| 质粒名称: | pmU6-gRNA |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 53187 |
| 存储实验室: | Charles Gersbach |
|
载体骨架基本信息 |
|
| 载体名称: | pzDonor |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 3542 bp |
| 载体类型: | 哺乳细胞表达，CRISPR |
| 高拷贝/低拷贝: | 未知 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 启动子 |
mouse U6
|
| 克隆方法: | 限制性内切酶 |
| 3' 测序引物 | M13 Reverse |
|
文献引用方法 |
|
| 材料和方法部分: | pmU6-gRNA was a gift from Charles Gersbach (爱迪基因 plasmid # 53187) |
| 参考文献部分: | Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Nucleic Acids Res. 2014 Aug 13. pii: gku749. 10.1093/nar/gku749 PubMed 25122746 |
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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 pmU6-gRNA 3542 bp ds-DNA circular SYN 10-JAN-2018
DEFINITION Expresses the S. pyogenes sgRNA from the mouse U6 promoter.
ACCESSION .
VERSION .
KEYWORDS pmU6-gRNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3542)
AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
TITLE Multiplex CRISPR/Cas9-based genome engineering from a single
lentiviral vector.
JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749.
PUBMED 25122746
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTNAME|pmU6-gRNA|
FEATURES Location/Qualifiers
source 1..3542
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin complement(3..458)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(140..161)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 352..371
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 585..607
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 599..616
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 600..616
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 626..645
/label=T7
/note="T7 promoter, forward primer"
promoter 626..644
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 670..983
/label=U6 promoter
/note="RNA polymerase III promoter for mouse U6 snRNA (Das
et al., 1988)"
primer_bind 902..922
/label=mU6-F
/note="Mouse U6 promoter, forward primer"
misc_RNA 1001..1076
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind complement(1121..1141)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(1121..1139)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(1160..1176)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1160..1176)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1173..1195)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1184..1200
/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(1208..1238)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1253..1274
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1391..1408)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1562..2150)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1642..1661)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2227..3021)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(2338..2357)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 2948..2967
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
promoter complement(3056..3413)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3070..3205
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind complement(3124..3143)
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind 3370..3390
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter complement(3415..3519)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccg
661 tctcagatag atccgacgcc gccatctcta ggcccgcgcc ggccccctcg cacagacttg
721 tgggagaagc tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac
781 tatagaggct taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat
841 tttacatgat aggcttggat ttctataaga gatacaaata ctaaattatt attttaaaaa
901 acagcacaaa aggaaactca ccctaactgt aaagtaattg tgtgttttga gactataaat
961 atcccttgga gaaaagcctt gtttgtcttc gagaagacct gttttagagc tagaaatagc
1021 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
1081 tttgtactga gacgccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg
1141 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
1201 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
1261 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
1321 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
1381 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
1441 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
1501 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
1561 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
1621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
1681 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
1741 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
1801 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
1861 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
1921 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
1981 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
2041 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
2101 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
2161 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
2221 tcatgatcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
2281 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
2341 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
2401 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg
2461 ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga acagttcggc
2521 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
2581 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
2641 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
2701 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
2761 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
2821 tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
2881 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
2941 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
3001 caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cgatctttgc
3061 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg
3121 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
3181 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
3241 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
3301 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
3361 actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct caggactctt
3421 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
3481 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
3541 ac
//
