| 质粒名称: | pDYSCKO |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 120263 |
| 存储实验室: | Christian Landry |
|
载体骨架基本信息 |
|
| 载体名称: | p426 |
| 空载体大小: | 5500bp |
| 完整质粒大小: | 6459 bp |
| 载体类型: | CRISPR, Synthetic Biology |
| 筛选标记: | URA3 ; Canavanine (after mutagenesis) |
| 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: | DYSCKO cassette |
| 物种来源: | S. cerevisiae (budding yeast) |
| 启动子: | SNR52 |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | GCGCCGCTACAGGGCGCGT |
| 3’测序引物 | caggaaacagctatgac |
|
文献引用方法 |
|
| 材料和方法部分: | pDYSCKO was a gift from Christian Landry (Addgene plasmid # 120263 ; http://n2t.net/addgene:120263 ; RRID:Addgene_120263) |
| 参考文献部分: | Double Selection Enhances the Efficiency of Target-AID and Cas9-Based Genome Editing in Yeast. Despres PC, Dube AK, Nielly-Thibault L, Yachie N, Landry CR. G3 (Bethesda). 2018 Oct 3;8(10):3163-3171. doi: 10.1534/g3.118.200461. 10.1534/g3.118.200461 PubMed 30097473 |
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
载体简介
载体序列
LOCUS pDYSCKO 6459 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Empty backbone for cloning any gRNA to be used with the canavanine
co-selection system.
ACCESSION .
VERSION .
KEYWORDS pDYSCKO
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6459)
AUTHORS Despres PC, Dube AK, Nielly-Thibault L, Yachie N, Landry CR
TITLE Double Selection Enhances the Efficiency of Target-AID and
Cas9-Based Genome Editing in Yeast.
JOURNAL G3 (Bethesda). 2018 Oct 3;8(10):3163-3171. doi:
10.1534/g3.118.200461.
PUBMED 30097473
REFERENCE 2 (bases 1 to 6459)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6459
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 69..1411
/label=2u ori
/note="yeast 2u plasmid origin of replication"
protein_bind 401..448
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 1438..1542
/gene="bla"
/label=AmpR promoter
CDS 1543..2403
/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(1761..1780)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 2574..3161
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3062..3081
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3315..3332
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 3449..3470
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3485..3515
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3523..3539
/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)."
primer_bind 3528..3550
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 3547..3563
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 3547..3563
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 3582..3602
/label=T3
/note="T3 promoter, forward primer"
promoter 3584..3602
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 3620..3888
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
misc_RNA 3909..3983
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 3988..4007
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
terminator 4013..4260
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
terminator 4266..4285
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
misc_RNA complement(4290..4364)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(4384..4652)
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
rep_origin 4661..5116
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4748..4767)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4958..4979
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(5247..6050)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/label=URA3
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(6051..6266)
/gene="S. cerevisiae URA3"
/label=URA3 promoter
primer_bind complement(6290..6309)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6409..6431
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
ORIGIN
1 gacgaaaggg cctcggcatg cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt cccgcggtat cattgcagca
2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa accaccgcta ccagcggtgg
2641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
2701 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
2761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
2821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
2881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
2941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
3001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
3061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
3121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
3181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
3241 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
3301 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
3421 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
3481 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
3541 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
3601 gggaacaaaa gctggagctt ctttgaaaag ataatgtatg attatgcttt cactcatatt
3661 tatacagaaa cttgatgttt tctttcgagt atatacaagg tgattacatg tacgtttgaa
3721 gtacaactct agattttgta gtgccctctt gggctagcgg taaaggtgcg cattttttca
3781 caccctacaa tgttctgttc aaaagatttt ggtcaaacgc tgtagaagtg aaagttggtg
3841 cgcatgtttc ggcgttcgaa acttctccgc agtgaaagat aaatgatcat aacggaatcc
3901 aactgggcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
3961 gaaaaagtgg caccgagtcg gtggtgcttt ttttgttttt tatgtcttcg agtcatgtaa
4021 ttagttatgt cacgcttacg ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
4081 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
4141 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
4201 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
4261 ctcgaagaca taaaaaacaa aaaaagcacc accgactcgg tgccactttt tcaagttgat
4321 aacggactag ccttatttta acttgctatt tctagctcta aaactgagac cagatctggt
4381 ctcgatcatt tatctttcac tgcggagaag tttcgaacgc cgaaacatgc gcaccaactt
4441 tcacttctac agcgtttgac caaaatcttt tgaacagaac attgtagggt gtgaaaaaat
4501 gcgcaccttt accgctagcc caagagggca ctacaaaatc tagagttgta cttcaaacgt
4561 acatgtaatc accttgtata tactcgaaag aaaacatcaa gtttctgtat aaatatgagt
4621 gaaagcataa tcatacatta tcttttcaaa gaatggcgcg acgcgccctg tagcggcgca
4681 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
4741 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
4801 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
4861 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
4921 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
4981 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
5041 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
5101 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
5161 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
5221 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct
5281 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat
5341 agtcctcttc caacaataat aatgtcagat cctgtagata ccacatcatc cacggttcta
5401 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac
5461 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa
5521 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag
5581 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct
5641 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg
5701 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca
5761 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt
5821 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt
5881 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca
5941 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca
6001 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt
6061 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt
6121 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct
6181 tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa
6241 gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt acaatctgct
6301 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
6361 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
6421 tgtgtcagag gttttcaccg tcatcaccga aacgcgcga
//
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