| 质粒名称: | pET28a-MH6-uvsY |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 163914 |
| 存储实验室: | Paul Freemont |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Novagen |
| 空载体大小: | 5369 bp |
| 完整质粒大小: | 5653 bp |
| 载体修饰: | 移除C端子His标签 |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 卡那霉素，50μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | uvsY |
| 物种来源: | Enterobacteria phage T4 |
| 插入大小: | 411 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | T7 promoter |
| 标签/融合蛋白: | His (N terminal on backbone) |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | TAATACGACTCACTATAGGG |
| 3’测序引物 | GCTAGTTATTGCTCAGCGG |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-MH6-uvsY was a gift from Paul Freemont (Addgene plasmid # 163914 ; http://n2t.net/addgene:163914 ; RRID:Addgene_163914) |
| 参考文献部分: | A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance. Patchsung M, Homchan A, Aphicho K, Suraritdechachai S, Wanitchanon T, Pattama A, Sappakhaw K, Meesawat P, Wongsatit T, Athipanyasilp A, Jantarug K, Athipanyasilp N, Buahom J, Visanpattanasin S, Niljianskul N, Chaiyen P, Tinikul R, Wichukchinda N, Mahasirimongkol S, Sirijatuphat R, Angkasekwinai N, Crone MA, Freemont PS, Joung J, Ladha A, Abudayyeh O, Gootenberg J, Zhang F, Chewapreecha C, Chanarat S, Horthongkham N, Pakotiprapha D, Uttamapinant C. CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048. 10.1089/crispr.2022.0048 PubMed 36367987 |
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-uvsY 5653 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expresses uvsY for bacterial expression and affinity purification.
ACCESSION .
VERSION .
KEYWORDS pET28a-MH6-uvsY
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5653)
TITLE One Pot CRISPR RPA
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 5653)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5653
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(646..1728)
/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 1690..1709
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1729..1806)
/gene="lacI"
/label=lacI promoter
primer_bind 2012..2031
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 2115..2134
/label=T7
/note="T7 promoter, forward primer"
promoter 2115..2133
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 2134..2158
/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 2167..2196
/label=lac promoter
/note="promoter for the E. coli lac operon"
RBS 2216..2238
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2270..2287
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2702..2720)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2716..2763
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2800..3255
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2887..2906)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3097..3118
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(3347..4162)
/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 4070..4089
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 4154..4173
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 4284..4872
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4773..4792
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5026..5043
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5058..5197
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5123..5142)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5283..5305
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5299..5490)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
61 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
121 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
181 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
241 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
301 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
361 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
421 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
481 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
541 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
601 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
661 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
721 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
781 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
841 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
901 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
961 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
1021 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
1081 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
1141 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
1201 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
1261 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
1321 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
1381 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
1441 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
1501 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
1561 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
1621 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
1681 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
1741 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
1801 ggtgtccggg atctcgacgc tctcccttat gcggctcctg cattaggaag cagcccagta
1861 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
1921 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
1981 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
2041 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
2101 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
2161 tctagattta cactttatgc ttccggctcg tatgtttcta gagaattcaa ataattttgt
2221 ttaactttaa gaaggagatt taaatatgaa aatcgaagaa ggtaaaggtc accatcacca
2281 tcaccacatg cgtctggaag atctgcaaga ggaactgaaa aaagatgtgt ttatcgacag
2341 caccaaactg cagtatgaag cagcaaataa tgtgatgctg tatagcaaat ggctgaacaa
2401 acatagcagc atcaaaaaag aaatgctgcg tattgaggcc cagaaaaaag ttgcactgaa
2461 agcacgtctg gattattaca gcggtcgtgg tgatggtgat gaatttagca tggatcgtta
2521 tgagaaaagc gagatgaaaa ccgttctgag cgcagataaa gatgttctga aagttgatac
2581 cagcctgcag tattggggta ttctgctgga tttttgtagc ggtgcactgg atgcaattaa
2641 aagccgtggt tttgccatta aacatatcca ggacatgcgt gcatttgaag ccggtaaata
2701 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
2761 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
2821 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
2881 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
2941 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
3001 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
3061 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
3121 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
3181 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
3241 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
3301 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
3361 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
3421 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
3481 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
3541 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
3601 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
3661 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
3721 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
3781 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
3841 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
3901 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
3961 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
4021 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
4081 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
4141 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
4201 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
4261 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
4321 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
4381 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
4441 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
4501 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
4561 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
4621 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
4681 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
4741 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
4801 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
4861 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
4921 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
4981 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
5041 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
5101 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
5161 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
5221 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
5281 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
5341 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
5401 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
5461 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
5521 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
5581 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
5641 ggaccagaga aaa
//
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