| 质粒名称: | AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 60226 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | AAV |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 2597 bp |
| 完整质粒大小: | 6763 bp |
| 载体类型: | 哺乳动物表达，小鼠靶向，AAV，Cre/Lox，CRISPR，荧光素酶 |
| 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: | sgRNA,single guide RNA,guide RNA,gRNA |
| 插入大小: | 100 bp |
| 启动子 | U6 |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | MluI(未破坏) |
| 3' 克隆位点: | XbaI(未破坏) |
| 5' 测序引物 | GAGGGCCTATTTCCCATGATTC |
| 3' 测序引物 | GACTACTGCACTTATATACGGTTCTCCC |
|
文献引用方法 |
|
| 材料和方法部分: | AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR was a gift from Feng Zhang (Addgene plasmid # 60226 ; http://n2t.net/addgene:60226 ; RRID:Addgene_60226) |
| 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(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR
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
-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR质粒图谱.png)
载体简介
载体序列
LOCUS AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR 6752 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses Cre recombinase and Renilla luciferase from the EFS
promoter and one U6-driven sgRNA. AAV backbone with SapI spacer for
sgRNA cloning..
ACCESSION .
VERSION .
KEYWORDS AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6752)
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 6752)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6752
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 145..385
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 145..165
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 316..335
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 412..487
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 548..759
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 773..1705
/codon_start=1
/product="luciferase from the anthozoan coelenterate
Renilla reniformis (sea pansy)"
/label=Rluc
/translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
CDS 1721..1777
/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 1778..1798
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2825..2851
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
misc_feature 2879..3467
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2932..2952)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3350..3361)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 3499..3975
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(3698..3717)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 4015..4155
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4015..4144
/label=AAV2 ITR
rep_origin 4230..4685
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4317..4336)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4527..4548
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4702..4721)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4821..4843
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4881..4899)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4967..5071
/gene="bla"
/label=AmpR promoter
CDS 5072..5932
/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(5290..5309)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6103..6691
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6592..6611
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
481 tcggtgcttt ttttctagag gtaccaggtc ttgaaaggag tgggaattgg ctccggtgcc
541 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
601 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
661 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
721 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg cgtacggcca ccatgacttc
781 gaaagtttat gatccagaac aaaggaaacg gatgataact ggtccgcagt ggtgggccag
841 atgtaaacaa atgaatgttc ttgattcatt tattaattat tatgattcag aaaaacatgc
901 agaaaatgct gttatttttt tacatggtaa cgcggcctct tcttatttat ggcgacatgt
961 tgtgccacat attgagccag tagcgcggtg tattatacca gaccttattg gtatgggcaa
1021 atcaggcaaa tctggtaatg gttcttatag gttacttgat cattacaaat atcttactgc
1081 atggtttgaa cttcttaatt taccaaagaa gatcattttt gtcggccatg attggggtgc
1141 ttgtttggca tttcattata gctatgagca tcaagataag atcaaagcaa tagttcacgc
1201 tgaaagtgta gtagatgtga ttgaatcatg ggatgaatgg cctgatattg aagaagatat
1261 tgcgttgatc aaatctgaag aaggagaaaa aatggttttg gagaataact tcttcgtgga
1321 aaccatgttg ccatcaaaaa tcatgagaaa gttagaacca gaagaatttg cagcatatct
1381 tgaaccattc aaagagaaag gtgaagttcg tcgtccaaca ttatcatggc ctcgtgaaat
1441 cccgttagta aaaggtggta aacctgacgt tgtacaaatt gttaggaatt ataatgctta
1501 tctacgtgca agtgatgatt taccaaaaat gtttattgaa tcggacccag gattcttttc
1561 caatgctatt gttgaaggtg ccaagaagtt tcctaatact gaatttgtca aagtaaaagg
1621 tcttcatttt tcgcaagaag atgcacctga tgaaatggga aaatatatca aatcgttcgt
1681 tgagcgagtt ctcaaaaatg aacaagctag cggaagcgga gccactaact tctccctgtt
1741 gaaacaagca ggggatgtcg aagagaatcc cgggccaccc aagaagaaga ggaaggtgtc
1801 caatctcctg actgttcacc agaacctccc tgcgctgcca gtagatgcca ctagcgatga
1861 ggtcaggaaa aatctcatgg atatgtttag ggatagacag gcgttttctg aacacacctg
1921 gaaaatgctg cttagcgtgt gccgatcctg ggcagcctgg tgtaagctga acaatcgcaa
1981 atggttcccc gccgagccgg aggacgtgcg cgattacctg ctgtatctcc aggcaagagg
2041 gctggctgtc aagactatcc agcagcactt gggccaactg aatatgctgc atcgacgcag
2101 cgggctcccc cggcctagcg attcaaacgc agtctccctt gttatgagga gaattagaaa
2161 ggaaaacgta gatgcgggtg agagggctaa gcaggctctc gcttttgagc ggactgattt
2221 cgaccaggtc agatccctga tggagaacag cgatcggtgc caggacatca ggaacctcgc
2281 atttctggga attgcatata acacacttct gcgcatagct gagatcgccc ggatcagagt
2341 gaaagacatc agtcgaacgg acggcggccg gatgcttatt catattggac gcacaaagac
2401 attggtcagc accgctggcg ttgaaaaggc cttgtccctg ggcgtaacga agctggtgga
2461 aagatggatc tcagtgtccg gcgtggctga cgaccctaat aattacttgt tctgtcgagt
2521 gagaaaaaac ggagtcgccg cgccctctgc caccagccaa ttgagtacac gggcccttga
2581 agggatcttt gaggcaaccc accgactcat atacggagcc aaggatgaca gtggccagag
2641 gtatctcgcc tggtcaggtc attctgctag ggtgggggcc gcacgagaca tggcgcgggc
2701 aggagtctcc ataccagaga ttatgcaagc tggaggttgg acaaatgtga acatcgttat
2761 gaactatatc cgcaatcttg actctgaaac cggggccatg gtgagactgc tcgaagatgg
2821 tgactaccca tacgatgttc cagattacgc ttaagaattc gatatcaagc ttatcgataa
2881 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
2941 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
3001 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
3061 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
3121 ttggggcatt gccaccacct gtcagctcct ttccggaact ttcgctttcc ccctccctat
3181 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
3241 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
3301 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
3361 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
3421 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc
3481 gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
3541 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
3601 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
3661 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
3721 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
3781 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
3841 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
3901 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
3961 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac
4021 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
4081 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
4141 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
4201 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
4261 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
4321 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
4381 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
4441 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
4501 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
4561 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
4621 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
4681 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
4741 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
4801 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
4861 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
4921 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
4981 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
5041 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
5101 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
5161 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
5221 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
5281 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
5341 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
5401 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
5461 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
5521 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
5581 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
5641 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
5701 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
5761 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
5821 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
5881 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
5941 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
6001 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
6061 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
6121 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
6181 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
6241 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
6301 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
6361 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
6421 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
6481 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
6541 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
6601 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
6661 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
6721 cctggccttt tgctggcctt ttgctcacat gt
//
