| 质粒名称: | pET-6His-MBP-MpAgo |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 79249 |
| 存储实验室: | Jennifer Doudna |
|
载体骨架基本信息 |
|
| 载体名称: | pET 2ct |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5942bp |
| 完整质粒大小: | 7856 bp |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Marinitoga piezophila Argonaute |
| 物种来源: | 光合细菌 Rhodopseudomonas palustris |
| 插入大小: | 1914bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AgeI(未破坏) |
| 3' 克隆位点: | NotI (未破坏) |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: | pET-6His-MBP-MpAgo was a gift from Jennifer Doudna (Addgene plasmid # 79249 ; http://n2t.net/addgene:79249 ; RRID:Addgene_79249) |
| 参考文献部分: | A bacterial Argonaute with noncanonical guide RNA specificity. Kaya E, Doxzen KW, Knoll KR, Wilson RC, Strutt SC, Kranzusch PJ, Doudna JA. Proc Natl Acad Sci U S A. 2016 Mar 30. pii: 201524385. 10.1073/pnas.1524385113 PubMed 27035975 |
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 pET-6His-MBP-MpAgo 7868 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION This plasmid encodes the Argonaute protein from Marinitoga
Piezophila and is codon optimized for E. coli expression.
ACCESSION .
VERSION .
KEYWORDS pET-6His-MBP-MpAgo
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7868)
AUTHORS Kaya E, Doxzen KW, Knoll KR, Wilson RC, Strutt SC, Kranzusch PJ,
Doudna JA
TITLE A bacterial Argonaute with noncanonical guide RNA specificity.
JOURNAL Proc Natl Acad Sci U S A. 2016 Mar 30. pii: 201524385.
PUBMED 27035975
REFERENCE 2 (bases 1 to 7868)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7868
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(18..36)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 104..208
/gene="bla"
/label=AmpR promoter
CDS 209..1069
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(427..446)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1240..1828
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1729..1748
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 1982..1999
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 2014..2156
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(2082..2101)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2242..2264
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2258..2449)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind 3907..3926
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 4010..4029
/label=T7
/note="T7 promoter, forward primer"
promoter 4010..4028
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 4117..4139
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 4159..4188
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 4198..5298
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/label=MBP
/note="This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol."
/translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
primer_bind 5272..5295
/label=MBP-F
/note="Maltose binding protein, forward primer"
primer_bind complement(7404..7422)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 7418..7465
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(7831..7859)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
ORIGIN
1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt
3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc
3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa
4081 gcggccgccc tgcaggactc gagttctaga aatatttttg tttaacttta agaaggagat
4141 atacatatga aatcttctca ccatcaccat caccatcacc atcaccatgg ttcttctatg
4201 aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
4261 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
4321 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
4381 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
4441 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
4501 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
4561 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
4621 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
4681 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
4741 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
4801 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
4861 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
4921 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
4981 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
5041 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
5101 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact
5161 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
5221 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
5281 ctgaaagacg cgcagactaa ttcgagctcg gattacgata tcccaacgac cctagaagtg
5341 ctttttcagg gcgccaatgc atacctgaat ctgtataaaa tcgacatccc gaagaaaatc
5401 aaacgcttgt atttctataa tccggatatg gagccaaagc tcttcgcacg taatctgtcc
5461 cgtgttaaca actttaagtt ccaggactcg aatgaccttg tgtggattga aatccctgac
5521 atcgattttc aaatcacacc taaaaatgta tttcagtaca aagttgagaa agaagaaatt
5581 atcaaagaag aagaagataa aaagctgttt gtcaagacac tgtataaata catcaagaag
5641 ctgttcctcg ataacgattt ctattttaaa aaaggcaaca actttattag caactcggag
5701 gtcttcagct tagatagcaa tgaaaatgta aacgcccatt taacttataa aatcaagatt
5761 cataacattt cgaatgagta ctatctgtca attcttccga aatttacctt tctttcgaag
5821 gaaccggcat tggaaagcgc aatcaaaagc ggttatttgt ataatatcaa gtcgggcaag
5881 tctttcccat atattagcgg cctggacgga atcctgaaaa ttgacatcgg aaataatcag
5941 atcgtggaag tggcgtatcc ggaaaattat ctgtttaact ttaccacgcg cgatgctgaa
6001 aaatatggct ttagcaaaga agtacatgag atctataaaa acaaagtctt cgaaggcttt
6061 aaaaaaatcc cgaagactct gggcttctta aataaaatta caaatctgaa cgagaattac
6121 cagttgaaag acggctacaa aatcttcatt aacgtgatct ataaattcaa aaacggcgag
6181 agccgttatg ccaaagatgt gtttaaatac tccttttata aaaatgaaca gccattaaaa
6241 gccatcttct ttttctcttc gaaaaaacaa ttttttgaag tgcagaaatc gcttaaagaa
6301 ctgtttcata acaaacactc ggtgttctat cgcgccgccg cggagctggg cttctctaaa
6361 gtcgaatttt tacgtgatag caaaacgaaa tcctctgcct tcctgtacaa tccggaggag
6421 ttcaccgtga aaaatacgga atttattaac caaatcgaag ataatgttat ggccatcgtc
6481 ttactggaca aatacattgg taatatcgat ccgctcgtac gtaattttcc agataattta
6541 atccttcaac cgatcctgaa agaaaaattg gaagatatta aaccttttat catcaaatca
6601 tatgtgtata aaatgggaaa ctttatccca gaatgcaagc ctttcatcct gaaaaaaatg
6661 gaagataaag agaaaaactt gtatattggg attgatcttt cccatgatac gtacgcacgt
6721 aaaactaacc tgtgtattgc ggccgttgat aacaccggcg atatcttgta tattggcaaa
6781 cataaaaacc tggaattgaa cgaaaagatg aatttggata ttctcgaaaa agaatatatt
6841 aaggcttttg aaaagtatat tgagaaattc aacgtgtccc cggaaaatgt atttattttg
6901 cgcgatggcc ggtttatcga agacattgag attattaaaa atttcatctc atataatgat
6961 actaagtata cactggtgga agtaaataaa aatacgaaca ttaatagtta tgacgatctc
7021 aaagaatgga ttattaaact ggacgaaaac acctatatct attacccgaa aacttttctg
7081 aatcaaaaag gggttgaagt taaaatcctg gagaacaaca ccgattatac gattgaagaa
7141 atcattgagc agatctatct gctgacccgt gtggcgcaca gcaccccgta cactaactat
7201 aaattgccgt acccgctcca catcgcaaat aaggtggcgt tgacggatta cgagtggaag
7261 ctgtatattc cgtactaata acattggaag tggataacgg atccgcgatc gcggcgcgcc
7321 acctggtggc cggccggtac cacgcgtgcg cgctgatccg gctgctaaca aagcccgaaa
7381 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
7441 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atccacagga
7501 cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga
7561 ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat agaaattgca
7621 tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg gaatggacga
7681 tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg
7741 tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc
7801 gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa
7861 catgagaa
//
