BBa_K911004 1 BBa_K911004 Synthesised Ratiometric Luciferase construct in non-standard plasmid 2012-09-20T11:00:00Z 2015-05-08T01:13:45Z Commercial synthesis by DNA 2.0 This part was designed as a ratiometric luciferase reporter. The first promoter, hyperSpank, is LacI - repressed and controls the transcription of a luxA gene that has been fused at the N-terminus to an mOrange gene via a flexible linker. This was described by Dachuan Ke and Shiao-Chun Tu (2011) as having an additional peak in its emission spectrum at 560 nm, whereas the normal peak is at 490 nm. This is terminated by b0015. Downstream, pVEG controls the translation of the entire normal lux operon, which is again terminated by b0015. This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus. During and after assembly of this sequence, unexpected toxicity issues were observed. This necessitated its assembly in a low copy number vector. This part is submitted to the registry as is, in the suitable backbone provided by the synthesis company. We strongly recommend that you do not attempt to assemble it in psB1C3, as it will kill your cells. The toxicity of the construct causes strong selective pressure against it, and characterisation has been hampered by the tendency of cells to lose parts of the insert. We suspect that the repeated terminator may facilitate recombination, and another team might investigate whether replacing the second terminator aids stability. The construct is not behaving entirely as expected, as the e.coli colonies are initially orange, despite mOrange being lacI-repressed. This is probably due to leakage, as the RBSes are very strong in both e.coli and bacillus. Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent) We ran out of time before attempting to insert this into bacillus. Bacillus homology regions would need to be added, but on the upside, the much lower copy number would likely counteract the toxicity issues. Further characterisation to follow false false _1176_ 0 12683 9 It's complicated true Sequence designed to be compatible with bacillus and e.coli. As such, all genes codon-optimised for bacillus, all RBSes are at bacillus consensus, all promoters are documented as having the same function in e.coli and bacillus. false Thomas Whittaker annotation2191354 1 Bacillus consensus RBS + Spacers range2191354 1 3623 3651 annotation2191358 1 Bacillus consensus RBS + Spacers range2191358 1 5667 5695 annotation2191359 1 LuxB range2191359 1 5696 6670 annotation2191349 1 LuxA (fused to mOrange) range2191349 1 865 1932 annotation2191360 1 Bacillus consensus RBS + Spacers range2191360 1 6671 6699 annotation2191362 1 Bacillus consensus RBS + Spacers range2191362 1 7837 7865 annotation2191345 1 HyperSpank range2191345 1 1 97 annotation2191346 1 Bacillus consensus RBS + Spacers range2191346 1 98 126 annotation2191363 1 LuxG range2191363 1 7866 8585 annotation2191364 1 B0015 terminator range2191364 1 8586 8715 annotation2191350 1 B0015 terminator range2191350 1 1933 2062 annotation2191348 1 GS Linker range2191348 1 835 864 annotation2191353 1 LuxC range2191353 1 2189 3622 annotation2191347 1 mOrange no stop range2191347 1 127 834 annotation2191352 1 Bacillus consensus RBS + Spacers range2191352 1 2160 2188 annotation2191357 1 LuxA (normal) range2191357 1 4599 5666 annotation2191356 1 Bacillus consensus RBS +Spacers range2191356 1 4570 4598 annotation2191355 1 LuxD range2191355 1 3652 4569 annotation2191351 1 pVEG range2191351 1 2063 2159 annotation2191361 1 LuxE range2191361 1 6700 7836 BBa_K911004_sequence 1 caacctaattgtgagcgctcacaattttttgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtgtaattgtgagcggctcacaatttactagaggggaaaaggaggtgaactactatggtgtctaaaggcgaagaaaacaacatggcgataatcaaagagtttatgcgctttaaagtcaggatggaaggctccgtcaatggtcacgaatttgaaattgagggagaaggtgaaggacggccgtacgagggatttcaaacagctaaacttaaagtaacaaaaggcggaccgcttccgtttgcatgggacattttgagtcctcagtttacgtatggttctaaagcgtatgtcaaacatcccgcggatattccggattactttaagctctcgtttcctgaagggttcaagtgggagagagtaatgaatttcgaggatggtggagtagttaccgtcacgcaagactcatcattacaagacggagagttcatttacaaagttaaactgcgaggtacaaacttcccgtccgatgggcctgtgatgcagaagaaaactatgggatgggaagcatctagcgaaagaatgtatccagaggatggcgctctgaaaggcgagatcaaaatgcggctaaaactgaaagacggcggacattatacgtcagaagttaaaaccacttacaaagccaagaagcctgtgcaattaccaggggcatatatcgtggggattaagcttgatattacaagccataatgaagattatacgatcgttgaacagtatgaacgtgccgaaggccgccacagcacaggcggaatggatgaattgtataagggcggcggcggctcaggcggcggcggctcaatgaagtttggtaactttctgttgacgtatcagccgccagaactcagtcaaacagaggttatgaaacgcctcgtcaatttaggaaaagcttccgaagggtgcggctttgacacagtatggttgctggaacaccattttaccgaatttggcctgctgggcaatccatatgtcgcggcagctcatcttcttggagcgacggaaaaactcaacgtaggaactgccgccattgtattacctaccgctcatccggttagacaggcagaggatgttaatcttttagaccagatgtctaaaggtaggttcagatttggcatttgccgcggtttatacgataaagattttcgggtgttcggaacggatatggacaatagcagagcgttaatggattgctggtacgatttgatgaaagagggatttaacgaaggctatattgccgcagacaatgaacatatccaatttcctaaaatccagttaaatccgtcagcatatacgcaaggcggtgcacctgtgtatgtcgtcgcggagtcggcttctactacagaatgggctgccgaacgaggtttgccgatgattctttcctggatcattaacactcacgaaaagaaagcacaattagatctgtataatgaggttgctacagagcatggatatgacgtgactaagattgaccactgtctttcttacattacaagcgtcgatcacgattcaaacaaagccaaagatatttgtcgtaactttcttggccattggtacgactcatatgttaatgcgacaaaaatcttcgacgattcggaccaaaccaagggctatgatttcaacaaggggcagtggcgggattttgtgctgaaggggcataaagatacaaatagacgtattgattattcttacgagataaatccggtggggacacctgaagaatgtattgcgatcatacaacaggatatcgacgccacgggcataaacaacatttgctgtggatttgaggcgaatggaagcgaagaagaaatcatcgcatcaatgaaattgttccagagcgatgtaatgccctaccttaaagaaaaacaataagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataaattttgtcaaaataattttattgacaacgtcttattaacgttgatataatttaaattttatttgacaaaaatgggctcgtgttgtacaataaatgttactagaggggaaaaggaggtgaactactatggagaagcatctaccactcattatcaatggccaaattgtatttaccgaagaaaaccgttttgagatatcttttgaagaaaagaaagtcaagattgactcattcaatcatgtccatctgacacaaatggttactcatgattatctcaacgacataaacattaacaatatcatcaacttcttatacacgaccggccagcggtggaaaagcgaagaatatagccgaaggcgggcttacattagatcgcttgtcacatacctgggatatagtcctcaaatggctaaactcgaagccaattggatcgcgatgatactttgcagtaagtctgcactgtatgatatcatcgacaccgaacttggatcgacacacattcaggatgagtggttgccgcaaggggaatgctacgtccgcgcgttcccaaaaggtcgtacaatgcacttactagccgggaatgtcccgctgagcggcgtgacatctattttgcgaggtatcttaacgcggaatcagtgcattgtgaggatgtccgcaagcgatccttttacggcacatgcgctcgccatgtcgtttatcgatgtggacccgaaccatccaattagtcgctctatttcggtactttattggcctcacgctagtgacacaaccttggcagaagaactgctcaatcacatggacgctgtcgttgcctggggtggccgtgacgcgatcgattgggcggttaaacattcaccgtcccatatagatgttctgaaatttggacccaagaagtccttcacggtccttgatcacccggcagatttagaagaggctgcgtccggtgttgcacatgacatatgtttttacgatcagaacgcatgctttagcacacagaacatttacttttctggcgataactatgaagaatttaagagcaaattggttgaaaaactgaacctctatcaagaggtccttcctaaatctaagcagaattttgatgatgaggcgctcttttcaatgacgcgattggagtgtcagtttagcggactcaaagtaatatcagaaccggaaaacaattggatgattatagattcagaaccgggcgtagagtacaatcatccgttgagccgttgtgtctatgtacacaaaatcaataaagttgacgacgttgtgcagtatatcgaaaaacatcaaacgcaaacgatttctttctatccctgggaaagttcgaaaaagtacagagatgcgttcgccgcgaaaggcgttgagaggattgttgagtcgggaatgaataacatttttcgcgttggtggtgcacacgatgctatgcgccctctccaacggttagtccgcttcgtatcacacgaaagaccttatcattttacaaccaaagatgttagcgtggagatcgaacaaacgcggtttctggaagaagataagtttttagtgtttgtgccttaatactagaggggaaaaggaggtgaactactatgtcaaaccaatgtaaaacaatagctcatgtgctcccagtgaataatggacaagaaattcacgtctgggaaactccgccaaaagaaaatgctccgtctaagaatagcacgattctgattgcctctggatttgccagacgcatggatcattttgcaggccttgcagaatacttatctgagaacggtttccacgttttcaggtacgattcgctacatcatgtaggacttagtagcggctcaattgatgaatttacaatgacgactggtaagaactccctgtgcaccgtgtatcattggttgcagaccaaagggacgcagaatataggcctcatcgccgctagcctgagtgcgcgggtagcctatgaagtaatcagcgatttagagctcagctttctcattacggcagtcggggttgttaacttgcgcgacacacttgagaaagcgctggggtttgactatctctcattgccgattaatgaactgcctaatgatctggatttcgaaggccataagttgggttccgaagtgttcgttcgggactgttttgagcaccattgggatacattagattcaacactagacaaagtcgcaaatactagcgtgcctcttatcgcatttacagccaacaatgacgattgggtcaagcaagaagaagtgtacgacatgttagctcatatcagatctggacactgcaaactttattcacttctgggatcaagtcatgatttaggcgaaaacttagtcgtattgagaaacttctatcaatccgttacaaaagcggcgattgcgatggatggcggatctctggagattgatgtcgactttattgaacccgattttgagcagttaaccatcgcaacggttaacgaacgtcgacttaaagcggagatcgaatcgcgtgctccggaaatggcataatactagaggggaaaaggaggtgaactactatgaaattcggaaactttttactgacctatcagccaccagaattatcccagactgaagttatgaaaagattagtaaatctgggcaaagcttcagaaggctgcggatttgatacagtgtggcttctcgaacatcattttacggaatttgggctactcggtaatccgtacgtcgcagccgcacacttactgggcgcaacggaaaagctgaatgtcggaacagcagcgattgtacttccgaccgcccatcctgttcgccaggcagaagatgttaacttactggatcaaatgagtaaaggtcggtttcgtttcggtatctgccgcggactatacgacaaagattttagggtgtttggcactgatatggataacagcagggcgttgatggattgttggtatgatctg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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