基本信息
| 别名: | pLKO.1-TRC,pLKO.1/TRC,pLKO.1 TRC |
| 启动子: | U6 promoter |
| 复制子: | SV40 Ori，F1 Ori |
| 终止子: | SV40 poly(A) signal |
| 质粒分类: | 病毒系列，慢病毒干扰载体 |
| 质粒大小: | 8901bp |
| 原核抗性: | 氨苄青霉素Amp |
| 筛选标记: | 嘌呤霉素Puro |
| 克隆菌株: | 大肠杆菌Stbl3 |
| 培养条件: | 37℃，有氧 LB |
| 表达宿主: | 哺乳细胞 |
| 诱导方式: | 无须诱导，瞬时表达 |
| 5'测序引物: | U6:ATGGACTATCATATGCTTACCGTA |
| 3'测序引物: | Sv40-polyA-R：GAAATTTGTGATGCTATTGC |
质粒简介
Lentiviral expression vector for shRNA expression (U6 promoter); amp resistance (puromycin for mammalian selection); restriction cloning.质粒图谱
质粒序列
LOCUS Exported 8901 bp ds-DNA circular SYN 08-JAN-2014
DEFINITION Stuffer-containing lentiviral vector for expression of shRNA. This
plasmid is the official TRC cloning vector.
KEYWORDS pLKO.1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8901)
AUTHORS The RNAi Consortium
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8901
/organism="synthetic DNA construct"
/lab_host="Mammalian Cells"
/mol_type="other DNA"
CDS 529..1128
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 1256..1489
/note="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 1561..1682
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 1722..1857
/note="SV40 ori"
/note="SV40 origin of replication"
rep_origin 2064..2519
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2545..2649
/gene="bla"
/note="AmpR promoter"
CDS 2650..3510
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3681..4269
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 4965..5145
/note="5' LTR"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 5192..5317
/note="HIV-1 psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 5810..6043
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 6570..6810
/label=U6 Promoter
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 6816..8696
/note="Stuffer"
/note="can be removed with AgeI + EcoRI and replaced with
an shRNA"
misc_feature 8732..8849
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 8898..507
/note="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
ORIGIN
1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
481 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac
541 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
601 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
661 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
721 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
781 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
841 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
901 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
961 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
1021 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
1081 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
1141 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca
1201 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
1261 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
1321 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
1381 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
1441 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
1501 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
1561 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
1621 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
1681 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
1741 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
1801 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
1861 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
1921 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
1981 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
2041 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
2401 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
2461 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
2521 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
2581 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
2641 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
2701 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
2761 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
2821 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
2881 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
2941 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
3001 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
3061 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
3121 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
3181 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
3241 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
3301 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
3361 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
3421 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
3481 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
3541 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
3601 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
3661 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
3721 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
3781 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
3841 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
3901 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
3961 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
4021 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
4081 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
4141 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
4201 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
4261 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
4321 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
4381 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
4441 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
4501 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
4561 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
4621 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
4681 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt
4741 agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct
4801 tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg
4861 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc
4921 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag
4981 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
5041 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
5101 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
5161 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
5221 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
5281 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
5341 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
5401 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
5461 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
5521 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
5581 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
5641 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
5701 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
5761 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
5821 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
5881 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
5941 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
6001 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
6061 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
6121 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
6181 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
6241 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
6301 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
6361 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
6421 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag
6481 agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag
6541 actagcctcg agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt
6601 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
6661 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
6721 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
6781 tcttggcttt atatatcttg tggaaaggac gaaacaccgg tgtgttgtaa atgagcacac
6841 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
6901 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
6961 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
7021 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
7081 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
7141 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
7201 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
7261 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
7321 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
7381 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
7441 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
7501 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
7561 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
7621 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
7681 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
7741 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
7801 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
7861 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
7921 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
7981 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
8041 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
8101 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
8161 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
8221 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
8281 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
8341 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
8401 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
8461 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
8521 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
8581 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
8641 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta gaattctcga
8701 cctcgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
8761 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
8821 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
8881 tttggccgcg gctcgagggg g
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