首页技术支持质粒载体

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 pCC1BAC is a single copy vector that is inducible with 0.04% Arabinose or 1x CopyControl Induction Solution (CopyControl Fosmid clones grown in TransforMax EPI300 cells can be amplified to 10-50 copies per cell). An EPI400/EPI300 strain is recommended. The protocol is as below:
a) Add 4 ml LB media into each test tube. Inoculate each tube with bacterial culture with antibiotic at the proper concentration.
b) Incubate the tubes at 37°C, shaking overnight.
c) Dilute the starting culture (from step b) 1:10 into antibiotic-supplemented fresh media.
d) Supplement induction solution with a ratio of 1:1000, and grow the culture at 37°C for 4 h with vigorous shaking (approx. 250 rpm).
e) Isolate DNA from the induced culture cells as per the protocol provided.
The pCC1BAC vector has two origins of replication – a single copy E. coli F-factor replicon (ori2 or oriS) and a high-copy origin of replication called "oriV". Initially, replication of CopyControl clones can be controlled by the F-factor replicon so the vector is present at one copy per cell. Maintaining clones at single copy ensures insert stability and allows cloning of toxic gene products. Initiation of replication from oriV requires the trfA gene product. CopyControl Vectors use a specifically engineered E. coli host strain, EPI300/EPI400, which contains a mutant trfA gene under tight control of an inducible promoter (araBAD promoter). Addition of the CopyControl Induction Solution to the growth medium induces expression of trfA and subsequent amplification of the clone to high-copy number. Induction of CopyControl BAC clones from singlecopy up to 10-20 copies per cell greatly improves the yield and purity of BAC DNA for sequencing, fingerprinting and other applications. The ParA ATPase of F plasmid, SopA, is stimulated by SopB that assembles into a partition complex on the centromere-like locus, sopC, on the plasmid cargo. Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells.

载体名称:
pCC1BAC
载体抗性:
Chloramphenicol
载体长度:
8139 bp
载体类型:
Cloning vector
复制子:
ori2
载体来源:
EPICENTRE Biotechnologies.
拷贝数:
Single copy
感受态:
EPI300/400
培养温度:
37℃

pCC1BAC 载体图谱

pCC1BAC8139 bp400800120016002000240028003200360040004400480052005600600064006800720076008000lacZ-alphalac operatorlac promoterCAP binding siteCmRcat promoterredForiVori2repEincCsopAsopCcosloxP

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。

pCC1BAC 载体序列

LOCUS       Exported                8139 bp DNA     circular SYN 26-DEC-2024
DEFINITION  Cloning vector pCC1FOS, complete sequence.
ACCESSION   EU140751
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8139)
  AUTHORS   EPICENTRE Biotechnologies.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-AUG-2007) 726 Post Road, Madison, WI 53713, USA
REFERENCE   2  (bases 1 to 8139)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (23-AUG-2007) 726 Post Road, Madison, WI 53713, USA"
FEATURES             Location/Qualifiers
     source          1..8139
                     /mol_type="other DNA"
                     /db_xref="taxon:468516"
                     /organism="Cloning vector pCC1FOS"
     CDS             complement(160..447)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGETIEYSSLHACRSTLEDPTWDPRVPSSNSPYSESY
                     YNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRS"
     primer_bind     288..304
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        311..329
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(443..459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    467..483
                     /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(491..521)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    536..557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(777..1436)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1437..1539)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1655..2002
                     /codon_start=1
                     /label=redF
                     /translation="MERRNRRTGRTEKARIWEVTDRTVRTWIGEAVAAAAADGVTFSVP
                     VTPHTFRHSYAMHMLYAGIPLKVLQSLMGHKSISSTEVYTKVFALDVAARHRVQFAMPE
                     SDAVAMLKQLS"
     rep_origin      2397..3011
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     rep_origin      3087..3306
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             3397..4152
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     misc_feature    4155..4405
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             4731..5906
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     CDS             5906..6877
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     misc_feature    6950..7423
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
     misc_feature    7683..8081
                     /label=cos
                     /note="lambda cos site; allows packaging into phage lambda 
                     particles"
     protein_bind    8099..8132
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."