Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010658 | pMonAID_nCas9-PmCDA_Hyg_ALS | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pMonAID_nCas9-PmCDA_Hyg_ALS
- Antibiotic Resistance:
- Spectinomycin
- Length:
- 19008 bp
- Type:
- Plant Expression
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Promoter:
- CaMV35S(enhanced)
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- ggaagttcatttcatttggagag
- 3' Primer:
- ccatttgcattttgatgtccg
pMonAID_nCas9-PmCDA_Hyg_ALS vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
pMonAID_nCas9-PmCDA_Hyg_ALS vector Sequence
LOCUS 40924_31240 19008 bp DNA circular SYN 13-MAY-2021
DEFINITION Monocot Target-AID vector expressing rice-optimized nCas9-PmCDA1
with sgRNA targeting OsAls.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 19008)
AUTHORS Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H,
Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K,
Ariizumi T, Kondo A
TITLE Targeted base editing in rice and tomato using a CRISPR-Cas9
cytidine deaminase fusion.
JOURNAL Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3833.
PUBMED 28346401
REFERENCE 2 (bases 1 to 19008)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 19008)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3833."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..19008
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(113..137)
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
protein_bind 295..316
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 331..361
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 369..385
/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)."
primer_bind 374..396
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 393..409
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 393..409
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
protein_bind 486..506
/label=attB1
/note="core recombination site for the Gateway(R) BP
reaction"
primer_bind complement(559..575)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 596..695
/label=attL1
/note="recombination site for the Gateway(R) LR reaction"
misc_RNA 1104..1179
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind complement(1191..1215)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
protein_bind complement(1221..1320)
/label=attL2
/note="recombination site for the Gateway(R) LR reaction"
promoter 1489..2156
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
5'UTR 2167..2267
/label=OsADH 5'-UTR
/note="translational enhancer from the 5'-UTR of the Oryza
sativa alcohol dehydrogenase gene (Sugio et al., 2008)"
CDS 2277..2297
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 2322..6425
/codon_start=1
/label=Cas9(D10A)
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 6447..6467
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 6660..6725
/codon_start=1
/label=3xFLAG
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 6732..7355
/codon_start=1
/label=PmCDA1
/note="cytidine deaminase from the sea lamprey Petromyzon
marinus (Rogozin et al., 2007)"
/translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER
RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI
LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF
IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV"
CDS 7359..7379
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
promoter 9657..9998
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 10017..11039
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
protein_bind complement(12194..12318)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
primer_bind complement(12358..12375)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(12358..12374)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12367..12389)
/label=M13/pUC Forward
/note="In lacZ gene"
misc_feature complement(12589..12613)
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS 13134..13922
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
rep_origin 14171..14759
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 14913..14930
/label=L4440
/note="L4440 vector, forward primer"
misc_feature complement(14945..15085)
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 15171..15193
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
rep_origin complement(15429..15623)
/direction=LEFT
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
CDS complement(15692..16762)
/codon_start=1
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/translation="VSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
LVMRYRNLIEGEASAGS"
CDS complement(17194..17820)
/codon_start=1
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"