质粒名称: | pAtetO_6xHis-Klentaq |
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出品公司: | Addgene |
目录编号: | 157862 |
存储实验室: | Andrew Ellington |
载体骨架基本信息 |
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载体名称: | pAtetO |
载体出品公司: | modified from pASK-IBA37plus (IBA GmbH) |
载体骨架修饰: | 从载体pASK-IBA37plus中,去除了His标签,多克隆位点以及Rop基因。 |
载体抗性: | 氨苄霉素 |
载体类型: | 细菌表达 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Klentaq |
基因又名: | Klentaq polymerase |
物种来源: | Thermus aquaticus |
插入大小: | 1710 bp |
基因突变: | T2A, S13L, A92T, V104T, V114I, E161K, Y193K, F198Y, D202T, I203V, Y258F, A283V, K288T, N290Y relative to RpBphP2 |
GenBank编号 | JN247409 |
启动子 | tet PA |
标签/融合蛋白: | N-6xHis |
克隆方法: | Gibson克隆连接 |
5’测序引物 |
TTTGTTTAACTTTAAGAAGGAGATATACATATGGCTAGCAGAGGATCGC ATCACCATCACCATCACATCGAAGGGCGCCTTCTTCACGAGTTCGGAC |
3’测序引物 |
GAAGATTGGTTATCTGCGAAAGAATGATAATTGCCT CTGCCAAAATTCTGTCCTCAAGCG |
文献引用方法 |
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材料和方法部分: |
pAtetO_6xHis-Klentaq was a gift from Andrew Ellington (Addgene plasmid # 157862 ; http://n2t.net/addgene:157862 ; RRID:Addgene_157862) |
参考文献部分: |
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 |
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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 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 http://www.biofeng.com 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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