| 质粒名称: | PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 154002 |
| 存储实验室: | Hui Yang |
|
载体骨架基本信息 |
|
| 载体名称: | PB |
| 载体出品公司: | Clontech |
| 载体类型: | CRISPR |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SgRNA expression system |
| 物种来源: | Synthetic |
| 插入大小: | 1769bp |
| 启动子: | U6, EF1a |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | unknown (unknown if destroyed) |
| 3’克隆位点: | unknown (unknown if destroyed) |
|
文献引用方法 |
|
| 材料和方法部分: | PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40 was a gift from Hui Yang (Addgene plasmid # 154002 ; http://n2t.net/addgene:154002 ; RRID:Addgene_154002) |
| 参考文献部分: | Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. He B, Peng W, Huang J, Zhang H, Zhou Y, Yang X, Liu J, Li Z, Xu C, Xue M, Yang H, Huang P. Protein Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi: 10.1007/s13238-020-00700-2. 10.1007/s13238-020-00700-2 PubMed 32185621 |
买家导航
PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40载体质粒基本信息
PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40质粒图谱载体图谱和PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40载体序列质粒序列多克隆位点信息
PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40质粒载体简介
PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40质粒序列载体序列
LOCUS PB-U6-DR-SgRNA-DR-EF1a-mCherry-SV40 6255 bp DNA circular SYN 25-AUG-2024
DEFINITION Empty SgRNA expression system matching CasRx.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6255)
AUTHORS He B, Peng W, Huang J, Zhang H, Zhou Y, Yang X, Liu J, Li Z, Xu C,
Xue M, Yang H, Huang P
TITLE Modulation of metabolic functions through Cas13d-mediated gene
knockdown in liver.
JOURNAL Protein Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi:
10.1007/s13238-020-00700-2.
PUBMED 32185621
REFERENCE 2 (bases 1 to 6255)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Protein
Cell. 2020 Mar 18. pii: 10.1007/s13238-020-00700-2. doi:
10.1007/s13238-020-00700-2."
FEATURES Location/Qualifiers
source 1..6255
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 105..126
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 141..171
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 179..195
/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)."
primer_bind 184..206
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 203..219
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 203..219
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
repeat_region 248..282
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter 593..841
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA
(Domitrovich & Kunkel, 2003)"
primer_bind 593..613
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 764..783
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 843..878
/label=RfxCas13d DR36
/note="36-base direct repeat for an RfxCas13d pre-gRNA
(Konermann et al., 2018)"
misc_RNA 897..932
/label=RfxCas13d DR36
/note="36-base direct repeat for an RfxCas13d pre-gRNA
(Konermann et al., 2018)"
promoter 1003..1214
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 1227..1495
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
regulatory 1523..1532
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1529..2239
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(1674..1692)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 1942..1961
/label=mCherry-F
/note="mCherry, forward primer"
polyA_signal 2240..2361
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2277..2296)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2331..2350
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 2539..2601
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(2686..2705)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2687..2705)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2712..2729)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2712..2728)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2721..2743)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2869..3297
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2956..2975)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3166..3187
/label=F1ori-F
/note="F1 origin, forward primer"
CDS 3641..4435
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3695..3714)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4305..4324
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
CDS 4453..5313
/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 complement(4671..4690)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5484..6072
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5973..5992
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6226..6243
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
61 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
121 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
181 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcttggt
241 accttaaccc tagaaagata gtctgcgtaa aattgacgca tgcattcttg aaatattgct
301 ctctctttct aaatagcgcg aatccgtcgc tgtgcattta ggacatctca gtcgccgctt
361 ggagctcccg tgaggcgtgc ttgtcaatgc ggtaagtgtc actgattttg aactataacg
421 accgcgtgag tcaaaatgac gcatgattat cttttacgtg acttttaaga tttaactcat
481 acgataatta tattgttatt tcatgttcta cttacgtgat aacttattat atatatattt
541 tcttgttata gatatcatta atatcgatgg gctggccttt tgctcagcta gcgagggcct
601 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
661 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
721 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
781 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
841 cgcaagtaaa cccctaccaa ctggtcgggg tttgaaacgg gtcttcgaga agacctcaag
901 taaaccccta ccaactggtc ggggtttgaa actttttttg aattctgatg cggtattttc
961 tccttacgca aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc
1021 ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt
1081 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
1141 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
1201 ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc
1261 ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg
1321 cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt
1381 gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc
1441 tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc
1501 tgtgaccggc gcctacaccg gtgccaccat ggtgagcaag ggcgaggagg ataacatggc
1561 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
1621 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
1681 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
1741 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
1801 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
1861 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
1921 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
1981 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
2041 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
2101 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
2161 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
2221 ggacgagctg tacaagtaaa acttgtttat tgcagcttat aatggttaca aataaagcaa
2281 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
2341 caaactcatc aatgtatctt acctaaaagt tttgttactt tatagaagaa attttgagtt
2401 tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag
2461 tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat
2521 atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac
2581 aatatgatta tctttctagg gttaaggccg gccaataagg gcgaattctg cagatatcca
2641 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg
2701 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
2761 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
2821 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta
2881 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
2941 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
3001 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
3061 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
3121 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
3181 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
3241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
3301 acaaaattca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca
3361 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc
3421 aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc
3481 ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg
3541 gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg
3601 atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
3661 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
3721 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
3781 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
3841 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
3901 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catcccacct
3961 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
4021 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
4081 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
4141 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
4201 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
4261 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
4321 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
4381 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgaattga
4441 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
4501 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
4561 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
4621 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
4681 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
4741 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
4801 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
4861 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
4921 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
4981 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
5041 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
5101 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
5161 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
5221 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
5281 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
5341 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
5401 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
5461 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
5521 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
5581 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
5641 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
5701 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
5761 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
5821 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
5881 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
5941 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
6001 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
6061 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
6121 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
6181 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
6241 gaggaagcgg aagag
//
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