BBa_K1744000
1
BBa_K1744000
araC-PBAD-vcrx028-ampR
2015-09-13T11:00:00Z
2015-09-18T12:43:05Z
The part was constructed using pVCR94's toxin coding sequence and cloning it in the plasmid pBAD30. The RBS was selected through rational design and added with the primer.
This part is designed for clean deletion recombineering experiments. It has an arabinose induced toxin for negative selection and an ampicillin resistance gene for positive selection. This part is designed to be amplified using primer with homology to the targeted region and then integrated in that region using lambda red recombineering systems available commercially such as the heat-inducible pSIM plasmid family. The toxin vcrx028 is toxic for the cell. To repress its expression you must put glucose in the medium (we use 5%w/v). To activate the arabinose killswitch, we use 1 %w/v arabinose in the medium. To do the clean deletion (without DNA scar in the sequence) through recombineering, you must first insert the cassette in the genome and select the recombinants with ampicillin without forgetting to use medium containing glucose to avoid unwanted toxin expression. Then you make it pop out using a fusion PCR of both adjacent regions to the one you want to delete and counterselect the cells that did not lose the part with arabinose (triggering the killswitch).
false
false
_2166_
26754
26915
9
true
The protein induced is a toxin, so you MUST repress this arabinose killswitch by adding glucose. Otherwise, you will get either no positive clones or highly mutated toxin that won't work well. In fact, the toxin sequence used is mutated from the native one, but proven good killing efficiency when induced. The part could not be BioBrick standardized for XbaI and PstI. It can still be digested using NotI or EcoRI + SpeI.
false
Frederic Grenier
annotation2454798
1
forward priming site
range2454798
1
1
22
annotation2454820
1
vcrx028
range2454820
1
1246
1623
annotation2454809
1
araC promoter
range2454809
1
1052
1080
annotation2454833
1
bla promoter and RBS
range2454833
1
2167
2364
annotation2454819
1
RBS
range2454819
1
1234
1245
annotation2454812
1
operator I2 and I1
range2454812
1
1140
1178
annotation2454835
1
reverse priming site
range2454835
1
3028
3047
annotation2454810
1
operator O1
range2454810
1
1088
1109
annotation2454817
1
TSS
range2454817
1
1213
1213
annotation2454800
1
operator O2
range2454800
1
930
947
annotation2454818
1
T > G
range2454818
1
1205
1205
annotation2454829
1
rrnB terminator T1 T2
range2454829
1
1649
2074
annotation2454823
1
A > T, was the native stop codon of vcrx028
range2454823
1
1596
1596
annotation2454799
1
araC
range2454799
1
23
901
annotation2454830
1
bla
range2454830
1
2365
3027
annotation2454811
1
CAP site
range2454811
1
1131
1144
annotation2454813
1
PBAD promoter
range2454813
1
1177
1204
BBa_K1744000_sequence
1
caattgtctgattcgttaccaattatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtgtctccatacccgtttttttgggctagcgtaggaggcaaaaatgtgggtcatcgagacaaccgacacctttgatgagtggttcgatgctctggatgataccgatagagcaaacgtgctggcttcgatgatggtgctgcgagatagaggccccatgctgtcgaggccatacgcggatactgttaacggttcatcctacagcaacatgaaagagcttcgggtccaaagcaaaggagatcctatcagagcgttctttgcgttcgatccaaagcgtaaggggattcttctctgcgccggtaacaagaccggggacgagaaaaggttttatgaagtaatgatcccaattgcagaccgtgagtttgcggcgcacttggataaattgaagaaggagtgtcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttggctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactc
igem2sbol
1
iGEM to SBOL conversion
Conversion of the iGEM parts registry to SBOL2.1
James Alastair McLaughlin
Chris J. Myers
2017-03-06T15:00:00.000Z