质粒名称: | pJL-SaKKH-BE3 |
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出品公司: | 爱迪基因 |
目录编号: | 85170 |
存储实验室: | David Liu |
载体骨架基本信息 |
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载体名称: | CMV |
载体出品公司: | origene |
载体抗性: | 氨苄青霉素 |
空载体大小: | 3399 bp |
完整质粒大小: | 7689 bp |
载体类型: | 哺乳细胞表达 |
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: | SaKKH-BE3,rAPOBEC1-XTEN-SaKKHCas9n-UGI-NLS |
物种来源: | R. norvegicus (rat); S. aureus |
插入大小: | 4290 bp |
启动子 | CMV |
克隆方法: | Ligation Independent克隆 |
5' 测序引物 | CMV |
3' 测序引物 | TGGTTCTTTCCGCCTCAGAAGC |
文献引用方法 |
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材料和方法部分: | pJL-SaKKH-BE3 was a gift from David Liu (Addgene plasmid # 85170 ; http://n2t.net/addgene:85170 ; RRID:Addgene_85170) |
参考文献部分: | Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803. 10.1038/nbt.3803 PubMed 28191901 |
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载体基本信息
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载体序列
LOCUS pJL-SaKKH-BE3 7689 bp ds-DNA circular SYN 15-FEB-2019 DEFINITION Expresses SaKKH-BE3 in mammalian cells [Edit]. ACCESSION . VERSION . KEYWORDS pJL-SaKKH-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7689) AUTHORS Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR TITLE Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. JOURNAL Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803. PUBMED 28191901 REFERENCE 2 (bases 1 to 7689) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 15, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7689 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 179..558 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 559..762 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 713..733 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 804..823 /label=T7 /note="T7 promoter, forward primer" promoter 804..822 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 836..1522 /codon_start=1 /product="cytidine deaminase (C to U editing enzyme) from rat" /label=APOBEC-1 /note="can use ssDNA as a substrate (Komor et al., 2016)" /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI LWATGLK" CDS 4736..4756 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4763..4828 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4841..5089 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5102..5122 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5131..5148 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5171..5188) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5177..5401 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5472..5488) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5472..5488) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5485..5507) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5496..5512 /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)." promoter complement(5520..5550) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5565..5586 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5703..5720) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5874..6462) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5954..5973) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6633..7493) /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 7256..7275 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7494..7598) /gene="bla" /label=AmpR promoter primer_bind complement(7677..7) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag 61 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat 121 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga 181 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 241 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 301 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 361 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 421 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 481 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 541 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 601 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 661 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 721 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag 781 atccgctaga gatccgcggc cgctaatacg actcactata gggagagccg ccaccatgag 841 ctcagagact ggcccagtgg ctgtggaccc cacattgaga cggcggatcg agccccatga 901 gtttgaggta ttcttcgatc cgagagagct ccgcaaggag acctgcctgc tttacgaaat 961 taattggggg ggccggcact ccatttggcg acatacatca cagaacacta acaagcacgt 1021 cgaagtcaac ttcatcgaga agttcacgac agaaagatat ttctgtccga acacaaggtg 1081 cagcattacc tggtttctca gctggagccc atgcggcgaa tgtagtaggg ccatcactga 1141 attcctgtca aggtatcccc acgtcactct gtttatttac atcgcaaggc tgtaccacca 1201 cgctgacccc cgcaatcgac aaggcctgcg ggatttgatc tcttcaggtg tgactatcca 1261 aattatgact gagcaggagt caggatactg ctggagaaac tttgtgaatt atagcccgag 1321 taatgaagcc cactggccta ggtatcccca tctgtgggta cgactgtacg ttcttgaact 1381 gtactgcatc atactgggcc tgcctccttg tctcaacatt ctgagaagga agcagccaca 1441 gctgacattc tttaccatcg ctcttcagtc ttgtcattac cagcgactgc ccccacacat 1501 tctctgggcc accgggttga aaagcggcag cgagactccc gggacctcag agtccgccac 1561 acccgaaagt gggaaacgga actacatcct ggggcttgcc attgggataa ccagcgttgg 1621 ctacggaatt attgattatg agacacgcga tgtgattgac gccggggtta ggctgttcaa 1681 agaggccaac gttgaaaaca acgagggaag acggagtaag cgcggagcaa gaagactcaa 1741 gcgcagacgg agacatcgga ttcagagggt gaaaaagctg ctcttcgatt acaatctcct 1801 gaccgatcat agtgagctga gcggaatcaa cccctacgag gcgcgagtga aagggctttc 1861 ccagaagctg tccgaagagg agttctccgc cgcgttgctg cacctggcca aacggagggg 1921 ggttcacaat gtaaacgaag tggaggagga cacgggcaat gaacttagta cgaaagaaca 1981 gatcagtagg aactctaagg ctctcgaaga gaaatacgtc gctgagttgc agcttgagag 2041 actgaaaaaa gacggcgaag tacgcggatc tattaatagg ttcaagactt cagattacgt 2101 aaaggaagcc aagcagctcc tgaaagtaca gaaagcgtac catcagctcg atcagagctt 2161 catcgatacc tacatagatt tgctggagac acggaggaca tactacgagg gcccagggga 2221 aggatctcct tttgggtgga aggacatcaa ggaatggtac gagatgctta tgggacattg 2281 tacatatttt ccggaggagc tcaggagcgt caagtacgcc tacaatgccg acctgtacaa 2341 tgccctcaat gacctcaata acctcgtgat taccagggac gagaacgaga agctggagta 2401 ctatgaaaag ttccagatta tcgagaatgt gtttaagcag aagaagaagc cgacacttaa 2461 gcagattgca aaggaaatcc tcgtgaatga ggaagatatc aagggataca gagtgacaag 2521 tacaggcaag cccgagttca caaatctgaa ggtgtaccac gatattaagg acataaccgc 2581 acgaaaggag ataatcgaaa acgctgagct cctcgatcag atcgcaaaaa ttcttaccat 2641 ctaccagtct agtgaggaca ttcaggagga actgactaat ctgaacagtg agctcaccca 2701 agaggaaatt gagcagattt caaacctgaa aggctacacc gggacgcaca atctgagcct 2761 caaagcaatc aacctcattc tggatgaact ttggcacaca aatgacaacc aaattgccat 2821 attcaaccgc ctgaaactgg tgccaaaaaa agtggatctg tcacagcaaa aggaaatccc 2881 tacaaccttg gttgacgatt ttattctgtc ccccgttgtc aagcggagct tcatccagtc 2941 aatcaaggtg atcaatgcca tcattaaaaa atacggattg ccaaacgata taattatcga 3001 gcttgcacga gagaagaact caaaggacgc ccagaagatg attaacgaaa tgcagaagcg 3061 caaccgccag acaaacgaac gcatagagga aattataaga acaaccggca aagagaatgc 3121 caagtatctg atcgagaaaa tcaagctgca cgacatgcaa gaaggcaagt gcctgtactc 3181 tctggaagct atcccactcg aagatctgct gaataatcca ttcaattacg aggtggacca 3241 catcatccct agatccgtaa gctttgacaa ttccttcaat aacaaagttc tggttaaaca 3301 ggaggaaaat tctaaaaaag ggaaccggac cccgttccag tacctgagct ccagtgacag 3361 caagattagc tacgagactt ttaagaaaca tattctgaat ctggccaaag gcaaaggcag 3421 gatcagcaag accaagaagg agtacctcct cgaagaacgc gacattaaca gatttagtgt 3481 gcagaaagat ttcatcaacc gaaaccttgt cgatactcgg tacgccacga gaggcctgat 3541 gaatctcctc aggagctact tccgcgtcaa taatctggac gttaaagtca agagcataaa 3601 tgggggattc accagctttc tgaggagaaa gtggaagttt aagaaggaac gaaacaaagg 3661 atacaagcac catgctgagg atgctttgat catcgctaac gcggacttta tctttaagga 3721 atggaaaaag ctggataagg caaagaaagt gatggaaaac cagatgttcg aggagaagca 3781 ggcagagtca atgcctgaga tcgagacaga gcaggaatac aaggaaattt tcatcacccc 3841 tcatcagatt aaacacataa aggacttcaa agactataaa tactctcata gggtggacaa 3901 aaaacccaat cgcaagctca ttaatgacac cctgtactca acacggaagg atgataaagg 3961 taataccttg attgtgaata atcttaatgg attgtatgac aaagataacg acaagctcaa 4021 gaagctgatc aacaagtctc cagagaagct ccttatgtat caccacgacc cacagactta 4081 tcagaaattg aaactgatca tggagcaata cggggatgag aagaacccac tctacaaata 4141 ttatgaggaa acaggtaatt acctgaccaa gtactccaag aaggataacg gaccagtgat 4201 caaaaagata aagtactatg gcaacaaact taatgcgcat ttggacataa ctgacgatta 4261 ccccaattct cgaaacaagg ttgtgaagct ctccctgaag ccttatagat ttgacgtgta 4321 cctggataat ggggtttata aattcgtcac cgtgaaaaat ctggacgtga tcaaaaagga 4381 gaactattat gaagtaaact caaagtgcta tgaggaggcg aagaagctga agaagatctc 4441 caatcaggcc gagttcatcg cttccttcta taagaacgat ctcatcaaga tcaatggaga 4501 gctttatcgc gtcattggtg tgaacaatga cttgctgaac aggatcgaag tcaatatgat 4561 agacattacc taccgggagt atctcgaaaa catgaatgat aaacggccgc ctcacatcat 4621 caagacaatc gcatctaaaa ctcagtcaat aaaaaagtac tctaccgata tcctggggaa 4681 tctctatgaa gtgaagtcaa agaagcaccc acaaatcatt aaaaaaggtg gatcccccaa 4741 gaagaagagg aaagtctcga gcgactacaa agaccatgac ggtgattata aagatcatga 4801 catcgattac aaggatgacg atgacaagtc tggtggttct actaatctgt cagatattat 4861 tgaaaaggag accggtaagc aactggttat ccaggaatcc atcctcatgc tcccagagga 4921 ggtggaagaa gtcattggga acaagccgga aagcgatata ctcgtgcaca ccgcctacga 4981 cgagagcacc gacgagaatg tcatgcttct gactagcgac gcccctgaat acaagccttg 5041 ggctctggtc atacaggata gcaacggtga gaacaagatt aagatgctct ctggtggttc 5101 tcccaagaag aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc 5161 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 5221 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 5281 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 5341 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 5401 gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg 5461 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 5521 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc 5581 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 5641 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 5701 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 5761 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 5821 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5881 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5941 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 6001 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 6061 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 6121 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 6181 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 6241 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 6301 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 6361 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 6421 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 6481 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 6541 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 6601 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 6661 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 6721 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 6781 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 6841 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 6901 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 6961 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 7021 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 7081 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 7141 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 7201 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 7261 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 7321 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 7381 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 7441 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 7501 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 7561 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 7621 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca 7681 atctgctct //
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