| 质粒名称: | pMEL10 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 107916 |
| 存储实验室: | Jean-Marc Daran |
|
载体骨架基本信息 |
|
| 载体名称: | p426-SNR52p-gRNA.CAN1.Y-SUP4t |
| 载体出品公司: | George Church (Addgene plasmid # 43803) |
| 空载体大小: | 6109bp |
| 完整质粒大小: | 4989 bp |
| 载体类型: | Yeast Expression, CRISPR |
| 筛选标记: | URA3 |
| 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: | gRNA-CAN1.Y |
| 物种来源: | S. cerevisiae (budding yeast) |
| 插入大小: | 20bp |
| 克隆方法: | Gibson Cloning |
|
文献引用方法 |
|
| 材料和方法部分: |
pMEL10 was a gift from Jean-Marc Daran (Addgene plasmid # 107916 ; http://n2t.net/addgene:107916 ; RRID:Addgene_107916)
|
| 参考文献部分: | CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, van den Broek M, Daran-Lapujade P, Pronk JT, van Maris AJ, Daran JM. FEMS Yeast Res. 2015 Mar;15(2). pii: fov004. doi: 10.1093/femsyr/fov004. Epub 2015 Mar 4. 10.1093/femsyr/fov004 PubMed 25743786 |
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 pMEL10 6103 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION URA3 based Yeast/E.coli shuttle vector designed to clone and express
Cas9 programming spacer.
ACCESSION .
VERSION .
KEYWORDS pMEL10
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6103)
AUTHORS Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, van den
Broek M, Daran-Lapujade P, Pronk JT, van Maris AJ, Daran JM
TITLE CRISPR/Cas9: a molecular Swiss army knife for simultaneous
introduction of multiple genetic modifications in Saccharomyces
cerevisiae.
JOURNAL FEMS Yeast Res. 2015 Mar;15(2). pii: fov004. doi:
10.1093/femsyr/fov004. Epub 2015 Mar 4.
PUBMED 25743786
REFERENCE 2 (bases 1 to 6103)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6103
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 98..145
/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)."
rep_origin 126..1108
/label=2u ori
/note="yeast 2u plasmid origin of replication"
promoter 1135..1239
/gene="bla"
/label=AmpR promoter
CDS 1240..2100
/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(1458..1477)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 2271..2859
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2760..2779
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3013..3030
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 3147..3168
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3183..3213
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3221..3237
/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 3226..3248
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 3245..3261
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 3245..3261
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 3280..3300
/label=T3
/note="T3 promoter, forward primer"
promoter 3282..3300
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 3318..3586
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
misc_RNA 3607..3681
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 3686..3705
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
terminator 3711..3958
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
primer_bind complement(3976..3995)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(3977..3995)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4005..4022)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4005..4021)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4014..4036)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(4249..4268)
/label=F1ori-R
/note="F1 origin, reverse primer"
protein_bind 4494..4527
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
protein_bind 5979..6012
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
ORIGIN
1 acaggcaaca cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg
61 ttgcattttc ggaagcgctc gttttcggaa acgctttgaa gttcctattc cgaagttcct
121 attctctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg ctctgaagac
181 gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat attgcgaata
241 ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt gctatatccc
301 tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc gacctctaca
361 ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta agaactattc
421 atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag tatatagaga
481 caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa cgctatcact
541 ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg gatgccttta
601 tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa gtggagtcag
661 gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc ttaacggacc
721 tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag aaaaaaagta
781 atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta gaacaaaaaa
841 gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa
901 atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca
961 aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg
1021 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
1081 ctacaaaatg aagcacagat gcttcgttca ggtggcactt ttcggggaaa tgtgcgcgga
1141 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
1201 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
1261 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
1321 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
1381 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
1441 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
1501 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
1561 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
1621 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
1681 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
1741 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
1801 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
1861 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
1921 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
1981 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
2041 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
2101 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
2161 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
2221 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
2281 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
2341 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
2401 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
2461 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
2521 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
2581 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
2641 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
2701 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
2761 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
2821 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
2881 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
2941 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
3001 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
3061 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
3121 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
3181 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
3241 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
3301 gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca ctcatattta
3361 tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta cgtttgaagt
3421 acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca ttttttcaca
3481 ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa agttggtgcg
3541 catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcgata cgttctctat
3601 ggaggagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
3661 aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag tcatgtaatt
3721 agttatgtca cgcttacgtt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg
3781 agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag
3841 aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa
3901 cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcgg
3961 ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
4021 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
4081 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
4141 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
4201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
4261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
4321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
4381 gtgatggttc acgtagtggg ccgttgaaca ttcttaggct ggtcgaatca tttagacacg
4441 ggcatcgtcc tctcgaaagg tggctaggcc actagtggat ctgatatcac ctaataactt
4501 cgtatagcat acattatacg aagttatatt aagggttctc gagagctcgt tttatttagg
4561 ttctatcgag gagaaaaagc gacaagaaga gatagaccat ggataaatga ttatgttcta
4621 aacactcctc agaagctcat cgaactgtca tcctgcgtga agattaaaat ccaacttaga
4681 aatttcgagc ttacggagac aatcatatgg gagaagcaat tggaagatag aaaaaaggta
4741 ctcggtacat aaatatatgt gattctgggt agaagatcgg tctgcattgg atggtggtaa
4801 cgcatttttt tacacacatt acttgcctcg agcatcaaat ggtggttatt cgtggatcta
4861 tatcacgtga tttgcttaag aattgtcgtt catggtgaca cttttagctt tgacatgatt
4921 aagctcatct caattgatgt tatctaaagt catttcaact atctaagatg tggttgtgat
4981 tgggccattt tgtgaaagcc agtacgccag cgtcaataca ctcccgtcaa ttagttgcac
5041 catgtccaca aaatcatata ccagtagagc tgagactcat gcaagtccgg ttgcatcgaa
5101 acttttacgt ttaatggatg aaaagaagac caatttgtgt gcttctcttg acgttcgttc
5161 gactgatgag ctattgaaac ttgttgaaac gttgggtcca tacatttgcc ttttgaaaac
5221 acacgttgat atcttggatg atttcagtta tgagggtact gtcgttccat tgaaagcatt
5281 ggcagagaaa tacaagttct tgatatttga ggacagaaaa ttcgccgata tcggtaacac
5341 agtcaaatta caatatacat cgggcgttta ccgtatcgca gaatggtctg atatcaccaa
5401 cgcccacggg gttactggtg ctggtattgt tgctggcttg aaacaaggtg cgcaagaggt
5461 caccaaagaa ccaaggggat tattgatgct tgctgaattg tcttccaagg gttctctagc
5521 acacggtgaa tatactaagg gtaccgttga tattgcaaag agtgataaag atttcgttat
5581 tgggttcatt gctcagaacg atatgggagg aagagaagaa gggtttgatt ggctaatcat
5641 gaccccaggt gtaggtttag acgacaaagg cgatgcattg ggtcagcagt acagaaccgt
5701 cgacgaagtt gtaagtggtg gatcagatat catcattgtt ggcagaggac ttttcgccaa
5761 gggtagagat cctaaggttg aaggtgaaag atacagaaat gctggatggg aagcgtacca
5821 aaagagaatc agcgctcccc attaattata caggaaactt aatagaacaa atcacatatt
5881 taatctaata gccacctgca ttggcacggt gcaacactac ttcaacttca tcttacaaaa
5941 agatcacgtg atctgttgta ttgggatctc tagacctaat aacttcgtat agcatacatt
6001 atacgaagtt atattaaggg ttgtcgacct gcagcgtacg aaggtgccta ttgatgatct
6061 ggcggaatgt ctgccgtgcc atagccatgc cttcacatat agt
//
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