not possible (15). pBR322 DNA
contains two separate regions on opposite strands and close to the
origin of replication which, when in single-stranded form, can act
as effectors for the ATPase activity of E.coli replication factor Y
(5). Small fragments of DNA containing these sites when cloned in
an f1 phage vector act as origins of DNA replication allowing the
formation of complementary double-stranded DNA in
rifampicin-resistant, dna(B,G,C)-dependent fashion in vitro (5). The
biological activity of echinomycin is thought to be related to the
formation of complexes by intercalating with cellular DNA (20).
Complete source information:
Plasmid pBR322 from E.coli (2),(1),(3),(6),(11),(8),(5),(7),(12),
(13),(10),(9),(14),(18),(19),(15),(20),(16); pBR322 DNA in pXf3 (4).
The following data and their annotation were supplied by Will
Gilbert under the auspices of the Curator Program.
CROSSREFERENCE
#parent
GenBank(50):pSC101C, GenBank(50):Trn3
#offspring
VecBase(3):pBR325, VecBase(3):pBR327,
VecBase(3):pBR328,
VecBase(3):pAT153, VecBase(3):pUC7,
VecBase(3):pJRD158,
VecBase(3):PiVX, VecBase(3):PiAN7,
VecBase(3):pSP64,
VecBase(3):pSP65, VecBase(3):pGEM1,
VecBase(3):pGEM2,
VecBase(3):pGEM3, VecBase(3):pGEM4,
VecBase(3):pKK223,
VecBase(3):pLBU3, VecBase(3):pTrS3,
VecBase(3):pRSVNeo,
VecBase(3):pSV2Cat, VecBase(3):M13mp9,
VecBase(3):pHC79,
VecBase(3):pV34, VecBase(3):pKTH601,
VecBase(3):pKTH604,
VecBase(3):pKTH605, VecBase(3):pKTH606,
VecBase(3):YEp24,
VecBase(3):YIp5, VecBase(3):YRp17,
VecBase(3):pSP18,
VecBase(3):pSP19,
VecBase(3):pSP6T3, VecBase(3):pSP6T719,
VecBase(3):pT712,
VecBase(3):pT713, VecBase(3):pT7T318,
VecBase(3):pT7T319,
VecBase(3):pT7T3A18, VecBase(3):pT7T3A19,
VecBase(3):pEX1,
VecBase(3):pEX2, VecBase(3):pEX3,
VecBase(3):pCKSP6,
VecBase(3):pACYC177, VecBase(3):pKO1,
VecBase(3):pKO2,
VecBase(3):pKM1,
VecBase(3):pKM2, VecBase(3):pMBL1,
VecBase(3):pMBL604,
VecBase(3):pMC1511, VecBase(3):pMC1871,
VecBase(3):pAA37X,
VecBase(3):pUR278, VecBase(3):pUR288,
VecBase(3):pUR289,
VecBase(3):pUR290, VecBase(3):pUR291,
VecBase(3):pUR292,
VecBase(3):pUR222.
FEATURES Location/Qualifiers
source 1..4361
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 10..38
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
misc_binding 39..42
/label=echinomycin binding site
/bound_moiety="echinomycin"
regulatory 43..49
/label=promoter P2 (6)
/note="promoter P2 (6)"
/regulatory_class="promoter"
misc_binding 53..56
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding 67..70
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding 80..83
/label=echinomycin binding site
/bound_moiety="echinomycin"
CDS 86..1273
/label=TcR
/note="tetracycline efflux protein"
repeat_region 1515..1519
/label=gamma-delta insertion target sequence
/note="gamma-delta insertion target sequence"
misc_feature 1636..1762
/label=from pSC101 (bp 1860-1986)
/note="from pSC101 (bp 1860-1986)"
repeat_region 1788..1792
/label=gamma-delta insertion target sequence
/note="gamma-delta insertion target sequence"
misc_difference 1891..1892
/replace="att"
/label=conflict
/note="conflict"
/citation=[23]
old_sequence 1892..1893
/citation=[2]
/citation=[22]
regulatory 1905..1910
/regulatory_class="ribosome_binding_site"
regulatory 1905..1909
/label=Shine-Dalgarno sequence
/note="Shine-Dalgarno sequence"
/regulatory_class="ribosome_binding_site"
misc_difference 1913..1914
/replace="caa"
/label=conflict
/note="conflict"
/citation=[23]
CDS 1915..2103
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 2208..2348
/label=bom
/note="basis of mobility region from pBR322"
misc_feature complement(2351..2414)
/label=L-strand Y effector site
/note="L-strand Y effector site"
/citation=[5]
misc_binding 2439..2447
/label=dnaA binding site
/bound_moiety="dnaA"
rep_origin complement(2534..3122)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
repeat_region 3148..3185
/note="corresponds to one of the 38bp repeats found in Tn3
(bp 1-38 and complement (4920-4957))"
CDS complement(3296..4153)
/label=AmpR
/note="beta-lactamase"
promoter complement(4154..4258)
/label=AmpR promoter
misc_binding complement(4268..4271)
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding complement(4280..4283)
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding complement(4285..4288)
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding complement(4296..4299)
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding complement(4311..4314)
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding complement(4317..4320)
/label=echinomycin binding site
/bound_moiety="echinomycin"
misc_binding complement(4331..4334)
/label=echinomycin binding site
/bound_moiety="echinomycin"