质粒编号
质粒名称
规格
价格
V005935
pCasPA
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。
- 载体名称:
- pCasPA
- 载体抗性:
- Tetracycline
- 载体长度:
- 17571 bp
- 载体类型:
- Bacterial Expression, CRISPR, Synthetic Biology
- 复制子:
- oriV
- 启动子:
- araBAD
- 感受态:
- DH10B
pCasPA 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。
pCasPA 载体序列
LOCUS 40924_9061 17571 bp DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression of Cas9 nuclease and lambda-Red system in
Pseudomonas aeruginosa.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17571)
AUTHORS Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, Wang Y, Ji Q
TITLE CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and
Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species.
JOURNAL iScience. 2018 Aug 31;6:222-231. doi: 10.1016/j.isci.2018.07.024.
Epub 2018 Aug 1.
PUBMED 30240613
REFERENCE 2 (bases 1 to 17571)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 17571)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "iScience.";
date: "2018-08-31"; pages: "
10.1016/j.isci.2018.07.024. Epub 2018 Aug 1"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..17571
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 3..712
/label=oriV
/note="incP origin of replication"
primer_bind complement(2109..2128)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2319..2340
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2603..2625
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 2617..2634
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 2618..2634
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(2745..3620)
/codon_start=1
/label=araC
/note="L-arabinose regulatory protein"
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 3647..3931
/gene="araBAD"
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
RBS 3958..3966
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 3975..4388
/codon_start=1
/label=Gam
/note="inhibitor of the host RecBCD nuclease in the lambda
Red system"
/translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
CDS 4397..5179
/codon_start=1
/label=Beta
/note="single-stranded DNA binding recombinase in the
lambda Red system"
/translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
CDS 5179..5856
/codon_start=1
/label=Exo
/note="5' to 3' double-stranded DNA exonuclease in the
lambda
Red system"
/translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
EQWR"
terminator 5860..6104
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
regulatory 6120..6129
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
primer_bind 6145..6164
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
CDS complement(6206..7624)
/codon_start=1
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
promoter 8030..8314
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
RBS 8341..8349
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 8358..12461
/codon_start=1
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
promoter complement(12520..12538)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(12552..12568)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12552..12568)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12565..12587)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12576..12592
/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(12600..12630)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(12645..12666)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 13024..14220
/codon_start=1
/label=TcR
/note="tetracycline efflux protein"
/translation="VKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
"
CDS complement(15499..16644)
/codon_start=1
/label=trfA
/note="trans-acting replication protein that binds to and
activates oriV"
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(16919..17287)
/codon_start=1
/label=traJ
/note="oriT-recognizing protein"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT complement(17320..17429)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"