| 质粒名称: | pBluescriptSKII+ U6-sgRNA(F+E) empty |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 74707 |
| 存储实验室: | Eugene Yeo |
|
载体骨架基本信息 |
|
| 载体名称: | pBlueScript II SK(+) |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: |
U6 promoter driving sgRNA with Bbsi sites for insertion of spacer sequence
gRNA/shRNA sequenceempty
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文献引用方法 |
|
| 材料和方法部分: | pBluescriptSKII+ U6-sgRNA(F+E) empty was a gift from Eugene Yeo (Addgene plasmid # 74707 ; http://n2t.net/addgene:74707 ; RRID:Addgene_74707) |
| 参考文献部分: | Programmable RNA Tracking in Live Cells with CRISPR/Cas9. Nelles DA, Fang MY, O'Connell MR, Xu JL, Markmiller SJ, Doudna JA, Yeo GW. Cell. 2016 Mar 16. pii: S0092-8674(16)30204-5. doi: 10.1016/j.cell.2016.02.054. 10.1016/j.cell.2016.02.054 PubMed 26997482 |
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
 empty质粒图谱.png)
载体简介
载体序列
LOCUS pBluescriptSKII+ U6-sgRNA(F+E) empty 3386 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Encodes an sgRNA for spCas9 driven by hU6 promoter with a modified
scaffold (Chen et al. Cell 2013) and BbsI sites that allow insertion
of a spacer sequence.
ACCESSION .
VERSION .
KEYWORDS pBluescriptSKII+ U6-sgRNA(F+E) empty
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3386)
AUTHORS Nelles DA, Fang MY, O'Connell MR, Xu JL, Markmiller SJ, Doudna JA,
Yeo GW
TITLE Programmable RNA Tracking in Live Cells with CRISPR/Cas9.
JOURNAL Cell. 2016 Mar 16. pii: S0092-8674(16)30204-5. doi:
10.1016/j.cell.2016.02.054.
PUBMED 26997482
REFERENCE 2 (bases 1 to 3386)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..3386
/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"
primer_bind 669..685
/label=pBluescriptKS
/note="For pBluescript vector"
primer_bind 670..686
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
promoter 771..1011
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 771..791
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 942..961
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(1145..1161)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1145..1161)
/label=pBluescriptSK
/note="For pBluescript vector"
primer_bind complement(1198..1218)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(1198..1216)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(1237..1253)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1237..1253)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1250..1272)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1261..1277
/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(1285..1315)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1330..1351
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1468..1485)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1639..2227)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1719..1738)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2398..3258)
/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 3021..3040
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3259..3363)
/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 tgggtaccgg
661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gtgtacaaaa aagcaggctt
721 taaaggaacc aattcagtcg actggatccg gtaccaaggt cgggcaggaa gagggcctat
781 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
841 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
901 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
961 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
1021 ggtcttcgag aagacctgtt taagagctat gctggaaaca gcatagcaag tttaaataag
1081 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt cctgcagccc
1141 gggggatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
1201 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
1261 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
1321 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
1381 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
1441 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
1501 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
1561 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
1621 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
1681 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
1741 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
1801 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
1861 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
1921 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
1981 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
2041 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
2101 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
2161 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
2221 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
2281 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
2341 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
2401 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
2461 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
2521 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
2581 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
2641 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
2701 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
2761 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
2821 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
2881 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
2941 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
3001 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
3061 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
3121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
3181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
3241 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
3301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
3361 gcgcacattt ccccgaaaag tgccac
//
