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 annotation2454809 1 araC promoter range2454809 1 1052 1080 annotation2454820 1 vcrx028 range2454820 1 1246 1623 annotation2454833 1 bla promoter and RBS range2454833 1 2167 2364 annotation2454830 1 bla range2454830 1 2365 3027 annotation2454812 1 operator I2 and I1 range2454812 1 1140 1178 annotation2454817 1 TSS range2454817 1 1213 1213 annotation2454818 1 T > G range2454818 1 1205 1205 annotation2454800 1 operator O2 range2454800 1 930 947 annotation2454819 1 RBS range2454819 1 1234 1245 annotation2454823 1 A > T, was the native stop codon of vcrx028 range2454823 1 1596 1596 annotation2454813 1 PBAD promoter range2454813 1 1177 1204 annotation2454810 1 operator O1 range2454810 1 1088 1109 annotation2454799 1 araC range2454799 1 23 901 annotation2454835 1 reverse priming site range2454835 1 3028 3047 annotation2454811 1 CAP site range2454811 1 1131 1144 annotation2454829 1 rrnB terminator T1 T2 range2454829 1 1649 2074 annotation2454798 1 forward priming site range2454798 1 1 22 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