| 质粒名称: | pET-28a_6H-MMLV_RT_D524N-6H |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 166945 |
| 存储实验室: | Robert Tjian |
|
载体骨架基本信息 |
|
| 载体名称: | pET-28a(+) |
| 载体出品公司: | Novagen (EMD Millipore) |
| 空载体大小: | 5369 bp |
| 完整质粒大小: | 7306 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | Kanamycin, 50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | gag-pol、Pr180、M-MLV Reverse Transcriptase |
| 物种来源: | Moloney murine leukemia virus |
| 插入大小: | 2100 bp |
| 基因突变: | D524N, H8Y |
| GenBank编号 | 2193424 AAC82568 |
| 启动子: | T7 |
| 标签/融合蛋白: |
6X His (N terminal on backbone)
6X His (C terminal on backbone)
|
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NdeI (not destroyed) |
| 3’克隆位点: | XhoI (not destroyed) |
| 5’测序引物 | taatacgactcactataggg |
| 3’测序引物 |
TAGTTATTGCTCAGCGGTGG
(Common Sequencing Primers)
|
|
文献引用方法 |
|
| 材料和方法部分: | pET-28a_6H-MMLV_RT_D524N-6H was a gift from Robert Tjian (Addgene plasmid # 166945 ; http://n2t.net/addgene:166945 ; RRID:Addgene_166945) |
| 参考文献部分: | Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection. Graham TGW, Dugast-Darzacq C, Dailey GM, Nguyenla XH, Van Dis E, Esbin MN, Abidi A, Stanley SA, Darzacq X, Tjian R. PLoS One. 2021 Feb 3;16(2):e0246647. doi: 10.1371/journal.pone.0246647. eCollection 2021. 10.1371/journal.pone.0246647 PubMed 33534838 |
买家导航
pET-28a_6H-MMLV_RT_D524N-6H载体质粒基本信息
pET-28a_6H-MMLV_RT_D524N-6H质粒图谱载体图谱和pET-28a_6H-MMLV_RT_D524N-6H载体序列质粒序列多克隆位点信息
pET-28a_6H-MMLV_RT_D524N-6H质粒载体简介
pET-28a_6H-MMLV_RT_D524N-6H质粒序列载体序列
LOCUS pET-28a_6H-MMLV_RT_D524N-6H 7302 bp DNA circular SYN 14-APR-2025
DEFINITION Bacterial expression of M-MLV reverse transcriptase.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7302)
AUTHORS Graham TGW, Dugast-Darzacq C, Dailey GM, Nguyenla XH, Van Dis E,
Esbin MN, Abidi A, Stanley SA, Darzacq X, Tjian R
TITLE Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2
detection.
JOURNAL PLoS One. 2021 Feb 3;16(2):e0246647. doi:
10.1371/journal.pone.0246647. eCollection 2021.
PUBMED 33534838
REFERENCE 2 (bases 1 to 7302)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
date: "2021-02-3"; volume: "16(2):e0246647. doi"; pages: "
10.1371/journal.pone.0246647. eCollection 2021"
COMMENT Sequence Label: pET-28a_6H-MMLV_RT_D524N-6H
FEATURES Location/Qualifiers
source 1..7302
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 973..992
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1012..1089)
/gene="lacI"
/label=lacI promoter
primer_bind 1295..1314
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 1398..1417
/label=T7
/note="T7 promoter, forward primer"
promoter 1398..1416
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1417..1441
/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)."
RBS 1456..1478
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 1497..1514
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1524..1541
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 3564..3581
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3634..3652)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3648..3695
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 3732..4187
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3819..3838)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4029..4050
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(4279..5094)
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
CDS complement(4279..5094)
/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 5002..5021
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 5086..5105
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 5216..5804
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5705..5724
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5958..5975
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5990..6129
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(6055..6074)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6215..6237
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(6231..6422)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(join(7231..7302,1..1011))
/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"
ORIGIN
1 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
1501 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatgacc ctaaatatag
1561 aagatgagta tcggctacat gagacctcaa aagagccaga tgtttctcta gggtccacat
1621 ggctgtctga ttttcctcag gcctgggcgg aaaccggggg catgggactg gcagttcgcc
1681 aagctcctct gatcatacct ctgaaagcaa cctctacccc cgtgtccata aaacaatacc
1741 ccatgtcaca agaagccaga ctggggatca agccccacat acagagactg ttggaccagg
1801 gaatactggt accctgccag tccccctgga acacgcccct gctacccgtt aagaaaccag
1861 ggactaatga ttataggcct gtccaggatc tgagagaagt caacaagcgg gtggaagaca
1921 tccaccccac cgtgcccaac ccttacaacc tcttgagcgg gctcccaccg tcccaccagt
1981 ggtacactgt gcttgattta aaggatgcct ttttctgcct gagactccac cccaccagtc
2041 agcctctctt cgcctttgag tggagagatc cagagatggg aatctcagga caattgacct
2101 ggaccagact cccacagggt ttcaaaaaca gtcccaccct gtttgatgag gcactgcaca
2161 gagacctagc agacttccgg atccagcacc cagacttgat cctgctacag tacgtggatg
2221 acttactgct ggccgccact tctgagctag actgccaaca aggtactcgg gccctgttac
2281 aaaccctagg gaacctcggg tatcgggcct cggccaagaa agcccaaatt tgccagaaac
2341 aggtcaagta tctggggtat cttctaaaag agggtcagag atggctgact gaggccagaa
2401 aagagactgt gatggggcag cctactccga agacccctcg acaactaagg gagttcctag
2461 ggacggcagg cttctgtcgc ctctggatcc ctgggtttgc agaaatggca gcccccttgt
2521 accctctcac caaaacgggg actctgttta attggggccc agaccaacaa aaggcctatc
2581 aagaaatcaa gcaagctctt ctaactgccc cagccctggg gttgccagat ttgactaagc
2641 cctttgaact ctttgtcgac gagaagcagg gctacgccaa aggtgtccta acgcaaaaac
2701 tgggaccttg gcgtcggccg gtggcctacc tgtccaaaaa gctagaccca gtagcagctg
2761 ggtggccccc ttgcctacgg atggtagcag ccattgccgt actgacaaag gatgcaggca
2821 agctaaccat gggacagcca ctagtcattc tggcccccca tgcagtagag gcactagtca
2881 aacaaccccc cgaccgctgg ctttccaacg cccggatgac tcactatcag gccttgcttt
2941 tggacacgga ccgggtccag ttcggaccgg tggtagccct gaacccggct acgctgctcc
3001 cactgcctga ggaagggctg caacacaact gccttgatat cctggccgaa gcccacggaa
3061 cccgacccga cctaacggac cagccgctcc cagacgccga ccacacctgg tatacgaatg
3121 gaagcagtct cttacaagag ggacagcgta aggcgggagc tgcggtgacc accgagaccg
3181 aggtaatctg ggctaaagcc ctgccagccg ggacatccgc tcagcgggct gaactgatag
3241 cactcaccca ggccctaaag atggcagaag gtaagaagct aaatgtttat actgatagcc
3301 gttatgcttt tgctactgcc catatccatg gagaaatata cagaaggcgt gggttgctca
3361 catcagaagg caaagagatc aaaaataaag acgagatctt ggccctacta aaagccctct
3421 ttctgcccaa aagacttagc ataatccatt gtccaggaca tcaaaaggga cacagcgccg
3481 aggctagagg caaccggatg gctgaccaag cggcccgaaa ggcagccatc acagagactc
3541 cagacacctc taccctcctc gagcaccacc accaccacca ctgagatccg gctgctaaca
3601 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
3661 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
3721 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
3781 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
3841 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
3901 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
3961 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
4021 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
4081 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
4141 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
4201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
4261 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
4321 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
4381 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
4441 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
4501 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
4561 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
4621 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
4681 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
4741 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
4801 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
4861 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
4921 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
4981 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
5041 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
5101 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
5161 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
5221 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
5281 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
5341 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
5401 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
5461 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
5521 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
5581 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
5641 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
5701 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
5761 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
5821 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
5881 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
5941 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
6001 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
6061 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
6121 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
6181 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
6241 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
6301 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
6361 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
6421 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
6481 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
6541 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
6601 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
6661 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
6721 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
6781 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
6841 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
6901 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
6961 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
7021 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
7081 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
7141 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
7201 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
7261 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga ga
//
