| 质粒名称: | pCMV-dCpf1-BE |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 107685 |
| 存储实验室: | Jia Chen |
|
载体骨架基本信息 |
|
| 载体名称: | CMV |
| 完整质粒大小: | 8166bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dLbCpf1-BE/pCMV-SV40NLS-APOBEC1-XTEN-dLbCpf1(D832A/E1006A/D1125A)-SV40NLS-SGGS-UGI-SV40NLS |
| 物种来源: | R. norvegicus (rat); Lachnospiraceae bacterium, Bacteriophage PBS2 |
| 基因突变: | D832A/E925A/D1148A |
| 启动子: | CMV |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | CMV |
| 3’测序引物 | BGH poly reverse |
|
文献引用方法 |
|
| 材料和方法部分: | pCMV-dCpf1-BE was a gift from Jia Chen (Addgene plasmid # 107685 ; http://n2t.net/addgene:107685 ; RRID:Addgene_107685) |
| 参考文献部分: | Base editing with a Cpf1-cytidine deaminase fusion. Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J, Huang X, Yang L, Chen J. Nat Biotechnol. 2018 Mar 19. pii: nbt.4102. doi: 10.1038/nbt.4102. 10.1038/nbt.4102 PubMed 29553573 |
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 pCMV-dCpf1-BE 8166 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses dLbCpf1-BE in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pCMV-dCpf1-BE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8166)
AUTHORS Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J,
Huang X, Yang L, Chen J
TITLE Base editing with a Cpf1-cytidine deaminase fusion.
JOURNAL Nat Biotechnol. 2018 Mar 19. pii: nbt.4102. doi: 10.1038/nbt.4102.
PUBMED 29553573
REFERENCE 2 (bases 1 to 8166)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8166
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 39..418
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 419..622
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 573..593
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 664..683
/label=T7
/note="T7 promoter, forward primer"
promoter 664..682
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 699..719
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 720..1406
/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 5145..5165
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5178..5426
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label=UGI
/translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
CDS 5439..5459
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5468..5485
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5508..5525)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 5514..5738
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(5809..5825)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5809..5825)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5822..5844)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5833..5849
/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(5857..5887)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5902..5923
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6040..6057)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6211..6799)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6291..6310)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6970..7830)
/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 7593..7612
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7831..7935)
/gene="bla"
/label=AmpR promoter
primer_bind complement(8014..8033)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctaga gatccgcggc
661 cgctaatacg actcactata gggagagccg ccaccatgcc caagaagaag aggaaagtca
721 tgagctcaga gactggccca gtggctgtgg accccacatt gagacggcgg atcgagcccc
781 atgagtttga ggtattcttc gatccgagag agctccgcaa ggagacctgc ctgctttacg
841 aaattaattg ggggggccgg cactccattt ggcgacatac atcacagaac actaacaagc
901 acgtcgaagt caacttcatc gagaagttca cgacagaaag atatttctgt ccgaacacaa
961 ggtgcagcat tacctggttt ctcagctgga gcccatgcgg cgaatgtagt agggccatca
1021 ctgaattcct gtcaaggtat ccccacgtca ctctgtttat ttacatcgca aggctgtacc
1081 accacgctga cccccgcaat cgacaaggcc tgcgggattt gatctcttca ggtgtgacta
1141 tccaaattat gactgagcag gagtcaggat actgctggag aaactttgtg aattatagcc
1201 cgagtaatga agcccactgg cctaggtatc cccatctgtg ggtacgactg tacgttcttg
1261 aactgtactg catcatactg ggcctgcctc cttgtctcaa cattctgaga aggaagcagc
1321 cacagctgac attctttacc atcgctcttc agtcttgtca ttaccagcga ctgcccccac
1381 acattctctg ggccaccggg ttgaaaagcg gcagcgagac tcccgggacc tcagagtccg
1441 ccacacccga aagtatgtca aagctggaga aattcaccaa ctgttatagc ctgtctaaga
1501 ccctgcgctt caaggcaatc ccagtgggca agacacaaga gaacattgac aacaaacggc
1561 tcctggtgga ggatgagaag agggctgaag attacaaggg cgttaagaag ctgctggata
1621 ggtactatct gtcattcatc aacgatgtcc tccacagtat caagctgaag aatctgaaca
1681 attacatttc tctgttccgg aagaagacac ggaccgagaa ggagaacaaa gagctggaga
1741 atctggagat caacctgagg aaagaaatag ctaaggcttt caaagggaac gagggttaca
1801 agtccctgtt caagaaagac attatcgaga ctattctgcc tgagttcctg gacgataaag
1861 atgagatcgc cctcgtcaat tccttcaatg ggtttaccac agcctttacc ggcttcttcg
1921 acaatagaga gaatatgttc tctgaagagg ccaaatccac tagcatcgcc tttcgctgca
1981 taaacgagaa cctgactagg tacatcagca atatggacat ctttgagaaa gtcgatgcca
2041 tattcgacaa acatgaggtg caggagatta aggagaagat cctgaactca gattacgatg
2101 tcgaagattt cttcgaggga gagttcttca acttcgtgct cacacaagag ggcattgatg
2161 tgtacaatgc aatcattgga gggttcgtga cagagagtgg cgagaagata aagggcctga
2221 acgagtatat caacctctac aaccagaaaa ccaagcagaa actgcctaag ttcaagccac
2281 tgtacaaaca agtgctctca gatagggaaa gcctgagctt ctacggtgaa gggtatacat
2341 cagatgaaga agtgctcgaa gtgttccgca acaccctcaa taagaacagt gaaatcttct
2401 cttcaatcaa gaagctggag aaactgttca agaatttcga tgagtactcc tctgccggaa
2461 tctttgtgaa gaatggccct gcaatatcca ctattagcaa agacatcttt ggcgagtgga
2521 acgttatcag ggataagtgg aatgccgagt acgatgatat tcatctcaag aagaaagccg
2581 tggttacaga gaaatacgag gatgatagac gcaagagctt taagaagatt ggtagcttct
2641 ctctcgaaca gctgcaggag tacgccgacg ctgacctgtc agtcgtggag aaactcaagg
2701 agatcataat ccagaaggtg gatgaaatct acaaagtgta tggaagctct gagaaactct
2761 tcgatgcaga ctttgttctg gagaagagtc tgaagaagaa cgacgcagtg gttgctatca
2821 tgaaggacct gctggattct gttaagtctt tcgagaatta cattaaggca ttctttggtg
2881 aagggaagga gacaaatagg gacgagagct tctatggcga ctttgttctg gcctacgaca
2941 tcctcctcaa ggttgaccac atctatgacg ctatacggaa ttacgttacc cagaagccct
3001 atagcaaaga caagttcaag ctgtatttcc agaatccaca gtttatgggt gggtgggata
3061 aagacaaaga aacagattac agggccacta tcctgcggta cggcagcaaa tactatctgg
3121 ctatcatgga taagaagtac gccaaatgcc tccagaagat cgacaaggac gacgtgaacg
3181 gtaactacga gaagatcaat tacaagctcc tgccaggacc taacaagatg ctgcccaagg
3241 tgttcttctc caagaaatgg atggcctact ataacccaag cgaggacatt cagaagatat
3301 acaagaatgg gacattcaag aagggcgata tgttcaacct caacgactgc cacaagctga
3361 ttgatttctt caaggatagc atttctcgct atcccaagtg gtctaatgca tacgatttca
3421 acttcagcga gactgagaag tacaaagaca tcgctggctt ctaccgggag gtggaagagc
3481 aaggctataa ggtgtcattc gaatccgctt ctaagaagga agtggataag ctcgtggaag
3541 agggtaagct gtacatgttc cagatataca acaaagactt cagcgataag agccacggca
3601 ctccaaacct ccatactatg tatttcaagc tgctgtttga cgagaacaac cacggacaga
3661 ttaggctgtc aggaggcgca gaactcttca tgcgcagagc ttcactgaag aaggaggaac
3721 tcgttgtcca cccagccaat agccctatag ccaataagaa tccagacaat cctaagaaaa
3781 ccactactct gtcttacgat gtgtataagg ataagagatt ctctgaagat cagtacgaac
3841 tgcacatacc cattgccatt aacaagtgcc ctaagaacat cttcaagatt aacacagagg
3901 ttagagtgct cctgaaacac gacgataacc cttatgttat aggcattgct cgcggagaga
3961 gaaacctgct gtacatcgtc gtggtggacg gcaaaggcaa catcgtggaa cagtacagtc
4021 tcaatgaaat cattaacaat ttcaacggaa tccgcattaa gaccgactac cattctctcc
4081 tcgacaagaa ggagaaagaa aggttcgaag caagacagaa ttggacaagt atagagaata
4141 tcaaagaact gaaggctggg tacatctctc aggttgtgca caagatatgt gagctggtgg
4201 agaagtacga cgctgttatc gccctcgcgg acctgaatag cggcttcaag aactccaggg
4261 tgaaggtgga gaagcaggtg tatcagaagt tcgagaagat gctgatcgac aagctcaact
4321 atatggtgga caagaaatcc aatccttgcg ctactggtgg agccctgaag ggctatcaaa
4381 tcaccaataa gttcgaatct ttcaagtcta tgagcaccca gaatggcttc atcttctaca
4441 tacccgcatg gctgacatcc aagattgatc cctctaccgg atttgttaat ctgctcaaga
4501 ctaagtacac ctctattgct gactcaaaga agttcatatc atcatttgac cgcatcatgt
4561 acgtgccaga agaggacctg ttcgagtttg ccctggatta caagaatttc tctcggactg
4621 acgccgacta catcaagaag tggaagctct actcttatgg taatcggatt cgcatattcc
4681 gcaatcccaa gaagaataac gtgttcgatt gggaggaagt ttgcctcacc agcgcttaca
4741 aggagctgtt caataagtat gggattaact accagcaggg cgacataaga gccctgctgt
4801 gcgaacaatc tgataaggca ttctattcct ctttcatggc actgatgtca ctgatgctgc
4861 aaatgcgcaa ttccatcacc ggaagaacag acgtggcctt tctgatctct cctgtcaaga
4921 actcagatgg catcttctac gattcccgca actatgaagc acaggagaat gctatcctgc
4981 ctaagaatgc cgatgcaaat ggagcctata acatcgccag aaaggtcctc tgggccatag
5041 gacaattcaa gaaagctgaa gatgagaagc tggacaaggt gaagatcgcc atttcaaaca
5101 aagagtggct cgaatatgct cagacctcag tgaagcatgg atcacccaag aagaaacgga
5161 aagtgtctgg tggttctact aatctgtcag atattattga aaaggagacc ggtaagcaac
5221 tggttatcca ggaatccatc ctcatgctcc cagaggaggt ggaagaagtc attgggaaca
5281 agccggaaag cgatatactc gtgcacaccg cctacgacga gagcaccgac gagaatgtca
5341 tgcttctgac tagcgacgcc cctgaataca agccttgggc tctggtcata caggatagca
5401 acggtgagaa caagattaag atgctctctg gtggttctcc caagaagaag aggaaagtct
5461 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
5521 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
5581 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
5641 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
5701 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
5761 ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
5821 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
5881 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
5941 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
6001 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
6061 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
6121 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
6181 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
6241 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
6301 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
6361 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
6421 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
6481 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
6541 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
6601 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
6661 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
6721 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
6781 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
6841 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
6901 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
6961 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
7021 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
7081 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
7141 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
7201 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
7261 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
7321 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
7381 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
7441 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
7501 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
7561 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
7621 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
7681 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
7741 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
7801 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
7861 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
7921 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
7981 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
8041 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
8101 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
8161 gcgttt
//
