| 质粒名称: | pET28a-MH6-RspWYL1 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 108306 |
| 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 |
| 载体修饰: | -- |
| 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: | RspWYL1 |
| 物种来源: | Synthetic |
| 插入大小: | 1182 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: | BamHI(未破坏) |
| 3' 克隆位点: | BamHI(未破坏) |
| 5' 测序引物 | ATGCTGATTCCGCCTAGCAC |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-MH6-RspWYL1 was a gift from Arbor Biotechnologies (Addgene plasmid # 108306 ; http://n2t.net/addgene:108306 ; RRID:Addgene_108306) |
| 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-RspWYL1 6568 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses E. coli codon optimized RspWYL1 in the pET28a backbone..
ACCESSION .
VERSION .
KEYWORDS pET28a-MH6-RspWYL1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6568)
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 6568)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6568
/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 108..130
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 162..179
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
promoter 1374..1408
/label=J23119 promoter
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119)"
primer_bind complement(1503..1521)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 1517..1564
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 1601..2056
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(1688..1707)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 1898..1919
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(2148..2963)
/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 2871..2890
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 2955..2974
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 3085..3673
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3574..3593
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3827..3844
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 3859..3998
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(3924..3943)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4084..4106
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4100..4291)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(5100..6182)
/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 6144..6163
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(6183..6260)
/gene="lacI"
/label=lacI promoter
primer_bind 6466..6485
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gatttacact
61 ttatgcttcc ggctcgtatg tttctagaga attcggatcc aaataatttt gtttaacttt
121 aagaaggaga tttaaatatg aaaatcgaag aaggtaaagg tcaccatcac catcaccaca
181 tgctgattcc gcctagcacc tttctgccga aacgtgataa aaatgttccg tatattgcag
241 aggttcagag cattccgctg agcccgagcg catatagcgt tattatcaaa gataagagca
301 tctttgaaac cagcctgagt ccgaatggta gcgttagcat gagcagcttt ctgaccagca
361 tttttgatag cgcctatatc gcaagcctga aatacaaaag cgacgacaac tataaataca
421 ttggtattcc gctgctgaac gcctttgtta aatggcagat tgaagaaatt gatgatggcc
481 tggatgacaa gagcaaagaa atcattaaaa gctatctgat cagcaagctg agcgccaaat
541 atgaaaaaac caaaaccgaa aatgccgttc gtgttcgtct gagcatttgt cgtgatctgt
601 atgataccct gagcagcgac gatctgtatt atgaaaataa agtgtatagc agcaccctgc
661 gtcgttttct gaaagcagtt tatgaagatt atgcactgct gagcgattgt gaacgtgaac
721 gtctgatttt tgccgataac atcatcaaaa tcaacgaagt gattaagcag aatggcagcc
781 gttattatag cttcatttat gcctatagca acatgtacag ccgtgaaaaa cgtcgtattc
841 gcctgattcc gtatcgtatt gtttccgatg agtacaaaat gtataactac ctggtttgtc
901 tgagcgacga aaaaagcgca ggtaaagaat ttaaagcaga tagctgtcgt attagccgtc
961 tgagcggtct gagtattgca gaaaaactga gccagaaaga atatagcagc gtgaccgaat
1021 atgaacgcct gaaagaagtt cacgttaaaa gcgttaaaca cctgttaagc gatccgcgtt
1081 ttggtagtga tgaaagcgat attagcaaag tgtatctgac cgaaaaaggc gtggaaatgt
1141 ttggcaaaat tctgtatcag cgtccgattc tgaaaggcaa tgagaaaccg aaacctaatg
1201 ccgtgaatga atttatcagc cctccgattc aggtgaaata ctactttaac aaattcggca
1261 aagacggcgt tattctgagc ccgtctgata gctttgaaga gatgcgtacc ctgtatgttg
1321 aaggtgccga agcatataat cgtgaagttg aaatgtaata aggatccctc gagttgacag
1381 ctagctcagt cctaggtata atgctagcag agaccatggc caccggtctc tgctaaagat
1441 atgaggagct caaactcgag ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
1501 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
1561 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
1621 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
1681 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
1741 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
1801 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
1861 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
1921 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
1981 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
2041 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
2101 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
2161 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
2221 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
2281 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
2341 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
2401 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
2461 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
2521 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
2581 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
2641 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
2701 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
2761 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
2821 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
2881 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
2941 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
3001 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
3061 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
3121 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
3181 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
3241 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
3301 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
3361 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
3421 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
3481 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
3541 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
3601 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
3661 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
3721 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
3781 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
3841 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
3901 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
3961 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
4021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
4081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
4141 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
4201 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
4261 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
4321 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
4381 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
4441 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
4501 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
4561 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
4621 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
4681 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
4741 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
4801 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
4861 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
4921 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
4981 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
5041 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
5101 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
5161 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
5221 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
5281 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
5341 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
5401 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
5461 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
5521 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
5581 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
5641 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
5701 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
5761 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
5821 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
5881 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
5941 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
6001 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
6061 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
6121 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
6181 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
6241 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
6301 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
6361 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
6421 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
6481 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
6541 gaggatcgag atctcgatcc cgcgaaat
//
