| 质粒名称: | pET28a-MH6-Bsu |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 163911 |
| 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: | 6997 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: | Bsu聚合酶 |
| 物种来源: | 枯草芽孢杆菌 |
| 插入大小: | 1755 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | T7 promoter |
| 标签/融合蛋白: | His（主干上的N端） |
| 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-Bsu was a gift from Paul Freemont (Addgene plasmid # 163911 ; http://n2t.net/addgene:163911 ; RRID:Addgene_163911) |
| 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-Bsu 6997 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expresses Bsu large fragment for affinity purification.
ACCESSION .
VERSION .
KEYWORDS pET28a-MH6-Bsu
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6997)
TITLE One Pot CRISPR RPA
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6997)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6997
/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(4046..4064)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 4060..4107
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 4144..4599
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4231..4250)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4441..4462
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(4691..5506)
/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 5414..5433
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 5498..5517
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 5628..6216
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6117..6136
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6370..6387
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 6402..6541
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(6467..6486)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6627..6649
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(6643..6834)
/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 gcgactcctg 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 gatcagagcc tggaagatat taatgttaaa accgttaccg atgtgaccag
2341 cgatattctg gttagcccga gcgcatttgt tgttgagcag attggtgata actatcacga
2401 agaaccgatt ctgggtttta gcattgttaa tgaaaccggt gcctatttca tcccgaaaga
2461 tattgcagtt gaaagcgaag tgttcaaaga atgggttgaa aacgacgagc agaaaaaatg
2521 ggtgtttgat agcaaacgtg cagttgttgc actgcgttgg cagggtattg aactgaaagg
2581 tgcagaattt gataccctgc tggcagccta tattatcaat ccgggtaata gctatgatga
2641 tgttgcaagc gttgccaaag attatggtct gcatattgtt agcagtgatg aaagcgttta
2701 tggcaaaggt gcaaaacgtg ccgttccgag cgaagatgtt ctgagcgaac atctgggtcg
2761 taaagcactg gcaattcaga gtctgcgtga aaaactggtt caagaactgg aaaataacga
2821 tcagctggaa ctgtttgaag aattagaaat gccgctggca ctgattctgg gcgaaatgga
2881 aagcaccggt gttaaagttg atgttgatcg tctgaaacgt atgggtgaag aactgggtgc
2941 caaactgaaa gaatatgaag aaaaaatcca cgagattgcc ggtgaaccgt ttaatatcaa
3001 tagcccgaaa cagctgggtg tgatcctgtt tgaaaaaatt ggtctgccgg ttgtgaaaaa
3061 aaccaaaacc ggttatagca ccagcgcaga tgtgctggaa aaattagcag ataaacatga
3121 catcgtggac tacattctgc agtatcgtca gattggtaaa ctgcagagca cctatattga
3181 aggtctgctg aaagttaccc gtccggatag ccataaagtt catacccgtt ttaatcaggc
3241 actgacccag accggtcgtc tgagcagcac cgatccgaat ctgcagaata ttccgattcg
3301 tctggaagaa ggtcgtaaaa ttcgtcaggc atttgtgccg agcgaaaaag attggctgat
3361 ttttgcagca gattatagcc agattgaact gcgtgttctg gcacatatta gcaaagatga
3421 aaatctgatc gaagccttca ccaacgatat ggatattcat accaaaacag ccatggatgt
3481 gtttcatgtt gcgaaagatg aagttaccag cgcaatgcgt cgtcaggcca aagcagttaa
3541 ttttggtatt gtgtatggca tcagcgatta tggcctgagc cagaatctgg gtattacccg
3601 taaagaagca ggcgcattta ttgatcgcta tctggaaagt tttcagggcg ttaaagcata
3661 catggaagat agcgttcaag aggcaaaaca gaaaggttat gttaccacac tgatgcatcg
3721 tcgtcgttat attccggaac tgaccagccg taatttcaat attcgtagct ttgcagaacg
3781 taccgcaatg aataccccga ttcagggtag cgcagcagat atcatcaaaa aagcaatgat
3841 tgacatggca gcgaagctga aagaaaaaca gctgaaagca cgtctgctgc tgcaagtgca
3901 tgatgaactg atctttgaag caccgaaaga agaaatcgag atcctggaaa agctggttcc
3961 ggaagttatg gaacatgcac tggcactgga tgttccgctg aaagtggatt ttgcaagcgg
4021 tccgagctgg tatgatgcaa aataaccgct gagcaataac tagcataacc ccttggggcc
4081 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
4141 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
4201 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
4261 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
4321 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
4381 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
4441 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
4501 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
4561 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
4621 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
4681 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
4741 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
4801 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
4861 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
4921 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
4981 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
5041 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
5101 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
5161 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
5221 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
5281 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
5341 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
5401 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
5461 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
5521 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
5581 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
5641 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
5701 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
5761 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
5821 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
5881 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
5941 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
6001 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
6061 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
6121 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
6181 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
6241 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
6301 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
6361 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
6421 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
6481 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
6541 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
6601 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
6661 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
6721 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
6781 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
6841 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
6901 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
6961 acaactggcg gtatggatgc ggcgggacca gagaaaa
//
