| 质粒名称: | AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR (AAV-KPL) |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 60224 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | AAV |
| 空载体大小: | 2597bp |
| 完整质粒大小: | 7203bp |
| 载体类型: | 哺乳细胞表达、Mouse Targeting, AAV, Cre/Lox, CRISPR |
| 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1: | sgRNA / single guide RNA /guide RNA /chimeric RNA |
| 物种来源: | Synthetic |
| 插入大小: | 102 bp |
| 启动子: | U6 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | MluI/BamHI/XbaI(未破坏) |
| 3’克隆位点: | BamHI/XbaI/KpnI (未破坏) |
| 5’测序引物 | NA |
| 3’测序引物 | GACTACTGCACTTATATACGGTTCTCCC |
| 插入基因名称2: | Renilla luciferase / Rluc |
| 插入大小： | 933 |
| 启动子: | EFS |
| 标签/融合蛋白： | Rluc-P2A (C terminal on insert) |
| 克隆方法： | 限制性内切酶 |
| 5’克隆位点: | BsiWI (not destroyed) |
| 3’克隆位点: | NheI (not destroyed) |
| 5’测序引物 | GGGAAAGTGATGTCGTGTACTGG |
| 3’测序引物 | gcgcagggaggttctggtg |
| 插入基因名称3: | Cre recombinase / Cre |
| 插入大小 | 1047bp |
| 启动子: | EFS |
| 标签/融合蛋白： | Cre-HA (C terminal on insert) |
| 克隆方法 | 限制性内切酶 |
| 5’克隆位点: | NheI (not destroyed) |
| 3’克隆位点: | HindIII (not destroyed) |
| 5’测序引物 | CCAATGCTATTGTTGAAGGTGCC |
| 插入基因名称4: | KrasG12D HDR donor / Donor template /HDR template / KrasG12D donor |
| 物种来源: | M. musculus (mouse) |
| 插入大小 | 800bp |
| 启动子: | No promoter |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | HindIII (not destroyed) |
| 3’克隆位点: | BstEII (not destroyed) |
| 5’测序引物 | tccataccagagattatgcaagctg |
| 3’测序引物 | NA |
|
文献引用方法 |
|
| 材料和方法部分: | AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR (AAV-KPL) was a gift from Feng Zhang (Addgene plasmid # 60224 ; http://n2t.net/addgene:60224 ; RRID:Addgene_60224) |
| 参考文献部分: | CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F. Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 10.1016/j.cell.2014.09.014. 10.1016/j.cell.2014.09.014 PubMed 25263330 |
AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rlu
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
-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR (AAV-KPL)质粒图谱.png)
载体简介
载体序列
LOCUS AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR (AAV-KPL) 7203 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses Cre recombinase and Renilla luciferase from the EFS
promoter and three U6-driven sgRNAs targeting Kras, p53, and Lkb1.
Contains KrasG12D HDR donor. AAV backbone..
ACCESSION .
VERSION .
KEYWORDS
AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-...
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7203)
AUTHORS Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE,
Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S,
Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A,
Feng G, Sharp PA, Zhang F
TITLE CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.
JOURNAL Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi:
10.1016/j.cell.2014.09.014.
PUBMED 25263330
REFERENCE 2 (bases 1 to 7203)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7203
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..141
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 12..141
/label=AAV2 ITR
promoter 156..396
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 425..500
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 513..753
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 782..857
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 871..1111
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 1140..1215
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 1270..1481
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 1495..2427
/codon_start=1
/product="luciferase from the anthozoan coelenterate
Renilla reniformis (sea pansy)"
/label=Rluc
/translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
CDS 2443..2499
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 2500..2520
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3547..3573
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
repeat_region 4466..4606
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4466..4595
/label=AAV2 ITR
rep_origin 4681..5136
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4768..4787)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4978..4999
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5153..5172)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5272..5294
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5332..5350)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5418..5522
/gene="bla"
/label=AmpR promoter
CDS 5523..6383
/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(5741..5760)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6554..7142
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7043..7062
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcagcg ttacctctat
421 cgtagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
481 aagtggcacc gagtcggtgc ttttttggat ccgagggcct atttcccatg attccttcat
541 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
601 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
661 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
721 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgtgtaatag ctcctgcatg
781 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
841 tggcaccgag tcggtgcttt ttttctagaa gagggcctat ttcccatgat tccttcatat
901 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
961 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
1021 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
1081 ttcttggctt tatatatctt gtggaaagga cgaaacacca ctccgagacc ttatgccgcg
1141 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
1201 gcaccgagtc ggtgcttttt tggtaccagg tcttgaaagg agtgggaatt ggctccggtg
1261 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
1321 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
1381 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
1441 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggcgtacggc caccatgact
1501 tcgaaagttt atgatccaga acaaaggaaa cggatgataa ctggtccgca gtggtgggcc
1561 agatgtaaac aaatgaatgt tcttgattca tttattaatt attatgattc agaaaaacat
1621 gcagaaaatg ctgttatttt tttacatggt aacgcggcct cttcttattt atggcgacat
1681 gttgtgccac atattgagcc agtagcgcgg tgtattatac cagaccttat tggtatgggc
1741 aaatcaggca aatctggtaa tggttcttat aggttacttg atcattacaa atatcttact
1801 gcatggtttg aacttcttaa tttaccaaag aagatcattt ttgtcggcca tgattggggt
1861 gcttgtttgg catttcatta tagctatgag catcaagata agatcaaagc aatagttcac
1921 gctgaaagtg tagtagatgt gattgaatca tgggatgaat ggcctgatat tgaagaagat
1981 attgcgttga tcaaatctga agaaggagaa aaaatggttt tggagaataa cttcttcgtg
2041 gaaaccatgt tgccatcaaa aatcatgaga aagttagaac cagaagaatt tgcagcatat
2101 cttgaaccat tcaaagagaa aggtgaagtt cgtcgtccaa cattatcatg gcctcgtgaa
2161 atcccgttag taaaaggtgg taaacctgac gttgtacaaa ttgttaggaa ttataatgct
2221 tatctacgtg caagtgatga tttaccaaaa atgtttattg aatcggaccc aggattcttt
2281 tccaatgcta ttgttgaagg tgccaagaag tttcctaata ctgaatttgt caaagtaaaa
2341 ggtcttcatt tttcgcaaga agatgcacct gatgaaatgg gaaaatatat caaatcgttc
2401 gttgagcgag ttctcaaaaa tgaacaagct agcggaagcg gagccactaa cttctccctg
2461 ttgaaacaag caggggatgt cgaagagaat cccgggccac ccaagaagaa gaggaaggtg
2521 tccaatctcc tgactgttca ccagaacctc cctgcgctgc cagtagatgc cactagcgat
2581 gaggtcagga aaaatctcat ggatatgttt agggatagac aggcgttttc tgaacacacc
2641 tggaaaatgc tgcttagcgt gtgccgatcc tgggcagcct ggtgtaagct gaacaatcgc
2701 aaatggttcc ccgccgagcc ggaggacgtg cgcgattacc tgctgtatct ccaggcaaga
2761 gggctggctg tcaagactat ccagcagcac ttgggccaac tgaatatgct gcatcgacgc
2821 agcgggctcc cccggcctag cgattcaaac gcagtctccc ttgttatgag gagaattaga
2881 aaggaaaacg tagatgcggg tgagagggct aagcaggctc tcgcttttga gcggactgat
2941 ttcgaccagg tcagatccct gatggagaac agcgatcggt gccaggacat caggaacctc
3001 gcatttctgg gaattgcata taacacactt ctgcgcatag ctgagatcgc ccggatcaga
3061 gtgaaagaca tcagtcgaac ggacggcggc cggatgctta ttcatattgg acgcacaaag
3121 acattggtca gcaccgctgg cgttgaaaag gccttgtccc tgggcgtaac gaagctggtg
3181 gaaagatgga tctcagtgtc cggcgtggct gacgacccta ataattactt gttctgtcga
3241 gtgagaaaaa acggagtcgc cgcgccctct gccaccagcc aattgagtac acgggccctt
3301 gaagggatct ttgaggcaac ccaccgactc atatacggag ccaaggatga cagtggccag
3361 aggtatctcg cctggtcagg tcattctgct agggtggggg ccgcacgaga catggcgcgg
3421 gcaggagtct ccataccaga gattatgcaa gctggaggtt ggacaaatgt gaacatcgtt
3481 atgaactata tccgcaatct tgactctgaa accggggcca tggtgagact gctcgaagat
3541 ggtgactacc catacgatgt tccagattac gcttaaagcg ctaataaaag atctttattt
3601 tcattagatc tgtgtgttgg ttttttgtgt aagctttggc tccaacacag atgttcttag
3661 gctacctaac ttctaacttt taatatccag tcaacaaaga ataccgcaag ggtaggtgtt
3721 gggatagctg tcgacaagct catgcgggtg tgtccacagg gtatagcgta ctatgcagaa
3781 tatttgtact gagtgaagtc atgatacatt cctttgagag ccattagctg ctacaaaaca
3841 gtaatctggc tgtttagatc aacaagctaa atgatagaag atgaaagtac tggtttccat
3901 gtatttttat taagtgttga tgagaaagtt gtaagtgact tacaggttac tctgtacatc
3961 tgtagtcact gaattcggaa tatcttagag ttttacacac aaaggtgagt gttaaaatat
4021 tgataaagtt tttgataatc ttgtgtgaga catgttctaa tttagttgta ttttattatt
4081 tttattgtaa ggcctgctga aaatgactga gtataaactt gtggtcgtgg gcgccgacgg
4141 cgtgggcaag agcgctttga cgatacagct aattcagaat cactttgtgg atgagtatga
4201 tccaaccatc gaggtaacgc tgctctacag tctgcgtgcg cttgtaaagg acggcagcca
4261 gccgctttga aaaagatatc atttttatat ttattagaaa attatattga aagttatttc
4321 agttatatgt gatgtccttt agttccaagg ctttaaactg ggtgttaggg aaccataggt
4381 gcaagaaagt ccacttctca tgagagctca ccacagagaa agaaagtcca cttctcaggt
4441 aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
4501 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
4561 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
4621 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
4681 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
4741 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
4801 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
4861 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
4921 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
4981 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
5041 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
5101 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
5161 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
5221 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
5281 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
5341 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
5401 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
5461 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
5521 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
5581 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
5641 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
5701 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
5761 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
5821 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
5881 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
5941 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
6001 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
6061 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
6121 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
6181 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
6241 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
6301 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
6361 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
6421 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
6481 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
6541 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
6601 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
6661 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
6721 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
6781 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
6841 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
6901 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
6961 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
7021 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
7081 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
7141 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
7201 tgt
//
