BBa_K1744001 1 BBa_K1744001 P1U8-amilCP-kanR 2015-09-13T11:00:00Z 2015-09-18T12:41:04Z The part was constructed using a kanamycin resistance gene from plasmid pOK12 (available commercially) and amilCP from BBa_K592009. The promoter and RBS P1U8 were synthesised and come from Mutalik et al. (Nature 2013). This part is a positive selection system for recombineering. It contains a kanamycin resistance gene Aph(3???)-I (aminoglycoside 3???-phosphotransferase, truncated version) with its native promoter as well as amilCP, a deep blue chromoprotein. amilCP gene is under the control of a strong constitutive promoter with a strong RBS to make it more intense than previous versions. The cassette is designed to be a double positive selection marker for recombineering. Once amplified with primer bearing homology to the targeted region, the cassette can be integrated in the genome through lambda red recombination. Once integrated, the colonies should become resistant to kanamycin and become pale blue overtime. However, the blue color is actually faint and really slow to become apparent. false false _2166_ 26754 26754 9 false The amilCP chromoprotein synthesis could be improved for the blue color to be visible in single copy faster. It has been of great concern through-out all the project and that is why we have put the strongest combination of promoter and RBS we have found to be well characterised. Still, expression in single copy is not so intense. In our case, amilCP can still be useful as a marker for plasmid background detection during recombineering experiment. Using only the kanamycin marker for recombineering, the blue colonies would represent plasmid transformants and not good recombinants. This use is good for us though, since background is a great concern in recombineering techniques, where screening can be long. false Kevin Neil annotation2454746 1 Promoter-aph-3'-I range2454746 1 613 819 annotation2454758 1 amilCP range2454758 1 820 1488 annotation2454760 1 Promoter P1 range2454760 1 1510 1548 annotation2454759 1 U8 range2454759 1 1489 1509 annotation2454745 1 aph-3'-I range2454745 1 1 612 BBa_K1744001_sequence 1 ttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaaggttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccagggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcaatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgagcaagacgtttcccgttgaatatggctcatttattaggcgaccacaggtttgcgtgcaatggaaatttcacactgctcaaccgaagtgtaatccttgttgtgattggttacatccagtttgcggtcaacatagtgataccctggcatcttcacaggcttctttgccttgtaagtagttttaaattcacacaaatagtgaccgcctccttctaacttcagagccataaagttgtttcctagcagcattccatctcgtgcaaagagacgctcagtgttgggttcccagccctgtgtcttcttctgcatgacaggtccattgggaggaaagttcaaaccagagaacttgacatggtagatgaaacagttgccttggatgctggaatcattgctgacagtacacactgcaccatcttcaaagttcatgatcctctcccatgtatagccctccgggaatgactgctttacatagtcagggatgtcttcagggtacttggtgaatggtatgcttccgtactgacactgtggtgataaaatatcccaagcaaatggcagaggtccgcccttggtgacagtgagctttaccgtctgctccccctcgtagggcttaccttttccatcgccttcgacctcaaagtagtgtccattgaccgtgcctgacatataaaccttgtaggtcatttgtttagcgatcacactcattaatatatacctcttaattttcaaagctagcataatacctaggactgagctagctgtaaa igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z