Part:BBa_K256004:Design
NorR
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 655
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 430
Design Notes
-- Primer synthesis
- Using NCBI’s reference sequence NC_000913.2 and E.coli (strain: K-12; substrain: MG1655) as search organism, the DNA sequence for NorR was obtained. The 1515 bp NorR resides at genome position 2828797 – 2830311, and the sequence is as follows:
ATGAGTTTTTCCGTTGATGTGCTGGCGAATATCGCCATCGAATTGCAGCGTGGGATTGGTCACCAGGATC
GTTTTCAGCGCCTGATCACCACGCTACGTCAGGTGCTGGAGTGCGATGCGTCTGCGTTGCTACGTTACGA
TTCGCGGCAGTTTATTCCGCTTGCCATCGACGGTCTGGCAAAGGATGTACTCGGTAGACGCTTTGCGCTG
GAAGGGCATCCACGGCTGGAAGCGATTGCCCGCGCCGGGGATGTGGTGCGCTTTCCCGCAGACAGCGAAT
TGCCCGATCCCTATGACGGTTTGATTCCTGGGCAGGAGAGTCTGAAGGTTCACGCCTGCGTTGGTCTGCC
ATTGTTTGCCGGGCAAAACCTGATCGGCGCACTGACGCTCGACGGGATGCAGCCCGATCAGTTCGATGTT
TTCAGCGACGAAGAGCTACGGCTGATTGCTGCGCTGGCGGCGGGAGCGTTAAGCAATGCGTTGCTGATTG
AACAACTGGAAAGCCAGAATATGCTGCCAGGCGATGCCACGCCGTTTGAAGCGGTGAAACAGACGCAGAT
GATTGGCTTGTCCCCTGGCATGACGCAACTGAAAAAAGAGATTGAGATTGTGGCGGCGTCCGATCTCAAC
GTCCTGATCAGCGGTGAGACTGGAACCGGTAAGGAGCTGGTGGCGAAAGCGATTCATGAAGCCTCGCCAC
GGGCGGTGAATCCGCTGGTCTATCTCAACTGTGCTGCACTGCCGGAAAGTGTGGCGGAAAGTGAGTTGTT
CGGGCATGTGAAAGGAGCGTTTACTGGCGCTATCAGTAATCGCAGCGGGAAGTTTGAAATGGCGGATAAC
GGCACGCTGTTTCTGGATGAGATCGGCGAGTTGTCGTTGGCATTGCAGGCCAAGCTGCTGAGGGTGTTGC
AGTATGGCGATATTCAGCGCGTTGGCGATGACCGTTGTTTGCGGGTCGATGTGCGCGTGCTGGCGGCGAC
TAACCGCGATTTACGCGAAGAGGTGCTGGCAGGGCGATTCCGCGCCGATTTGTTTCATCGCCTGAGCGTG
TTTCCACTTTCGGTGCCGCCGCTGCGTGAGCGGGGCGATGATGTCATTCTGCTGGCGGGGTATTTCTGCG
AGCAGTGTCGTTTGCGGCAGGGGCTCTCCCGCGTGGTATTAAGTGCCGGAGCGCGAAATTTACTGCAACA
CTACAGTTTTCCGGGAAACGTGCGCGAACTGGAACATGCTATTCATCGGGCGGTAGTTCTGGCGAGAGCC
ACCCGCAGCGGCGATGAAGTGATTCTTGAGGCGCAACATTTTGCTTTTCCTGAGGTGACGTTGCCGACGC
CAGAAGTGGCGGCGGTGCCCGTTGTTAAGCAAAACCTGCGTGAAGCGACAGAAGCGTTCCAGCGTGAAAC
TATTCGTCAGGCACTGGCACAAAATCATCACAACTGGGCTGCCTGCGCGCGGATGCTGGAAACCGACGTC
GCCAACCTGCATCGGCTGGCGAAACGTCTGGGATTGAAGGATTAA
- To ensure that the endogenous NorR sequence can be directly converted into a biobrick part without any modification, the sequence was checked with Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2/) for any presence of EcoRI / NotI / XbaI / SpeI / PstI restriction sites. Since none of these restriction sites was found in the endogenouse NorR genome, direct PCR extraction can be performed. PCR primers were designed using NCBI’s PrimerBlast.
- The primer sequences for NorR Extraction :
- Forward Primer (27 bases): 5’-TTAATCCTTCAATCCCAGACGTTTCGC-3’
- Reverse Primer (26 bases):5’- ATGAGTTTTTCCGTTGATGTGCTGGC-3’
- Since NorR sequence is on the anti-sense strand of E.coli genome, the biobrick prefix has to be added to the reverse primer, rather than forward primer, and vice-versa.
- The primer sequences for Biobrick conversion :
- Addition of Preffix to Reverse primer – Forward Primer (50 bases) : 5’-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATGAGTTTTTCCGTTGATGT-3’
- Addition of Suffix to Forward primer – Reverse Primer (50 bases) : 5’-GTT TCT TCC TGC AGC GGC CGC TAC TAG TA TTAATCCTTCAATCCCAGACG-3’
The oligonucleotides were synthesized by one of our team’s sponsor, 1st Base Holdings, Singapore.
Source
-- NCBI’s reference sequence NC_000913.2.
The 1515 bp NorR resides at genome position 2828797 – 2830311
Organism: Escherichia coli K12
Risk group: Risk group 1