质粒名称: | 2Bc-T MBP_BoMoC(ed)_F753A_6xH |
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出品公司: | addgene |
目录编号: | 185710 |
存储实验室: | Kathleen Collins |
载体骨架基本信息 |
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载体名称: | pET His6 LIC cloning vector (2Bc-T) |
载体出品公司: | Scott Gradia |
空载体大小: | 5899 bp |
完整质粒大小: | 8416 bp |
载体类型: | 细菌细胞表达 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 低拷贝 |
插入基因信息 |
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插入基因名称: | B. mori truncated R2 RT / BoMoC |
物种来源: | Bombyx mori |
插入大小: | 2520 bp |
基因突变: | Changed F753 to A |
启动子: | T7 |
标签/融合蛋白: |
MBP (N terminal on insert)
6xHis (C terminal on insert)
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克隆方法: | Ligation Independent Cloning |
5’测序引物 | T7 promoter |
3’测序引物 | T7 terminator |
文献引用方法 |
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材料和方法部分: | 2Bc-T MBP_BoMoC(ed)_F753A_6xH was a gift from Kathleen Collins (Addgene plasmid # 185710 ; http://n2t.net/addgene:185710 ; RRID:Addgene_185710) |
参考文献部分: | Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase. Pimentel SC, Upton HE, Collins K. J Biol Chem. 2022 Mar;298(3):101624. doi: 10.1016/j.jbc.2022.101624. Epub 2022 Jan 21. 10.1016/j.jbc.2022.101624 PubMed 35065960 |
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LOCUS 2Bc-T MBP_BoMoC(ed)_F753A_6xH 8416 bp ds-DNA circular SYN 01-AUG-2022 DEFINITION Recombinant B. mori truncated R2 RT expression. ACCESSION . VERSION . KEYWORDS 2Bc-T MBP_BoMoC(ed)_F753A_6xH SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8416) AUTHORS Pimentel SC, Upton HE, Collins K TITLE Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase. JOURNAL J Biol Chem. 2022 Mar;298(3):101624. doi: 10.1016/j.jbc.2022.101624. Epub 2022 Jan 21. PUBMED 35065960 REFERENCE 2 (bases 1 to 8416) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 1, 2022 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..8416 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 168..187 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 421..438 /label=L4440 /note="L4440 vector, forward primer" misc_feature 453..592 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(518..537) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 678..700 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(694..885) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 2343..2362 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 2446..2465 /label=T7 /note="T7 promoter, forward primer" promoter 2446..2464 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 2553..2575 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 6223..6243 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 6250..6267 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6391..6409) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6405..6452 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(6818..6846) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(6873..6891) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6959..7063 /gene="bla" /label=AmpR promoter CDS 7064..7924 /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(7282..7301) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8095..267 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 61 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 121 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 181 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 241 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 301 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 361 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 421 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat 481 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat 541 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca 601 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 661 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 721 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc 781 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct 841 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta 901 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat 961 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc 1021 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa 1081 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat 1141 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac 1201 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc 1261 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct 1321 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg 1381 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg 1441 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt 1501 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc 1561 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat 1621 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg 1681 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg 1741 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg 1801 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc 1861 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg 1921 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc 1981 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct 2041 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc 2101 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta 2161 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc 2221 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac gcggcctgcc 2281 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca 2341 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc 2401 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta 2461 tagggagacc acaacggttt ccctctagtg ccggctccgg agagctcttt aattaagcgg 2521 ccgccctgca ggactcgagt tctagaaata attttgttta actttaagaa ggagatatag 2581 ttcatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 2641 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 2701 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 2761 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 2821 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 2881 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 2941 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 3001 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 3061 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 3121 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 3181 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 3241 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 3301 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 3361 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 3421 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 3481 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc 3541 gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 3601 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 3661 gaagccctgg cagccgcgca gactgctgcg gccgcagcta tgggtcgtcg tcgtgcgtgc 3721 cgtgcgatgc gtccgaagac cgcgggtcgt cgtaacgacc tgcacgacga tcgtaccgcg 3781 agcgcgcaca agaccagccg tcagaaacgt cgtgcggagt acgcgcgtgt gcaagaactg 3841 tataagaaat gccgtagccg tgcggcggcg gaagtgatcg atggtgcgtg cggtggcgtt 3901 ggtcacagcc tggaggaaat ggaaacctac tggcgtccga ttctggaacg tgtgagcgac 3961 gcgccgggtc cgaccccgga ggcgctgcac gcgctgggtc gtgcggaatg gcacggtggc 4021 aaccgtgatt atacccagct gtggaagccg atcagcgttg aggaaattaa agcgagccgt 4081 ttcgactggc gtaccagccc gggtccggat ggtatccgta gcggccagtg gcgtgcggtg 4141 ccggttcacc tgaaggcgga aatgttcaac gcgtggatgg cgcgtggcga gatcccggaa 4201 attctgcgtc aatgccgtac cgtgtttgtt ccgaaagttg agcgtccggg tggcccgggt 4261 gaataccgtc cgatcagcat tgcgagcatc ccgctgcgtc acttccacag cattctggcg 4321 cgtcgtctgc tggcgtgctg cccgccggac gcgcgtcagc gtggctttat ctgcgcggat 4381 ggtaccctgg agaacagcgc ggtgctggac gcggttctgg gtgatagccg taagaaactg 4441 cgtgaatgcc acgtggcggt tctggacttc gcgaaggcgt ttgataccgt gagccacgag 4501 gcgctggttg aactgctgcg tctgcgtggc atgccggagc agttctgcgg ttacattgcg 4561 cacctgtatg acaccgcgag caccaccctg gcggtgaaca acgaaatgag cagcccggtg 4621 aaagttggcc gtggtgttcg tcaaggcgac ccgctgagcc cgatcctgtt taacgtggtt 4681 atggatctga ttctggcgag cctgccggag cgtgtgggtt accgtctgga gatggaactg 4741 gttagcgcgc tggcgtatgc ggacgatctg gtgctgctgg cgggcagcaa ggttggtatg 4801 caggaaagca tcagcgcggt ggactgcgtt ggccgtcaaa tgggtctgcg tctgaactgc 4861 cgtaaaagcg cggtgctgag catgatcccg gatggtcacc gtaagaaaca ccactacctg 4921 accgagcgta ccttcaacat tggtggcaag ccgctgcgtc aggtgagctg cgttgaacgt 4981 tggcgttatc tgggcgtgga ctttgaggcg agcggttgcg ttaccctgga acacagcatc 5041 agcagcgcgc tgaacaacat tagccgtgcg ccgctgaaac cgcagcaacg tctggagatc 5101 ctgcgtgcgc acctgattcc gcgtttccag cacggcgccg ttctgggtaa catcagcgac 5161 gatcgtctgc gtatgctgga tgtgcagatt cgtaaggcgg ttggtcaatg gctgcgtctg 5221 ccggcggacg tgccgaaagc gtactatcac gcggcggttc aagatggtgg cctggcgatc 5281 ccgagcgtgc gtgcgaccat cccggacctg attgttcgtc gttttggtgg cctggatagc 5341 agcccgtgga gcgtggcgcg tgcggcggcg aagagcgaca aaattcgtaa gaaactgcgt 5401 tgggcgtgga agcagctgcg tcgtttcagc cgtgtggata gcaccaccca acgtccgagc 5461 gttcgtctgt tttggcgtga gcacctgcac gcgagcgttg acggtcgtga gctgcgtgaa 5521 agcacccgta ccccgaccag caccaaatgg attcgtgaac gttgcgcgca gattaccggt 5581 cgtgatttcg tgcaatttgt tcacacccac atcaacgcgc tgccgagccg tattcgtggc 5641 agccgtggcc gtcgtggtgg cggtgagagc agcctgacct gccgtgcggg ttgcaaagtg 5701 cgtgaaacca ccgcgcacat cctgcagcaa tgccaccgta cccacggcgg tcgtatcctg 5761 cgtcacaaca agattgtgag cttcgttgcg aaggcgatgg aggaaaacaa atggaccgtg 5821 gagctggaac cgcgtctgcg taccagcgtt ggcctgcgta aaccggcgat cattgcgagc 5881 cgtgatggcg tgggtgttat cgtggacgtt caggtggtta gcggtcaacg tagcctggat 5941 gagctgcacc gtgaaaagcg taacaaatac ggcaaccacg gtgagctggt tgagctggtt 6001 gcgggccgtc tgggtctgcc gaaagcggag tgcgtgcgtg cgaccagctg caccattagc 6061 tggcgtggcg tttggagcct gaccagctat aaagagctgc gtagcatcat tggtctgcgt 6121 gaaccgaccc tgcagatcgt gccgattctg gcgctgcgtg gcagccacat gaactggacc 6181 cgttttaacc aaatgaccag cgtgatgggt ggcggtgttg gtgagaacct ctacttccaa 6241 tccggctctc atcaccatca ccatcactaa taaccaactc cataaggatc cgcgatcgcg 6301 gcgcgccacc tggtggccgg ccggtaccac gcgtgcgcgc tgatccggct gctaacaaag 6361 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg 6421 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggacatc 6481 cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg 6541 agcaggactg ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga 6601 aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa 6661 tggacgacat cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca 6721 tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc 6781 tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc 6841 tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt 6901 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 6961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 7021 ataaccctga taaatgcttc aataacattg aaaaaggaag agtatgagta ttcaacattt 7081 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 7141 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 7201 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 7261 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 7321 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 7381 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 7441 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 7501 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 7561 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac 7621 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 7681 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 7741 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 7801 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 7861 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 7921 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7981 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 8041 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 8101 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 8161 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 8221 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 8281 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 8341 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 8401 gcggtcgggc tgaacg //
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