| 质粒名称: | pET28a-MH6-EsCas13d |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 108303 |
| 存储实验室: | Arbor Biotechnologies |
|
载体骨架基本信息 |
|
| 载体名称: | pET-28a(+) |
| 载体出品公司: | Novagen |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5390 bp |
| 完整质粒大小: | 8252 bp |
| 载体类型: | 细菌表达,CRISPR |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | EsCas13d |
| 物种来源: | Synthetic |
| 插入大小: | 2862 bp |
| 启动子 | lac |
| 标签/融合蛋白: | mH6 tag（插入件上的N端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | EcoRI(未破坏) |
| 3' 克隆位点: | EcoRI(未破坏) |
| 5' 测序引物 | atgggcaaaaaaatccatgcac |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-MH6-EsCas13d was a gift from Arbor Biotechnologies (Addgene plasmid # 108303 ; http://n2t.net/addgene:108303 ; RRID:Addgene_108303) |
| 参考文献部分: | Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein. Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA. Mol Cell. 2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 10.1016/j.molcel.2018.02.028. 10.1016/j.molcel.2018.02.028 PubMed 29551514 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pET28a-MH6-EsCas13d 8248 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses E. coli codon optimized EsCas13d in the pET28a backbone..
ACCESSION .
VERSION .
KEYWORDS pET28a-MH6-EsCas13d
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8248)
AUTHORS Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA
TITLE Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively
Modulated by a WYL-Domain-Containing Accessory Protein.
JOURNAL Mol Cell. 2018 Mar 9. pii: S1097-2765(18)30173-4. doi:
10.1016/j.molcel.2018.02.028.
PUBMED 29551514
REFERENCE 2 (bases 1 to 8248)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8248
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 20..44
/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 53..82
/label=lac promoter
/note="promoter for the E. coli lac operon"
RBS 102..124
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 156..173
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
promoter 3054..3088
/label=J23119 promoter
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119)"
primer_bind complement(3183..3201)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3197..3244
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 3281..3736
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3368..3387)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3578..3599
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(3828..4643)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 4551..4570
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 4635..4654
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 4765..5353
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5254..5273
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5507..5524
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5539..5678
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5604..5623)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5764..5786
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5780..5971)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(6780..7862)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 7824..7843
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(7863..7940)
/gene="lacI"
/label=lacI promoter
primer_bind 8146..8165
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gatttacact
61 ttatgcttcc ggctcgtatg tttctagaga attcaaataa ttttgtttaa ctttaagaag
121 gagatttaaa tatgaaaatc gaagaaggta aaggtcacca tcaccatcac cacatgggca
181 aaaaaatcca tgcacgtgat ctgcgtgaac agcgtaaaac cgatcgtacc gaaaaatttg
241 cagaccagaa caaaaaacgt gaagcagaac gtgcagttcc gaaaaaagat gcagcagtta
301 gcgttaaaag cgttagcagc gtgagcagca aaaaagataa tgttaccaaa agcatggcaa
361 aagcagccgg tgtgaaaagc gtttttgcag ttggtaatac cgtttacatg accagctttg
421 gtcgtggtaa tgatgcagtt ctggaacaga aaattgttga taccagccat gaaccgctga
481 atattgatga tccggcatat cagctgaatg tggttaccat gaatggttat agcgttaccg
541 gtcatcgtgg tgaaaccgtt agcgcagtta ccgataatcc gctgcgtcgt tttaatggtc
601 gtaaaaaaga cgaaccggaa cagagcgttc cgaccgatat gctgtgtctg aaaccgacac
661 tggaaaaaaa gttttttggc aaagagttcg acgacaacat tcatatccag ctgatttata
721 acatcctgga catcgaaaaa atcctggcag tttatagcac caatgcaatt tatgccctga
781 ataacatgag cgcagatgaa aacattgaga acagcgactt ttttatgaaa cgcaccaccg
841 atgaaacctt cgatgatttc gaaaaaaaga aagaaagcac caacagccgt gaaaaagcag
901 attttgatgc cttcgaaaaa ttcatcggta attatcgcct ggcctatttt gcagatgcct
961 tttatgtcaa caaaaagaac ccgaaaggca aagccaaaaa tgtgctgcgt gaagataaag
1021 aactgtatag cgttctgacc ctgattggta aactgcgtca ttggtgtgtt catagcgaag
1081 aaggtcgcgc agaattttgg ctgtataaac tggatgaact gaaggacgat ttcaaaaacg
1141 ttctggatgt ggtttataat cgtccggtgg aagaaattaa caaccgcttt atcgaaaaca
1201 acaaagtgaa cattcagatt ctgggcagcg tgtataaaaa caccgatatt gcagaactgg
1261 tgcgcagcta ttatgaattt ctgatcacca aaaaatacaa aaacatgggc tttagcatca
1321 aaaaactgcg tgaaagcatg ctggaaggta aaggttatgc cgataaagaa tatgatagcg
1381 tgcgcaacaa actgtatcag atgaccgatt ttattctgta caccggctat atcaacgaag
1441 atagcgatcg tgcagatgat ctggttaata ccctgcgtag cagcctgaaa gaagatgata
1501 aaaccaccgt gtattgcaaa gaggcagatt acctttggaa aaagtatcgc gaaagcattc
1561 gtgaagttgc cgatgcactg gatggtgaca atattaagaa actgagcaaa agcaacatcg
1621 agatccaaga ggataaactg cgcaaatgct ttattagcta tgcagatagc gttagcgaat
1681 ttaccaaact gatttatctg ctgacccgtt ttctgagcgg taaagaaatc aacgatctgg
1741 tgaccacact gatcaacaaa tttgataaca ttcgcagctt tctggaaatc atggatgaat
1801 taggtctgga tcgtaccttt accgcagaat atagcttttt tgaaggcagc accaaatatc
1861 tggccgaact ggtggaactg aatagctttg ttaaatcctg cagcttcgat attaatgcca
1921 aacgtaccat gtatcgtgat gccctggata ttctgggtat tgaatcagat aaaaccgaag
1981 aggatatcga gaaaatgatc gacaatatcc tgcagattga tgccaacggt gataaaaagc
2041 tgaaaaaaaa caatggcctg cgcaacttta ttgccagcaa tgttattgat agcaaccgct
2101 tcaaatacct ggtgcgttat ggtaacccga agaaaattcg tgaaaccgca aaatgtaaac
2161 cggcagttcg ttttgtgctg aatgaaattc cggatgcaca gatcgaacgt tattatgaag
2221 catgttgtcc gaaaaatacc gcactgtgta gcgcaaataa acgtcgtgaa aaactggcag
2281 atatgatcgc cgaaatcaaa ttcgaaaact tttccgatgc aggcaactat cagaaagcaa
2341 atgtgaccag ccgtaccagc gaagcagaaa ttaaacgtaa aaaccaggcc attattcgcc
2401 tgtatctgac cgttatgtat atcatgctga aaaacctggt gaatgtgaat gcccgttatg
2461 tgattgcatt tcattgtgtt gaacgcgaca caaaactgta tgccgaaagc ggtctggaag
2521 tgggtaatat tgaaaagaat aaaaccaacc tgaccatggc cgttatgggt gttaaactgg
2581 aaaatggcat tatcaaaacc gagttcgata agagctttgc agaaaatgca gcaaatcgtt
2641 atctgcgtaa tgcgcgttgg tataagctga ttctggataa tctgaagaaa agcgaacgtg
2701 ccgttgtgaa tgaatttcgc aataccgtgt gtcatctgaa tgccattcgc aacattaaca
2761 tcaacattaa agagatcaaa gaggtcgaga actatttcgc cctgtatcat tatctgatcc
2821 agaaacatct ggaaaaccgc ttcgcagaca aaaaagttga acgtgatacc ggtgatttca
2881 tcagcaaact ggaagaacat aaaacctact gcaaagactt cgtgaaagcc tattgtaccc
2941 cgtttggtta taatctggtt cgctataaaa acctgacgat cgatggtctg ttcgacaaaa
3001 actatccggg taaagatgat agcgacgagc agaaataata agaattcctc gagttgacag
3061 ctagctcagt cctaggtata atgctagcag agaccatggc caccggtctc tgctaaagat
3121 atgaggagct caaactcgag ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
3181 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
3241 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
3301 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
3361 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
3421 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
3481 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
3541 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
3601 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
3661 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
3721 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
3781 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
3841 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
3901 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
3961 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
4021 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
4081 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
4141 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
4201 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
4261 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
4321 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
4381 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
4441 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
4501 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
4561 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
4621 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
4681 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
4741 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
4801 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
4861 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
4921 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
4981 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
5041 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
5101 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
5161 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
5221 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
5281 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
5341 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
5401 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
5461 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
5521 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
5581 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
5641 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
5701 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
5761 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
5821 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
5881 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
5941 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
6001 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
6061 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
6121 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
6181 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
6241 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
6301 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
6361 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
6421 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
6481 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
6541 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
6601 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
6661 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
6721 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
6781 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
6841 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
6901 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
6961 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
7021 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
7081 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
7141 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
7201 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
7261 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
7321 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
7381 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
7441 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
7501 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
7561 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
7621 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
7681 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
7741 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
7801 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
7861 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
7921 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
7981 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
8041 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
8101 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
8161 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
8221 gaggatcgag atctcgatcc cgcgaaat
//
