| 質粒名稱: | pAtetO_6xHis-Klentaq |
|---|---|
| 出品公司: | Addgene |
| 目錄編號: | 157862 |
| 存儲實驗室: | Andrew Ellington |
|
載體骨架基本信息 |
|
| 載體名稱: | pAtetO |
| 載體出品公司: | modified from pASK-IBA37plus (IBA GmbH) |
| 載體骨架修飾: | 從載體pASK-IBA37plus中，去除了His標簽，多克隆位點以及Rop基因。 |
| 載體抗性: | 氨芐霉素 |
| 載體類型: | 細菌表達 |
| 高拷貝/低拷貝: | 高拷貝 |
| 生長條件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名稱: | Klentaq |
| 基因又名: | Klentaq polymerase |
| 物種來源: | Thermus aquaticus |
| 插入大小: | 1710 bp |
| 基因突變: | T2A, S13L, A92T, V104T, V114I, E161K, Y193K, F198Y, D202T, I203V, Y258F, A283V, K288T, N290Y relative to RpBphP2 |
| GenBank編號 | JN247409 |
| 啟動子 | tet PA |
| 標簽/融合蛋白: | N-6xHis |
| 克隆方法: | Gibson克隆連接 |
| 5’測序引物 |
TTTGTTTAACTTTAAGAAGGAGATATACATATGGCTAGCAGAGGATCGC ATCACCATCACCATCACATCGAAGGGCGCCTTCTTCACGAGTTCGGAC |
| 3’測序引物 |
GAAGATTGGTTATCTGCGAAAGAATGATAATTGCCT CTGCCAAAATTCTGTCCTCAAGCG |
|
文獻引用方法 |
|
| 材料和方法部分: |
pAtetO_6xHis-Klentaq was a gift from Andrew Ellington (Addgene plasmid # 157862 ; //n2t.net/addgene:157862 ; RRID:Addgene_157862) |
| 參考文獻部分: |
Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication. Milligan JN, Shroff R, Garry DJ, Ellington AD. Biochemistry. 2018 Aug 7;57(31):4607-4619. doi: 10.1021/acs. biochem.8b00200. Epub 2018 Apr 18. 10.1021/acs.biochem.8b00200 PubMed 29629759 |
買家導航
pAtetO_6xHis-Klentaq載體質粒基本信息
pAtetO_6xHis-Klentaq質粒圖譜載體圖譜和pAtetO_6xHis-Klentaq載體序列質粒序列多克隆位點信息
pAtetO_6xHis-Klentaq質粒載體簡介
pAtetO_6xHis-Klentaq質粒序列載體序列
LOCUS pAtetO_6xHis-Klentaq 4504 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Plasmid for the aTc-inducible expression of the Klentaq.
ACCESSION .
VERSION .
KEYWORDS pAtetO_6xHis-Klentaq
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4504)
AUTHORS Milligan JN, Shroff R, Garry DJ, Ellington AD
TITLE Evolution of a Thermophilic Strand-Displacing Polymerase Using
High-Temperature Isothermal Compartmentalized Self-Replication.
JOURNAL Biochemistry. 2018 Aug 7;57(31):4607-4619. doi:
10.1021/acs.biochem.8b00200. Epub 2018 Apr 18.
PUBMED 29629759
REFERENCE 2 (bases 1 to 4504)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
//gt12365.cn
FEATURES Location/Qualifiers
source 1..4504
/organism="synthetic DNA construct"
/mol_type="other DNA"
RBS 14..36
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 62..79
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 80..91
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
rep_origin complement(1957..2545)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2037..2056)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2698..3324)
/codon_start=1
/gene="tetR"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRDNAKSFRCALLSHRDGAKVHLGT
RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
primer_bind 3229..3248
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
CDS complement(3331..4191)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 3954..3973
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4192..4296)
/gene="bla"
/label=AmpR promoter
promoter 4449..4504
/gene="tetR"
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
protein_bind 4455..4473
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O1 for the tetR and tetA genes"
protein_bind 4485..4503
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
1 aactagaaat aattttgttt aactttaaga aggagatata catatggcta gcagaggatc
61 gcatcaccat caccatcaca tcgaagggcg ccttcttcac gagttcggac ttctcgagtc
121 gcccaaggcc ctggaagagg cgccgtggcc tcctccagag ggggctttcg tgggatttgt
181 gctgagtcga aaagaaccca tgtgggctga tttactcgcg ctggctgcgg caagaggggg
241 acgtgtccat cgtgctcctg agccctataa agcactgcgc gatctcaagg aggctcgtgg
301 cctcctcgcc aaggatcttt ctgttttggc cctgcgggag ggattggggc ttcctccggg
361 cgacgatccc atgttacttg cgtatttatt ggaccccagt aacacgacac ctgaaggagt
421 agcccgtcgc tatggagggg aatggaccga agaagcggga gaacgtgcgg cactgtcgga
481 acgcctcttt gcgaacttat ggggccgtct tgaaggtgaa gagcgtcttc tgtggcttta
541 ccgcgaagtg gagcgtccgc tgtcggcagt tctcgctcat atggaagcta cgggtgttag
601 acttgacgtg gcatacctgc gtgcgttgtc gttagaagtg gccgaagaga ttgctagact
661 cgaagcagaa gtttttcggt tagcgggaca tccatttaat ctcaattccc gtgaccagct
721 ggagcgcgtt ctctttgatg aattggggct ccctgccatt gggaaaaccg aaaaaaccgg
781 taaacgctca accagtgctg ctgtgctgga ggcactgcgt gaggcccatc cgatcgtgga
841 gaaaattctg cagtaccggg agctcactaa gttgaaaagc acatatatcg accccctgcc
901 ggatttaatt cacccgcgca ccggaagact ccacacaaga tttaaccaga cggcgaccgc
961 aaccggacgc ctgtcctcat ccgatccgaa cttgcagaac atcccggtcc gtactccgtt
1021 gggtcagcgg atccggcggg catttatcgc cgaggaaggt tggcttctgg tcgcgctgga
1081 ctacagccaa atcgaattgc gtgtgttagc acacctttcg ggagacgaaa atttgattcg
1141 ggtgtttcaa gagggtcgtg atattcacac ggaaaccgcg agttggatgt ttggggtgcc
1201 tcgagaggca gtggatccac ttatgcgtcg tgcagcaaaa acaatcaatt tcggcgtcct
1261 ctatggtatg agcgctcacc gtctttctca ggaacttgcc atcccctacg aagaggccca
1321 ggcatttatt gagcgttatt tccagagctt tccgaaagtt agagcctgga ttgagaaaac
1381 cttggaagaa ggtcggcgtc gggggtatgt ggagactctt tttggtcgtc ggcgctacgt
1441 accggatctg gaagctcgtg tcaagtctgt gcgcgaagct gccgaacgta tggcatttaa
1501 tatgccggtc caggggaccg cagcagatct tatgaagtta gcaatggtca aattgttccc
1561 ccggcttgaa gagatggggg cgcgtatgct gcttcaggta catgatgagc ttgtcctgga
1621 agccccgaag gaacgagcgg aagctgtggc ccgtcttgct aaagaggtca tggaaggagt
1681 ctacccactg gccgtgccgt tggaagtgga ggtcggcatt ggagaagatt ggttatctgc
1741 gaaagaatga taattgcctc tgccaaaatt ctgtcctcaa gcgttttagt tcgtcggtca
1801 gtttcacctg atttacgtaa aaacccgctt cggcgggttt ttgcttttgg aggggcagaa
1861 agatgaatga ctgtcggcca ttcgatggtg tcgggtcatg tgagcaaaag gccagcaaaa
1921 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
1981 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
2041 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
2101 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
2161 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
2221 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
2281 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
2341 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
2401 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
2461 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
2521 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
2581 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
2641 cacctagatc cttttaaact agtgaagtta ccatcacgga aaaaggttat gctgctttta
2701 agacccactt tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa
2761 taagaaggct ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg
2821 cggcatacta tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc
2881 ttccaatacg caacctaaag taaaatgccc cactgcgctg agtgcatata atgcattctc
2941 tagtgaaaaa ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt
3001 ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca
3061 tctaaaactt ttagcgttat cacgtaaaaa atcttgccag ctttcccctt ctaaagggca
3121 aaagtgagta tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt
3181 attttttaca tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg
3241 ggttgttaaa ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt
3301 acttttatct aaacgagaca tcattaattc ctaccaatgc ttaatcagtg aggcacctat
3361 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
3421 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagagccacg
3481 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
3541 tggtcctgca actttatccg cctccatcca gtctatcaat tgttgccggg aagctagagt
3601 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
3661 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
3721 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
3781 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
3841 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
3901 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
3961 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
4021 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
4081 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
4141 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
4201 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
4261 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttcccggga actgccagac
4321 atcaaataaa acaaaaggct cagtcggaag actgggcctt ttgttttatc tgttgtttgt
4381 cggtgaacac tctcccgatt gcgatacaga ccctaatttc acatcatatg acactaatcc
4441 taatttttgt tgacactcta tcattgatag agttatttta cctctcccta tcagtgatag
4501 agaa
//
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