| 质粒名称: | pET28a-MH6-RspCas13d |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 108305 |
| 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: | 8153 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: | RspCas13d |
| 物种来源: | Synthetic |
| 插入大小: | 2763 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 | ATGGCCAAAAAGAACAAAATGAAACC |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-MH6-RspCas13d was a gift from Arbor Biotechnologies (Addgene plasmid # 108305 ; http://n2t.net/addgene:108305 ; RRID:Addgene_108305) |
| 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-RspCas13d 8149 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses E. coli codon optimized RspCas13d in the pET28a backbone..
ACCESSION .
VERSION .
KEYWORDS pET28a-MH6-RspCas13d
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8149)
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 8149)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8149
/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 2955..2989
/label=J23119 promoter
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119)"
primer_bind complement(3084..3102)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3098..3145
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 3182..3637
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3269..3288)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3479..3500
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(3729..4544)
/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 4452..4471
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 4536..4555
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 4666..5254
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5155..5174
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5408..5425
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5440..5579
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5505..5524)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5665..5687
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5681..5872)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(6681..7763)
/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 7725..7744
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(7764..7841)
/gene="lacI"
/label=lacI promoter
primer_bind 8047..8066
/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 cacatggcca
181 aaaagaacaa aatgaaaccg cgtgaactgc gtgaagcaca gaaaaaagca cgtcagctga
241 aagcagcaga aattaacaat aatgcagcac cggcaattgc agcaatgcct gcagcagaag
301 ttattgcacc ggttgcagaa aaaaagaaaa gcagcgttaa agcagccggt atgaaaagca
361 ttctggtgag caaaaacaaa atgtatatca ccagctttgg caaaggtaat agcgcagttc
421 tggaatatga agtggataac aacgattaca atcagaccca gctgagcagc aaaggtagca
481 gcaatattga gctgcgtggt gttaatgaag tgaacattac ctttagcagc aaacacggtt
541 ttgaaagcgg tgttgaaatc aataccagca atccgacaca tcgtagcggt gaaagcagtc
601 cggttcgtgg tgatatgctg ggcctgaaaa gcgaactgga aaaacgcttt tttggcaaaa
661 ccttcgatga caacattcat atccagctga tctataacat cctggacatc gaaaaaattc
721 tggccgttta tgtgaccaac attgtgtatg cactgaataa catgctgagc attaaagata
781 gcgagagcta tgatgatttc atgggttatc tgagcgcacg caatacctat gaagttttta
841 cccatccgga taaaagcaac ctgagcgata aagcaaaagg caacatcaaa aaatccttta
901 gcaccttcaa cgacctgctg aaaaccaaac gtctgggtta ttttggtctg gaagaaccga
961 aaacaaaaga tacccgtgtt agccaggcat acaaaaaacg tgtttatcac atgctggcaa
1021 ttgtgggtca gattcgtcag agcgtgtttc atgataaaag tagcaaactg gatgaggacc
1081 tgtatagctt tatcgatatc atcgattccg aatatcgtga aaccctggat tatctggttg
1141 atgaacgttt tgacagcatc aacaaaggtt ttatccaggg caacaaagtg aatattagcc
1201 tgctgatcga tatgatgaaa ggctatgaag ccgatgatat tatccgcctg tattatgatt
1261 ttatcgtgct gaaaagccag aagaatctgg gctttagtat caaaaaactg cgcgagaaaa
1321 tgctggatga atatggcttt cgcttcaaag ataaacagta tgatagcgtg cgtagcaaga
1381 tgtataaact gatggatttt ctgctgttct gcaactatta tcgcaatgat gttgttgccg
1441 gtgaagcact ggttcgtaaa ctgcgtttta gcatgaccga tgatgaaaaa gaaggtattt
1501 acgcagacga agcaagcaaa ctgtggggta aatttcgtaa cgattttgag aacattgccg
1561 accatatgaa cggtgatgtt attaaagaac tgggcaaagc cgatatggat ttcgatgaaa
1621 aaatactgga cagcgagaaa aaaaacgcaa gcgatctgct gtatttcagc aagatgattt
1681 atatgctgac ctatttcctg gatggcaaag aaattaatga tctgctgacc acgctgatta
1741 gcaaatttga taacatcaaa gaatttctga aaatcatgaa aagctcagcc gttgatgttg
1801 aatgtgaact gaccgcaggt tataaactgt ttaatgatag ccagcgcatt accaacgaac
1861 tgttcattgt taaaaacatt gcgagcatgc gtaaaccggc aagcagcgca aaactgacca
1921 tgtttcgtga tgcactgacc attctgggta ttgatgataa cattaccgat gatcgcatta
1981 gcgaaatcct gaagctgaaa gaaaaaggta aaggtattca tggcctgcgc aactttatta
2041 ccaataacgt gattgaaagc tcccgctttg tgtacctgat caaatatgca aatgcccaga
2101 aaattcgcaa agtggcgaaa aatgaaaagg tggtgatgtt tgtgttaggt ggtattccgg
2161 atacacagat tgaacgctat tacaaaagct gcgttgaatt tccggatatg aacagcagcc
2221 tggaagttaa acgtagtgaa ctggcacgta tgatcaaaaa catcagcttc gacgatttca
2281 agaacgttaa acagcaggca aaaggtcgtg aaaatgttgc aaaagaacgt gccaaagcag
2341 tgattggtct gtatctgacc gttatgtatc tgctggttaa aaatctggtt aacgtgaatg
2401 cccgttatgt gattgcaatt cattgtctgg aacgtgattt cggcctgtat aaagaaatca
2461 ttccggaact ggcaagcaag aacctgaaaa atgattatcg tattctgagt cagaccctgt
2521 gtgaactgtg tgataaaagc ccgaacctgt ttctgaagaa aaacgaacgt ctgcgcaaat
2581 gcgttgaggt ggatattaac aatgcagata gcagcatgac ccgcaaatat cgtaattgta
2641 ttgcacatct gacagttgtg cgcgaactga aagaatatat tggtgatatt cgtaccgtgg
2701 acagctactt tagcatctat cattatgtta tgcagcggtg tattaccaaa cgcgaaaatg
2761 ataccaaaca agaagagaaa atcaaatacg aggacgatct gcttaaaaac cacggctata
2821 ccaaagattt tgtgaaagcc ctgaatagcc cgttcggtta taacattccg cgtttcaaaa
2881 atctgagcat cgagcagctg tttgatcgta atgaatatct gacggaaaaa taataagaat
2941 tcggatccct cgagttgaca gctagctcag tcctaggtat aatgctagca gagaccatgg
3001 ccaccggtct ctgctaaaga tatgaggagc tcaaactcga gctgctaaca aagcccgaaa
3061 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
3121 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
3181 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
3241 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
3301 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
3361 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
3421 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
3481 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
3541 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
3601 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
3661 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
3721 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
3781 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
3841 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
3901 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
3961 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
4021 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
4081 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
4141 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
4201 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
4261 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
4321 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
4381 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
4441 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
4501 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
4561 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
4621 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
4681 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
4741 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
4801 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
4861 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
4921 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
4981 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
5041 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
5101 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
5161 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
5221 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
5281 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
5341 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
5401 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
5461 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
5521 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
5581 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
5641 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
5701 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
5761 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
5821 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
5881 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
5941 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
6001 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
6061 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
6121 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
6181 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
6241 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
6301 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
6361 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
6421 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
6481 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
6541 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
6601 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
6661 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
6721 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
6781 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
6841 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
6901 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
6961 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
7021 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
7081 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
7141 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
7201 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
7261 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
7321 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
7381 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
7441 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
7501 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
7561 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
7621 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
7681 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
7741 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
7801 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
7861 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
7921 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
7981 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
8041 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
8101 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
