| 质粒名称: | pgRNA-modified |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 84477 |
| 存储实验室: | Rudolf Jaenisch |
|
载体骨架基本信息 |
|
| 载体名称: | gRNA-humanized |
| 载体出品公司: | Addgene 44248 |
| 载体抗性: | 氨苄青霉素 |
| 完整质粒大小: | 8317 bp |
| 载体修饰: | engineered an AarI site |
| 载体类型: | 哺乳细胞表达,慢病毒 |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | guide RNA |
| 物种来源: | H. sapiens (human) |
| 启动子 | mU6 |
| 5' 测序引物 | gaaactcaccctaactg |
|
文献引用方法 |
|
| 材料和方法部分: | pgRNA-modified was a gift from Rudolf Jaenisch (Addgene plasmid # 84477 ; http://n2t.net/addgene:84477 ; RRID:Addgene_84477) |
| 参考文献部分: | Editing DNA Methylation in the Mammalian Genome. Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R. Cell. 2016 Sep 22;167(1):233-247.e17. doi: 10.1016/j.cell.2016.08.056. 10.1016/j.cell.2016.08.056 PubMed 27662091 |
买家导航
pgRNAmodified载体基本信息
pgRNAmodified载体图谱质粒图谱和多克隆位点信息
pgRNAmodified载体简介
pgRNAmodified载体序列
LOCUS pgRNA-modified 8306 bp ds-DNA circular SYN 21-JUL-2020
DEFINITION guide RNA expression lentiviral construct.
ACCESSION .
VERSION .
KEYWORDS pgRNA-modified
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8306)
AUTHORS Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D,
Young RA, Jaenisch R
TITLE Editing DNA Methylation in the Mammalian Genome.
JOURNAL Cell. 2016 Sep 22;167(1):233-247.e17. doi:
10.1016/j.cell.2016.08.056.
PUBMED 27662091
REFERENCE 2 (bases 1 to 8306)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jul 21, 2020 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8306
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 382..580
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 598..778
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1447..1680
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2207..2324
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 2577..2610
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind 2614..2634
/label=mU6-F
/note="Mouse U6 promoter, forward primer"
misc_RNA 2726..2801
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 2926..3229
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3230..3433
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 3467..4063
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(3467..3486)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 3963..3983
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
CDS 4073..4126
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
regulatory 4133..4142
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4139..4849
/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(4284..4302)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 4552..4571
/label=mCherry-F
/note="mCherry, forward primer"
protein_bind 4869..4902
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 4958..5546
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5011..5031)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(5429..5440)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(5549..5565)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5550..5566)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 6075..6255
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
rep_origin complement(6317..6905)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6397..6416)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7076..7936)
/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 7699..7718
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7937..8041)
/gene="bla"
/label=AmpR promoter
primer_bind complement(8116..8135)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
1261 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
1321 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
1381 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
1441 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
1501 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
1561 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
1621 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
1681 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
1741 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
1801 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
1861 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
1921 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
1981 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
2041 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
2101 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
2161 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
2221 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
2281 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
2341 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gagatccgac gcgccatctc
2401 taggcccgcg ccggccccct cgcacggact tgtgggagaa gctcggctac tcccctgccc
2461 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
2521 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
2581 cttcgtatag catacattat acgaagttat aaacagcaca aaaggaaact caccctaact
2641 gtaaagtaat tgtgtgtttt gagactataa gtatcccttg gagaaccacc ttgttggtct
2701 cgcaggtggg atcccacctg ctctcgtttt agagctagaa atagcaagtt aaaataaggc
2761 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct cgagtactag
2821 gatccattag gcggccgcgt ggataaccgt attaccgcca tgcattagtt attaatagta
2881 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
2941 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
3001 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
3061 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
3121 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
3181 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
3241 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
3301 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
3361 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
3421 tataagcaga gctggtttag tgaaccgtca gatccgctag cccaccatga ccgagtacaa
3481 gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc
3541 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg
3601 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
3661 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
3721 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
3781 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
3841 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
3901 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
3961 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
4021 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gccggatcgg gagagggcag
4081 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaccgg tcgccaccat
4141 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
4201 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
4261 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
4321 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
4381 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
4441 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
4501 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
4561 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
4621 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
4681 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
4741 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
4801 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag aattcgtcga
4861 gggacctaat aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg
4921 gttccactag gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat
4981 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
5041 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
5101 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
5161 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
5221 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
5281 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
5341 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
5401 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
5461 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
5521 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa
5581 aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag
5641 aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa
5701 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
5761 ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag
5821 gctacttccc tgattggcag aactacacac cagggccagg gatcagatat ccactgacct
5881 ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag
5941 gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag
6001 aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc
6061 atccggactg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
6121 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
6181 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
6241 ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
6301 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
6361 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
6421 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
6481 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
6541 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
6601 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
6661 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
6721 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
6781 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
6841 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
6901 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
6961 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
7021 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
7081 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
7141 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
7201 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
7261 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
7321 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
7381 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
7441 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
7501 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
7561 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
7621 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
7681 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
7741 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
7801 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
7861 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
7921 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
7981 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
8041 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
8101 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
8161 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
8221 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
8281 cgatgtacgg gccagatata cgcgtt
//
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