| 质粒名称: | pLKO5.sgRNA.EFS.tRFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 57823 |
| 存储实验室: | Benjamin Ebert |
|
载体骨架基本信息 |
|
| 载体名称: | pLKO.005 |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | 7408 bp |
| 载体修饰: | -- |
| 载体类型: | 慢病毒,CRISPR |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | Stbl3 |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | sgRNA |
| 物种来源: | -- |
| 插入大小: | 75 bp |
| 基因突变: | -- |
| 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: | pLKO5.sgRNA.EFS.tRFP was a gift from Benjamin Ebert (Addgene plasmid # 57823 ; http://n2t.net/addgene:57823 ; RRID:Addgene_57823) |
| 参考文献部分: | Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL. Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951. 10.1038/nbt.2951 PubMed 24952903 |
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pLKO5.sgRNA.EFS.tRFP载体质粒基本信息
pLKO5.sgRNA.EFS.tRFP质粒图谱载体图谱和pLKO5.sgRNA.EFS.tRFP载体序列质粒序列多克隆位点信息
pLKO5.sgRNA.EFS.tRFP质粒载体简介
pLKO5.sgRNA.EFS.tRFP质粒序列载体序列
LOCUS pLKO5.sgRNA.EFS.tRFP 7408 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION 3rd generation Lentiviral Vector for Sp sgRNA delivery without
SpCas9, tagRFP, EFS Promoter driven.
ACCESSION .
VERSION .
KEYWORDS pLKO5.sgRNA.EFS.tRFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7408)
AUTHORS Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey
ME, Thielke A, Aster JC, Regev A, Ebert BL
TITLE Generation of mouse models of myeloid malignancy with combinatorial
genetic lesions using CRISPR-Cas9 genome editing.
JOURNAL Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
PUBMED 24952903
REFERENCE 2 (bases 1 to 7408)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7408
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 98..115
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 232..253
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 268..298
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 306..322
/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 311..333
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 330..346
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 330..346
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 365..385
/label=T3
/note="T3 promoter, forward primer"
promoter 367..385
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 413..639
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 640..820
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 867..992
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1485..1718
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1903..1947
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
promoter 2125..2365
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 2125..2145
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2296..2315
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 2392..2467
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 2515..2632
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2701..2912
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 2932..2941
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2938..3651
/codon_start=1
/product="monomeric derivative of red fluorescent protein
from Entacmaea quadricolor (Merzlyak et al., 2007)"
/label=TagRFP
/note="mammalian codon-optimized"
/translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
ARYCDLPSKLGHKLN"
misc_feature 3673..4261
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3726..3746)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4144..4155)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 4333..4566
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 4644..4765
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4681..4700)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4735..4754
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 4805..4940
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4867..4886
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(4960..4979)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(4961..4979)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4989..5006)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4989..5005)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4998..5020)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 5147..5602
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5234..5253)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5444..5465
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 5628..5732
/gene="bla"
/label=AmpR promoter
CDS 5733..6593
/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(5951..5970)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6764..7352
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7253..7272
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
361 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
421 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
481 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
541 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
601 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
661 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
721 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
781 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
841 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
901 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
961 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
1021 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
1081 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
1141 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
1201 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
1261 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
1321 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
1381 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
1441 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
1501 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
1561 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
1621 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
1681 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
1741 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
1801 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
1861 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
1921 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
1981 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
2041 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
2101 ctcccaaccc cgaggggacc cagagagggc ctatttccca tgattccttc atatttgcat
2161 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
2221 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
2281 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
2341 gctttatata tcttgtggaa aggacgaaac accggagacg tgaccgtctc tgttttagag
2401 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
2461 tcggtgcttt tttgaattct agatcttgag acaaatggca gtattcatcc acaattttaa
2521 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
2581 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
2641 ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt gcccgtcagt
2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
2881 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc tgccaccatg
2941 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc
3001 accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
3061 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
3121 ctggctacca gcttcatgta cggcagcaga accttcatca accacaccca gggcatcccc
3181 gacttcttta agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
3241 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
3301 aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca
3361 ctcggctggg aggccaacac cgagatgctg taccccgctg acggcggcct ggaaggcaga
3421 agcgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt caagaccaca
3481 tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggaccac
3541 agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct
3601 gtggccagat actgcgacct ccctagcaaa ctggggcaca aacttaattg aggtaccacg
3661 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
3721 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
3781 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
3841 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
3901 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
3961 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
4021 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
4081 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
4141 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
4201 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
4261 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
4321 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
4381 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
4441 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
4501 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
4561 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
4621 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
4681 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
4741 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
4801 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
4861 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
4921 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
4981 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
5041 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
5101 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
5161 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
5221 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
5281 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
5341 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
5401 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
5461 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
5521 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
5581 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
5641 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
5701 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
5761 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
5821 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
5881 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
5941 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
6001 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
6061 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
6121 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
6181 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
6241 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
6301 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
6361 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
6421 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
6481 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
6541 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
6601 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
6661 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
6721 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
6781 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
6841 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
6901 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
6961 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
7021 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
7081 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
7141 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
7201 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
7261 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
7321 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
7381 tcctggcctt ttgctggcct tttgctca
//
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