Baza: |
Plazmidów |
Nr w bazie: |
2555 |
Nazwa plazmidu: |
pGJ100 |
Gatunek: |
Escherichia coli |
Szczep: |
DH5α |
Genotyp: |
φ80dlacZ Δ M15,recA1,endA1,gyrA96,thi-1,hsdR17(rk-,mk+),supE44,relA1,deoR, Δ(lacZYA-argF)U169 pGJ100
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Sekwencja plazmidu: |
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA
CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG
TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC
ACCATAAACGACATTACTATATATATAATATAGGAAGCATTTAATAGACAGCATCGTAAT
ATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAA
AAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAA
GAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTAT
TGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTA
ATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCG
CAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAA
AGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAA
ATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATG
TTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGT
GGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAG
ACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTG
ATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTG
GAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAA
ATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGG
AGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAAT
AGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCTGCGGTGTGAAA
TACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTT
GTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAAT
CGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGT
TTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGT
CTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGG
AAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGC
GCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCC
GCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC
GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAGGGGGGATGTGCTGCAAGGCGATT
AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATT
GTAATACGACTCACTATAGGGCGAATTGGAGCTCGACATAACACCGCAGGAAGAAAAGAA
GAAAGTTGAACTAAGAAATCGAAAAGCAGAGAATATGGATCTTCACCCGAGCAGGAAGAT
GCATATTAAGGAACTTCTACACTCTTTGAACTTAGATATGTGTAATGATGAGGAGGTTTA
TCAGAAAATATCACTTTACTTACAGAAAAATGAGGAAAGCAGGACTTCAGTGGGTGCCTC
GCAACAAAACCACGTAGATATCGATATAAACTCCCTAAAACGCTATCTACAAAATATTGA
AAAGAAGGCACGTCAAAAAAGTGCCATCGATAAACAAAAAAAGAATCAAGCTCGTATATA
TCAATGGAATACTCAATCCTTCTCCGAAATAGTACCCTTATCAGCTGGAAATATCTTATT
CAAAAGGGAACCCAATCGCTTATGGAAACGTTTACAAAATGGAATAAGTGTATTTTTAGG
TTCAAACGGAGGAGGAAAAAAGAGTAAAACTACTAAAAAAGTACTTCAAGGGAATAACAT
ATTGTTACACTCATTAGAAAACAATAAGGATATGACCCTGTCAAACAACTTTGATCACAG
TGTCTTTAACATCAATTTTACGGACTTATTTGGCGTTATTAATGCATCTGGGTCCCCACC
AGATAGAGTTTTGAACGAAATTAATGAGATCGAATTAAAAGGTTGGAAATGTGTGGGGAA
CTTATACGATAATAATAAAATTGTAGTTTTTCAAAGTAGCAATCCACTCTTAGAGGATAC
GAAGATTCCTCAAAAGTCTTTCACTAATTCAAAGAGATTCCTTATTTCACTCTCAGCATT
GCTGGCCTCCTTTTTTGCATATTATAGATACCGTCTATCTCAACGTCAAGAATCAAAGAA
ATGATTTTTTTCTTTCTTGTATATAACAACGTTATACATAGAAACAAATAACTACTCATC
ATTTTGCGAGACATATCTGTGTCTAGATTACCCGGGCCCAACAGTTGCGCAGCCTGAATG
GCGAATGGCGCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGC
GCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT
CCTTTCTCGCCACGTTCGCCGGGTTAATTAAGGCGCGCCAGATCTGTTTAGCTTGCCTTG
TCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTT
GACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCC
CATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTAC
GGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGT
CCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTC
TTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACAT
AAACAACCATGACAGTCAACACTAAGACCTATAGTGAGAGAGCAGAAACTCATGCCTCAC
CAGTAGCACAACGATTATTTCGATTAATGGAACTGAAGAAAACCAATTTATGTGCATCAA
TTGATGTTGATACCACTAAGGAATTCCTTGAATTAATTGATAAATTGGGTCCTTATGTAT
GCTTAATCAAGACTCATATTGATATAATCAATGATTTTTCCTATGAATCCACTATTGAAC
CATTATTAGAACTTTCACGTAAACATCAATTTATGATTTTTGAAGATAGAAAATTTGCTG
ATATTGGTAATACCGTGAAGAAACAATATATTGGTGGAGTTTATAAAATTAGTAGTTGGG
CAGATATTACTAATGCTCATGGTGTCACTGGGAATGGAGTAGTTGAAGGATTAAAACAGG
GAGCTAAAGAAACCACCACCAACCAAGAGCCAAGAGGGTTATTGATGTTAGCTGAATTAT
CATCAGTGGGATCATTAGCATATGGAGAATATTCTCAAAAAACTGTTGAAATTGCTAAAT
CCGATAAGGAATTTGTTATTGGATTTATTGCCCAACGTGATATGGGTGGACAAGAAGAAG
GATTTGATTGGCTTATTATGACACCTGGAGTTGGATTAGATGATAAAGGTGATGGATTAG
GACAACAATATAGAACTGTTGATGAAGTTGTTAGCACTGGAACTGATATTATCATTGTTG
GTAGAGGATTGTTTGGTAAAGGAAGAGATCCAGATATTGAAGGTAAAAGGTATAGAGATG
CTGGTTGGAATGCTTATTTGAAAAAGACTGGCCAATTATAATCAGTACTGACAATAAAAA
GATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATT
TTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGC
GAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTAT
ACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGGGCTTTCCCCGT
CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGAC
CCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT
TTTCGCCCTTTGACGTTGGAGTCCACGTTCTCTGCAGCATTTCCAAACATTTTATTTCGC
CACAACTTGTAGTTTTGTATTGACTCAAATACTTTCTCTTGTTTTGTGCTTCTATTACAG
AGATGGCTTTTTTCTTCAAGTACACTATTTCTAGACGCACTGCACTCGTCAAAAAAATGA
TTCAAGGTGAAAATAAAGGGGTGTTAGTGAATACTTATCAAGAAGAGACATACATAATTG
TACTGCTGTGCATTTTCCTACTTGACTTTCATGATTATGAATAGTACAAACACAGTTGTA
TATATCAAAGTTAAGGGTAGGAGACCACAGGGATTCTTGGATCCTCCCAAATTTGAGTGG
AATGGAACAAAGGAGCGACAGCTTTGGACAATGGTATCAAATTTGAATTATTCACAAGAT
CAAATAGATTGGCAGAATTTATCAAAAATATTTGAAACGCCTGAATTTTTTCTTAAAAAG
AGAACATATAAATTATTTGCTGAACACTTAGAGCTTTTACAACTACAATTGGAGAAAAAA
AGAGATCTTGAGAAGTATTCAAATGATCAAGTGAATGAAGGGATGTCTGATTTAATACAT
AAATATACCCCTACTTTACAAAATGATAATCTATTAAATGTATCCGCAAGCCCACTGACC
ACGGAAAGACAGGATTCTGAAGAAGTTGAGACAGAAGTAACAAATGAGGCGTTACAACAT
TTGCAAACTTCCAAAATTTTGAACATTCACAAGAAAACATCGGATAGTGAGAATAAGCCT
AATGACAAATTGGATAAGGATGGTATTAATAAAGAAATGGAGTGCGGTAGTTCAGATGAC
GATTTATCTTCGAGCTTAAGTGTTAGTAAATCTGCGTTGGAAGAAGCGCTAATGGACAGA
TTGCAATTCTGATTTACATCTTTCACAAGCACAATCAAAAAACCAACTGTCGGCCAAAAA
GGCCCGGCGTTTTACTGTTGGTAAGTCCAGTACCCCGGAATAATCGATACAGATCTGCTC
ATCCTTTTTTATATCTCTATTCATAGTGAATAGCATTGAATTACCTTTACGATATTTCGT
TATATTGGGGTTACAAGAGTGGTTGAAATAGGAGGCTTCTGGAAATACCCAGTATCCAAA
ATATTCTCTGCTGTCTGAGGCTTCGCCCTCTTGCCATAAACCAAAAGCATGTCGACCTCG
AGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTCCGAGCTTGGCG
TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC
ATAGGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACA
TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT
TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC
TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA
AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCA
AAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG
CTCGGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG
ACAGGACTATAAAGATACCAGGCGTTCCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT
CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT
TCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGC
TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT
GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATT
AGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC
TACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA
AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTT
TGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCT
ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA
TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA
AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATC
TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTGCCCGTCGTGTAGATAACT
ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGC
TCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT
GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA
AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTG
TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT
ACATGATCCCCCATGTTGTGAAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTC
AGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT
ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC
TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC
GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAAC
TGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAA
AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT
TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA
TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCT
GGGTCCTTTTCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATAT
ATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGA
AGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTATCTTGCTCTTCCTG
CTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAA
TCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTT
CTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTT
TATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAA
ATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAA
GATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCGTCCTAAGAAACCATTATTATCAT
GACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
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Konstrukcja: |
Construction of the disruption cassettes was a few steps procedure. First, the regions of homology, 5’ and 3’ REV3 flanking sequences was PCR amplified and cloned into pRS314. The 5’ REV3 flanking region, upstream of the REV3 ATG codon, was PCR amplified with pair of primers: 5’ CAGGTCGACATGCTCTCCATACCGCTATTG 3’ and 5’ CCGCTGCAGCATTTCCAAACATTTTATTTCGCC 3’. The resulting PCR product (1159 bp) was SalI/PstI digested and ligated with SalI/PstI digested pRS314, giving the pUPrev3 plasmid. Using primers: 5’ CTGCCCGGGTAATCTAGACACAGATATGTCTCGC 3’ and 5’ CTTGAGCTCGACATAACACCGCAGGAAGAA 3’ the 3’ REV3 flanking region, downstream of the REV3 STOP codon (TAA), was PCR amplified. The resulting PCR product (962 bp) was SmaI/SacI digested and cloned into SmaI/SacI digested pUPrev3. The resulting plasmid, named pUP-DWrev3, contained 5’ and 3’ REV3 flanking regions. Next, DNA fragment containing CaURA3 gene (URA3 from Candida albicans) was cloned between 5’ and 3’ REV3 flanking regions. The CaURA3 containing fragment was PCR amplified using primers: 5’ CGACTGCAGAGAACGTGGACTCCAAC 3’ (PstI) and 5’ GATCCCGGGCCCAACAGTTGCGCAG 3’ (SmaI) and pKF117 as a template (Jaszczur et al. 2008). The PCR product (1798 bp) was PstI/SmaI digested and cloned into PstI/SmaI digested pUP DWrev3. The resulting plasmid, named pGJ100, contain rev3::CaURA3 cassette for deletion of the chromosomal REV3 gene. To isolate rev3::CaURA3 (3922 bp)disruption cassette, the pGJ100 plasmids was SalI/SacI digested and corresponding fragment containing cassette was isolated. The integration of the rev3::CaURA3 cassette into REV3 chromosomal locus leads to deletion of the entire REV3 gene, from first ATG to TAA codons.
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Plasmid properties/Comments: |
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Mapa plazmidu: |
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Markery selekcyjne: |
AmpR |
Stężenie antybiotyku: |
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Wielkość plazmidu: |
8624 bp |
Origin of replication/Incompatibility group: |
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Host range: |
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Inne elementy genetyczne: |
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Właściwości szczepu/komentarz: |
pGJ100 contain rev3::CaURA3 disruption cassette |
Autor: |
Malgorzata Jaszczur |
Dostępność: |
restricted |
Patent: |
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Osoba deponująca: |
Malgorzata Jaszczur |
Instytucja/wydział: |
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Data zdeponowania: |
2009-03-21 17:53+01:00 |
Referencje: |
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