| 质粒名称: | pLKO.1 puro |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 8453 |
| 存储实验室: | Bob Weinberg |
|
载体骨架基本信息 |
|
| 载体名称: | pLKO.1 |
| 空载体大小: | 7032 bp |
| 完整质粒大小: | 7050bp |
| 载体类型: | 哺乳细胞表达, Lentiviral, RNAi |
| 筛选标记: | Puromycin |
| 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: | LKO.1 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | AgeI(未破坏) |
| 3’克隆位点: | EcoRI(未破坏) |
| 5’测序引物 | LKO.1 5' |
|
文献引用方法 |
|
| 材料和方法部分: |
pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453 ; http://n2t.net/addgene:8453 ; RRID:Addgene_8453)
|
| 参考文献部分: | Lentivirus-delivered stable gene silencing by RNAi in primary cells. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD. RNA 2003 Apr;9(4):493-501. 10.1261/rna.2192803 PubMed 12649500 |
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 pLKO.1 puro 7050 bp ds-DNA circular SYN 31-JAN-2024
DEFINITION 3rd gen lentiviral backbone for cloning and expression of new shRNA
sequences. Uses puromycin for selection..
ACCESSION .
VERSION .
KEYWORDS pLKO.1 puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7050)
AUTHORS Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS,
Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD
TITLE Lentivirus-delivered stable gene silencing by RNAi in primary cells.
JOURNAL RNA 2003 Apr;9(4):493-501.
PUBMED 12649500
REFERENCE 2 (bases 1 to 7050)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 31, 2024 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7050
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(312..911)
/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(395..415)
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind 892..911
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
promoter complement(933..1443)
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind complement(996..1014)
/label=hPGK-F
/note="Human PGK promoter, forward primer"
misc_feature 1492..1609
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter complement(1680..1920)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(1730..1749)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(1900..1920)
/label=hU6-F
/note="Human U6 promoter, forward primer"
CDS complement(2218..2262)
/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"
misc_feature 2447..2680
/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 3173..3298
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
LTR 3345..3525
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(3526..3752)
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
primer_bind complement(3780..3800)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(3780..3798)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3819..3835)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3819..3835)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3832..3854)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3843..3859
/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(3867..3897)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3912..3933
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4050..4067)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4221..4809)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4301..4320)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4980..5840)
/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 5603..5622
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5841..5945)
/gene="bla"
/label=AmpR promoter
rep_origin complement(5971..6426)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6108..6129)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 6320..6339
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 6553..6575
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 6567..6584
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 6568..6584
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 6594..6613
/label=T7
/note="T7 promoter, forward primer"
promoter 6594..6612
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin 6633..6768
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind complement(6687..6706)
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 6808..6929
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6819..6838)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 6873..6892
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
LTR 7001..184
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
ORIGIN
1 ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga
61 ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac
121 cagagagacc cagtacaagc aaaaagcaga tcttgtcttc gttgggagtg aattagccct
181 tccagtcccc ccttttcttt taaaaagtgg ctaagatcta cagctgcctt gtaagtcatt
241 ggtcttaaag gtaccgatgc atggggtcgt gcgctccttt cggtcgggcg ctgcgggtcg
301 tggggcgggc gtcaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc
361 acctcgacgt cggcggtgac ggtgaagccg agccgctcgt agaaggggag gttgcggggc
421 gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg ccgcctccac tccggggagc
481 acgacggcgc tgcccagacc cttgccctgg tggtcgggcg agacgccgac ggtggccagg
541 aaccacgcgg gctccttggg ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg
601 gccagccggg aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa caccgccccc
661 gcttcgacgc tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac
721 accttgccga tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc
781 tcgatgtggc ggtccggatc gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg
841 gcgagggtgc gtacggccct ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg
901 tactcggtca tggtaagctc cggtggatcc ccctggggag agaggtcggt gattcggtca
961 acgagggagc cgactgccga cgtgcgctcc ggaggcttgc agaatgcgga acaccgcgcg
1021 ggcaggaaca gggcccacac taccgcccca caccccgcct cccgcaccgc cccttcccgg
1081 ccgctgctct cggcgcgccc tgctgagcag ccgctattgg ccacagccca tcgcggtcgg
1141 cgcgctgcca ttgctccctg gcgctgtccg tctgcgaggg tactagtgag acgtgcggct
1201 tccgtttgtc acgtccggca cgccgcgaac cgcaaggaac cttcccgact taggggcgga
1261 gcaggaagcg tcgccggggg gcccacaagg gtagcggcga agatccgggt gacgctgcga
1321 acggacgtga agaatgtgcg agacccaggg tcggcgccgc tgcgtttccc ggaaccacgc
1381 ccagagcagc cgcgtccctg cgcaaaccca gggctgcctt ggaaaaggcg caaccccaac
1441 cccctcgagc cgcggccaaa gtggatctct gctgtccctg taataaaccc gaaaattttg
1501 aatttttgta atttgttttt gtaattcttt agtttgtatg tctgttgcta ttatgtctac
1561 tattctttcc cctgcactgt accccccaat cccccctttt cttttaaaat tgtggatgaa
1621 tactgccatt tgtctcgagg tcgagaattc acgcgtgcat acctgcggac cggtgtttcg
1681 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
1741 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
1801 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
1861 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
1921 ggtgaagggg gcggccgctc gaggctagtc tcgtgatcga taccgtcgag atccgttcac
1981 taatcgaatg gatctgtctc tgtctctctc tccaccttct tcttctattc cttcgggcct
2041 gtcgggtccc ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat atccctgcct
2101 aactctattc actatagaaa gtacagcaaa aactattctt aaacctacca agcctcctac
2161 tatcattatg aataatttta tataccacag ccaatttgtt atgttaaacc aattccacaa
2221 acttgcccat ttatctaatt ccaataattc ttgttcattc ttttcttgct ggttttgcga
2281 ttcttcaatt aaggagtgta ttaagcttgt gtaattgtta atttctctgt cccactccat
2341 ccaggtcgtg tgattccaaa tctgttccag agatttatta ctccaactag cattccaagg
2401 cacagcagtg gtgcaaatga gttttccaga gcaaccccaa atccccagga gctgttgatc
2461 ctttaggtat ctttccacag ccaggattct tgcctggagc tgcttgatgc cccagactgt
2521 gagttgcaac agatgctgtt gcgcctcaat agccctcagc aaattgttct gctgctgcac
2581 tataccagac aataattgtc tggcctgtac cgtcagcgtc attgacgctg cgcccatagt
2641 gcttcctgct gctcccaaga acccaaggaa caaagctcct attcccactg ctcttttttc
2701 tctctgcacc actcttctct ttgccttggt gggtgctact cctaatggtt caatttttac
2761 tactttatat ttatataatt cacttctcca attgtccctc atatctcctc ctccaggtct
2821 gaagatcagc ggccgcttgc tgtgcggtgg tcttactttt gttttgctct tcctctatct
2881 tgtctaaagc ttccttggtg tcttttatct ctatcctttg atgcacacaa tagagggttg
2941 ctactgtatt atataatgat ctaagttctt ctgatcctgt ctgaagggat ggttgtagct
3001 gtcccagtat ttgtctacag ccttctgatg tttctaacag gccaggatta actgcgaatc
3061 gttctagctc cctgcttgcc catactatat gttttaattt atattttttc tttccccctg
3121 gccttaaccg aattttttcc catcgcgatc taattctccc ccgcttaata ctgacgctct
3181 cgcacccatc tctctccttc tagcctccgc tagtcaaaat ttttggcgta ctcaccagtc
3241 gccgcccctc gcctcttgcc gtgcgcgctt cagcaagccg agtcctgcgt cgagagagct
3301 cctctggttt ccctttcgct ttcaagtccc tgttcgggcg ccactgctag agattttcca
3361 cactgactaa aagggtctga gggatctcta gttaccagag tcacacaaca gacgggcaca
3421 cactacttga agcactcaag gcaagcttta ttgaggctta agcagtgggt tccctagtta
3481 gccagagagc tcccaggctc agatctggtc taaccagaga gacccgttta tgtatcgagc
3541 taggcactta aatacaatat ctctgcaatg cggcaattca gtggttcgtc caatccatgt
3601 cagacccgtc tgttgccttc ctaataaggc acgatcgtac caccttactt ccaccaatcg
3661 gcatgcacgg tgctttttct ctccttgtaa ggcatgttgc taactcatcg ttaccatgtt
3721 gcaagactac aagagtattg cataagacta cattaagctt gcagctccag cttttgttcc
3781 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
3841 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
3901 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
3961 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
4021 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
4081 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
4141 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
4201 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
4261 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
4321 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
4381 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
4441 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
4501 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
4561 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
4621 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
4681 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
4741 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
4801 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
4861 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
4921 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
4981 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
5041 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
5101 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
5161 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
5221 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
5281 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
5341 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
5401 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
5461 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
5521 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
5581 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
5641 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
5701 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
5761 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
5821 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
5881 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
5941 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
6001 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
6061 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
6121 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
6181 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
6241 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
6301 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
6361 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
6421 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
6481 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
6541 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg
6601 actcactata gggcgaattg ggtacgtccc taggcctcca aaaaagcctc ctcactactt
6661 ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca
6721 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
6781 gggcgggata gctagagcca gacatgataa gatacattga tgagtttgga caaaccacaa
6841 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
6901 taaccattat aagctgcaat aaacaagttc ctctcactct ctgatattca tttctttgca
6961 agttataaat actgaataat aagatgacat gaactactac tgctagagat tttccacact
7021 gactaaaagg gtctgaggga tctctagtta
//
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