| 质粒名称: | pCMV-dCas13-M3M14nes |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 155367 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | pCMV with pBR322 origin |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达,CRISPR |
| 筛选标记: | -- |
| 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: | dCas13-M3M14nes |
| 物种来源: | Cas13b is from Prevotella sp. P5-125; METTL3 and METTL14 are from H. sapiens |
| 插入大小: | -- |
| 基因突变: | PspCas13b Δ984-1090 H133A |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | -- |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pCMV-dCas13-M3M14nes was a gift from David Liu (Addgene plasmid # 155367 ; http://n2t.net/addgene:155367 ; RRID:Addgene_155367) |
| 参考文献部分: | Programmable m(6)A modification of cellular RNAs with a Cas13-directed methyltransferase. Wilson C, Chen PJ, Miao Z, Liu DR. Nat Biotechnol. 2020 Jun 29. pii: 10.1038/s41587-020-0572-6. doi: 10.1038/s41587-020-0572-6. 10.1038/s41587-020-0572-6 PubMed 32601430 |
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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 pCMV-dCas13-M3M14nes 8280 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Targeted m6A RNA methylation in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pCMV-dCas13-M3M14nes
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8280)
AUTHORS Wilson C, Chen PJ, Miao Z, Liu DR
TITLE Programmable m(6)A modification of cellular RNAs with a
Cas13-directed methyltransferase.
JOURNAL Nat Biotechnol. 2020 Jun 29. pii: 10.1038/s41587-020-0572-6. doi:
10.1038/s41587-020-0572-6.
PUBMED 32601430
REFERENCE 2 (bases 1 to 8280)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8280
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..204
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 155..175
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 246..265
/label=T7
/note="T7 promoter, forward primer"
promoter 246..264
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 3239..3265
/codon_start=1
/product="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
/label=NES
/translation="LPPLERLTL"
CDS 5164..5181
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5204..5221)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 5210..5434
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(5505..5521)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5505..5521)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5518..5540)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5529..5545
/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(5553..5583)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5598..5619
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5736..5753)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5907..6495)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5987..6006)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6666..7526)
/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 7289..7308
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7527..7631)
/gene="bla"
/label=AmpR promoter
primer_bind complement(7710..7729)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 7901..8280
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
241 gccgctaata cgactcacta tagggagagc cgccaccatg aacatccccg ctctggtgga
301 aaaccagaag aagtactttg gcacctacag cgtgatggcc atgctgaacg ctcagaccgt
361 gctggaccac atccagaagg tggccgatat tgagggcgag cagaacgaga acaacgagaa
421 tctgtggttt caccccgtga tgagccacct gtacaacgcc aagaacggct acgacaagca
481 gcccgagaaa accatgttca tcatcgagcg gctgcagagc tacttcccat tcctgaagat
541 catggccgag aaccagagag agtacagcaa cggcaagtac aagcagaacc gcgtggaagt
601 gaacagcaac gacatcttcg aggtgctgaa gcgcgccttc ggcgtgctga agatgtacag
661 ggacctgacc aacgcataca agacctacga ggaaaagctg aacgacggct gcgagttcct
721 gaccagcaca gagcaacctc tgagcggcat gatcaacaac tactacacag tggccctgcg
781 gaacatgaac gagagatacg gctacaagac agaggacctg gccttcatcc aggacaagcg
841 gttcaagttc gtgaaggacg cctacggcaa gaaaaagtcc caagtgaata ccggattctt
901 cctgagcctg caggactaca acggcgacac acagaagaag ctgcacctga gcggagtggg
961 aatcgccctg ctgatctgcc tgttcctgga caagcagtac atcaacatct ttctgagcag
1021 gctgcccatc ttctccagct acaatgccca gagcgaggaa cggcggatca tcatcagatc
1081 cttcggcatc aacagcatca agctgcccaa ggaccggatc cacagcgaga agtccaacaa
1141 gagcgtggcc atggatatgc tcaacgaagt gaagcggtgc cccgacgagc tgttcacaac
1201 actgtctgcc gagaagcagt cccggttcag aatcatcagc gacgaccaca atgaagtgct
1261 gatgaagcgg agcagcgaca gattcgtgcc tctgctgctg cagtatatcg attacggcaa
1321 gctgttcgac cacatcaggt tccacgtgaa catgggcaag ctgagatacc tgctgaaggc
1381 cgacaagacc tgcatcgacg gccagaccag agtcagagtg atcgagcagc ccctgaacgg
1441 cttcggcaga ctggaagagg ccgagacaat gcggaagcaa gagaacggca ccttcggcaa
1501 cagcggcatc cggatcagag acttcgagaa catgaagcgg gacgacgcca atcctgccaa
1561 ctatccctac atcgtggaca cctacacaca ctacatcctg gaaaacaaca aggtcgagat
1621 gtttatcaac gacaaagagg acagcgcccc actgctgccc gtgatcgagg atgatagata
1681 cgtggtcaag acaatcccca gctgccggat gagcaccctg gaaattccag ccatggcctt
1741 ccacatgttt ctgttcggca gcaagaaaac cgagaagctg atcgtggacg tgcacaaccg
1801 gtacaagaga ctgttccagg ccatgcagaa agaagaagtg accgccgaga atatcgccag
1861 cttcggaatc gccgagagcg acctgcctca gaagatcctg gatctgatca gcggcaatgc
1921 ccacggcaag gatgtggacg ccttcatcag actgaccgtg gacgacatgc tgaccgacac
1981 cgagcggaga atcaagagat tcaaggacga ccggaagtcc attcggagcg ccgacaacaa
2041 gatgggaaag agaggcttca agcagatctc cacaggcaag ctggccgact tcctggccaa
2101 ggacatcgtg ctgtttcagc ccagcgtgaa cgatggcgag aacaagatca ccggcctgaa
2161 ctaccggatc atgcagagcg ccattgccgt gtacgatagc ggcgacgatt acgaggccaa
2221 gcagcagttc aagctgatgt tcgagaaggc ccggctgatc ggcaagggca caacagagcc
2281 tcatccattt ctgtacaagg tgttcgcccg cagcatcccc gccaatgccg tcgagttcta
2341 cgagcgctac ctgatcgagc ggaagttcta cctgaccggc ctgtccaacg agatcaagaa
2401 aggcaacaga gtggatgtgc ccttcatccg gcgggaccag aacaagtgga aaacacccgc
2461 catgaagacc ctgggcagaa tctacagcga ggatctgccc gtggaactgc ccagacagat
2521 gttcgacaat gagatcaagt cccacctgaa gtccctgcca cagatggaag gcatcgactt
2581 caacaatgcc aacgtgacct atctgatcgc cgagtacatg aagagagtgc tggacgacga
2641 cttccagacc ttctaccagt ggaaccgcaa ctaccggtac atggacatgc ttaagggcga
2701 gtacgacaga aagggctccc tgcagcactg cttcaccagc gtggaagaga gagaaggcct
2761 ctggaaagag cgggcctcca gaacagagcg gtacagaaag caggccagca acaagatccg
2821 cagcaaccgg cagatgagaa acgccagcag cgaagagatc gagacaatcc tggataagcg
2881 gctgagcaac agccggaacg agtaccagaa aagcgagaaa gtgatccggc gctacagagt
2941 gcaggatgcc ctgctgtttc tgctggccaa aaagaccctg accgaactgg ccgatttcga
3001 cggcgagagg ttcaaactga aagaaatcat gcccgacgcc gagaagggaa tcctgagcga
3061 gatcatgccc atgagcttca ccttcgagaa aggcggcaag aagtacacca tcaccagcga
3121 gggcatgaag ctgaagaact acggcgactt ctttgtgctg gctagcgaca agaggatcgg
3181 caacctgctg gaactcgtgg gcagcgacat cgtgtccaaa gaggatggat cccttcaact
3241 gcctccactt gaaagactga cactgtccgg aggatctagc ggaggctcct ctggctctga
3301 gacacctggc acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc
3361 agtgggtggc gactccagcg cggatcgtct gttcccgccg cagtggatct gctgcgacat
3421 tcgttacctg gatgtgagca tcctgggcaa gtttgctgtt gtgatggccg acccgccgtg
3481 ggatattcac atggagctgc cgtatggcac cctgaccgac gatgaaatgc gtcgcctgaa
3541 catcccggtg ctgcaggacg atggtttcct gtttctgtgg gtgacgggtc gtgctatgga
3601 gctgggtcgc gaatgcctga acctgtgggg ctacgagcgt gtggacgaaa tcatttgggt
3661 gaaaaccaac cagctgcagc gcatcattcg taccggtcgt accggtcact ggctgaacca
3721 cggcaaggag cactgcctgg tgggtgtgaa aggcaacccg cagggtttca accagggcct
3781 ggactgcgat gtgatcgtgg ctgaagtgcg tagcaccagc cacaagccgg acgagatcta
3841 tggtatgatt gaacgtctga gcccgggcac ccgcaaaatt gagctgtttg gtcgcccgca
3901 caacgtgcag ccgaactgga tcaccctggg caaccagctg gacggcattc acctgctgga
3961 cccggatgtg gtggcccgtt tcaagcagcg ttacccggat ggtatcatta gcaagccgaa
4021 aaacctgggt ggtagcggcg gcagcggtgg tagcggcggt tccggtggta gcggcggtag
4081 cggtggcagc ggcggtagcg gtggtagcgg tagcggtcag agcctgaacc cgcacaacga
4141 ctattgccag cacttcgtgg ataccggtca ccgtccgcag aactttatcc gcgacgtggg
4201 tctggcggat cgtttcgagg aatacccgaa gctgcgtgag ctgatccgcc tgaaggacga
4261 actgattgct aaaagcaaca ccccgccgat gtatctgcag gcggacatcg aggctttcga
4321 tattcgtgaa ctgaccccga aatttgatgt gatcctgctg gagccgccgc tggaggaata
4381 ctatcgcgaa accggcatca ccgcgaacga aaagtgctgg acctgggacg atattatgaa
4441 actggagatc gacgaaattg ccgcgccgcg tagcttcatt tttctgtggt gcggtagcgg
4501 cgagggcctg gacctgggtc gcgtgtgcct gcgtaaatgg ggctaccgtc gttgcgaaga
4561 tatctgctgg attaagacca acaaaaacaa cccgggcaag accaaaaccc tggatccgaa
4621 ggccgtgttt cagcgtacca aagagcactg cctgatgggt atcaagggca ccgtgaaacg
4681 cagcaccgac ggcgatttca tccacgcgaa cgtggacatt gatctgatca ttaccgagga
4741 accggagatc ggtaacattg agaagccggt ggaaatcttc cacatcattg aacacttttg
4801 cctgggccgt cgccgtctgc acctgtttgg tcgtgacagc accattcgtc caggttggct
4861 gaccgtgggt ccgaccctga ccaacagcaa ctacaacgcc gaaacctacg cgagctattt
4921 cagcgcgccg aacagctatc tgaccggctg caccgaggaa atcgaacgcc tgcgtccgaa
4981 aagcccacca ccaaagagca aaagcgatcg tggtggtggt gctccacgtg gtggcggtcg
5041 cggcggtacc agcgccggtc gcggccgtga gcgcaaccgt agcaacttcc gtggtgaacg
5101 tggtggtttt cgtggcggtc gcggtggtgc tcaccgcggc ggttttccac cacgctaacc
5161 ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct
5221 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
5281 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
5341 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
5401 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
5461 ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
5521 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
5581 aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
5641 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
5701 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
5761 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
5821 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
5881 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
5941 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
6001 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
6061 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
6121 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
6181 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
6241 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
6301 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
6361 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
6421 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
6481 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacac tcagtggaac
6541 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
6601 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
6661 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
6721 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
6781 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
6841 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
6901 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
6961 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
7021 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
7081 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
7141 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
7201 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
7261 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
7321 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
7381 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
7441 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
7501 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
7561 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
7621 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga
7681 tctcccgatc ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc
7741 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
7801 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
7861 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
7921 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
7981 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
8041 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
8101 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
8161 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
8221 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
//
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