| 质粒名称: | pT040 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 67640 |
| 存储实验室: | John Wyrick |
|
载体骨架基本信息 |
|
| 载体名称: | pCT3 |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4749 bp |
| 完整质粒大小: | 5187 bp |
| 载体修饰: | -- |
| 载体类型: | 酵母表达,CRISPR |
| 筛选标记: | URA3 |
| 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: | single guide RNA expression cassette |
| 物种来源: | S. cerevisiae (budding yeast), Synthetic |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | pSNR52 |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | SacII(未破坏) |
| 3' 克隆位点: | EagI (未破坏) |
| 5' 测序引物 | T3 |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pT040 was a gift from John Wyrick (Addgene plasmid # 67640 ; http://n2t.net/addgene:67640 ; RRID:Addgene_67640) |
| 参考文献部分: | New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T, Wyrick JJ. Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21. 10.1002/yea.3098 PubMed 26305040 |
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 pT040 5186 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION URA3 vector for cloning guide sequences using BclI-SwaI sites into
guide RNA expression cassette.
ACCESSION .
VERSION .
KEYWORDS pT040
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5186)
AUTHORS Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T,
Wyrick JJ
TITLE New vectors for simple and streamlined CRISPR-Cas9 genome editing in
Saccharomyces cerevisiae.
JOURNAL Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep
21.
PUBMED 26305040
REFERENCE 2 (bases 1 to 5186)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5186
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 27..131
/gene="bla"
/label=AmpR promoter
CDS 132..992
/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(350..369)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1163..1751
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1652..1671
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 1905..1922
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2039..2060
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2075..2105
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2113..2129
/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 2118..2140
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 2137..2153
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2137..2153
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 2174..2192
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 2224..2240
/label=pBluescriptKS
/note="For pBluescript vector"
primer_bind 2225..2241
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2287..2303)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2287..2303)
/label=pBluescriptSK
/note="For pBluescript vector"
terminator 2378..2397
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
promoter complement(2479..2747)
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
primer_bind complement(2773..2792)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2774..2792)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2799..2816)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2799..2815)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2808..2830)
/label=M13/pUC Forward
/note="In lacZ gene"
promoter 4089..4304
/gene="S. cerevisiae URA3"
/label=URA3 promoter
CDS 4305..5108
/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"
ORIGIN
1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
1801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
1861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
1921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
2161 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggtaccg ggccccccct
2221 cgatcgaggt cgacggtatc gataagcttg atatcgaatt cctgcagccc ccgctcgagc
2281 gggggggatc cactagttct agagcggccg tggtatcgtt tagattggca attacagtgt
2341 cttagctcac atgcttataa ctaattacat gactcgaaga cataaaaaac aaaaaaagca
2401 ccaccgactc ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta
2461 tttaaatgca tcgattctga tcatttatct ttcactgcgg agaagtttcg aacgccgaaa
2521 catgcgcacc aactttcact tctacagcgt ttgaccaaaa tcttttgaac agaacattgt
2581 agggtgtgaa aaaatgcgca cctttaccgc tagcccaaga gggcactaca aaatctagag
2641 ttgtacttca aacgtacatg taatcacctt gtatatactc gaaagaaaac atcaagtttc
2701 tgtataaata tgagtgaaag cataatcata cattatcttt tcaaagattg tccgcggtgg
2761 agctccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt
2821 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
2881 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
2941 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
3001 caccgcataa ctagctttta acttgtatcc taggttatct atgctgtctc accatagaga
3061 atattaccta tttcagaatg tatgtccatg attcgccggg taaatacata taatacacaa
3121 atctggctta ataaagtcta taatatatct cataaagaag tgctaaattg gctagtgcta
3181 tatattttta agaaaatttc ttttgactaa gtccatatcg actttgtaaa agttcacttt
3241 agcatacata tattacacga gccagaaatt gtaacttttg cctaaaatca caaattgcaa
3301 aatttaattg cttgcaaaag gtcacatgct tataatcaac ttttttaaaa atttaaaata
3361 cttttttatt ttttattttt aaacataaat gaaataattt atttattgtt tatgattacc
3421 gaaacataaa acctgctcaa gaaaaagaaa ctgttttgtc cttggaaaaa aagcactacc
3481 taggagcggc caaaatgccg aggctttcat agcttaaact ctttacagaa aataggcatt
3541 atagatcagt tcgagttttc ttattcttcc ttccggtttt atcgtcacag ttttacagta
3601 aataagtatc acctcttaga gttggcggtt gtttgcaaga ccgagaaaag gctagcaaga
3661 atcgggtcat tgtagcgtat gcgcctgtga acattctctt caacaagttt gattccattg
3721 cggtgaaatg gtaaaagtca accccctgcg atgtatattt tcctgtacaa tcaatcaaaa
3781 agccaaatga tttagcatta tctttacatc ttgttatttt acagatttta tgtttagatc
3841 ttttatgctt gcttttcaaa aggcctgcag gcaagtgcac aaacaatact taaataaata
3901 ctactcagta ataacctatt tcttagcatt tttgacgaaa tttgctattt tgttagagtc
3961 ttttacacca tttgtctcca cacctccgct tacatcaaca ccaataacgc catttaatct
4021 aagcgcatca ccaacatttt ctggcgtcag tccaccagct aacataaaat gtaagctaag
4081 cttttcaatt caattcatca tttttttttt attctttttt ttgatttcgg tttctttgaa
4141 atttttttga ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta
4201 gattggtata tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa
4261 cccaactgca cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat
4321 ataaggaacg tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc
4381 acgaaaagca aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg
4441 agttagttga agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga
4501 ctgatttttc catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt
4561 ttttactctt cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact
4621 ctgcgggtgt atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg
4681 gcccaggtat tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag
4741 gccttttgat gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta
4801 agggtactgt tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa
4861 gagacatggg tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt
4921 tagatgacaa gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta
4981 caggatctga cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg
5041 tagagggtga acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc
5101 aaaactaaaa aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt
5161 caatttaatt atatcagtta ttaccc
//
