α β γ δ ε ζ η θ ι κ λ μ ν ξ ο π ρ Σ σ τ υ φ χ ψ ω Δ Γ Ω Σ Λ Ψ Φ Ξ Θ Π °C Greek letters

Central Collection of Strains (IBB)

Base: Plasmid collection
Number in base: 2555
Plasmid name: pGJ100
Species: Escherichia coli
Strain: DH5α
Genotype: φ80dlacZ Δ M15,recA1,endA1,gyrA96,thi-1,hsdR17(rk-,mk+),supE44,relA1,deoR, Δ(lacZYA-argF)U169 pGJ100
Plasmid Sequence: 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
Construction: 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.
Plasmid properties/Comments:
Plasmid map:
Selectable Markers: AmpR
Antibiotic concetration:
Plasmid size: 8624 bp
Origin of replication/Incompatibility group:
Host range:
Other genetic elements:
Strain properties/Comments: pGJ100 contain rev3::CaURA3 disruption cassette
Author: Malgorzata Jaszczur
Availability: restricted
Patent:
Depositor: Malgorzata Jaszczur
Institution/Department:
Deposition date: 2009-03-21 17:53+01:00
References: