IGEM:Stanford/2009/Project Homeostasis/Primer

Anti-Immunosuppression Device

Primers

Added 7/1/09 - Mark

• HlyB (from E. coli str. CFT073)

Forward: TATAgaattcgcggccgcttctagagGGAGTCATAATGGATTCTTGTC

Reverse: TATAtactagtagcggccgctgcagGTTAGTCTGACTGTAACTG

• HlyD (from E. coli str. CFT073)

Forward: TATAgaattcgcggccgcttctagATGAAAACATGGTTAATGGGGTTCAGCG

Reverse: TATAtactagtagcggccgctgcagCTCTGAGACTTAACGCTCATG

• TolC (from E. coli str. CFT073)

Forward: TATAgaattcgcggccgcttctagagGCAAATGAAGAAATTGCTCC

Reverse: TATAtactagtagcggccgctgcagCGTCATCAGTTACGGAAAGGG

Added 7/1/09 - Ming

• HlyB (from CFT073)

"Forward": TATAgaattcgcggccgcttctag ATGGATTCTTGTCATAAAATTG

"Reverse": TATAtactagtagcggccgctgcag TTAGTCTGACTGTAACTG

• HlyD (from CFT073)

"Forward":TATA gaattcgcggccgcttctag ATGAAAACATGGTTAATG

"Reverse": TATAtactagtagcggccgctgcag TTAACGCTCATGTAAACTTTC

• TolC

"Forward": TATAgaattcgcggccgcttctagATGAAGAAATTGCTCCCCATTC

"Reverse": TATAtactagtagcggccgctgcagTCAGTTACGGAAAGGGTTATG'

Added 6/28/09 - Robert, Ariana

• IL-6 fusion gene

"Forward": TATAGAATTCGCGGCCGCTTCTAGAGGGATGCATCACCGGTTCCGC

"Reverse": ATATTACTAGTAGCGGCCGCTGCAGGGATGCATTCATTTGACGAAGAG

Added 7/1/09 - Anusuya

• TrpR (E. coli str. IAI1)

Forward: TATAgaattcgcggccgcttctagATGGCCCAACAATCACCCTATTCAGC

Reverse: 'TATAtactagtagcggccgctgcagTCAATCGCTTTTCAGCAACACCTCTTCCAGCCACTGGC

• Trp Promoter and Operator (E. coli: no specified strain )

Forward: TATAgaattcgcggccgcttctagagCTCAAGGCGCACTCCCGTTCTG

Reverse: TATAtactagtagcggccgctgcagTCGATACCCTTTTTACGTGAACTTGCGTACTAG

• Protease-binding sequence?

Gene Sequences

• HylB

ATGATCATCAATAAATTTTCCCTTAAATGGATGTTGGCTATTGCCGTCGCCATCCCTGCGATAGCACTGTTGTTTGTGGCTTTCACCAGTCTAAACACCATGTCAGTGATGCAAGCGCAGTCCAACAGCTTGTATGCCAACACGGCTGCACCAATGCGTGCCATGGCTGAAGCAACCTCACGTATTCCTCGGATGCGTGTCGGTATCGATATGATGCTACTGCAAGAAACGGCGCTCAAAGATGCGAAAGGGGTCCTCAAACGAGTCGAAGAGGCAAGAACCGAAGATATCCCAGAAATGCGTCAAGCAATGCAAGTTGCGGTTGATTCTCAGGTTAATCCGGAACTCAAAGAGCAGGCACGCAAACTTCAAGCTCGTTTTGAACAAATGGTACGTGAAGAGTTAGAGCCTATGCTGCAAGCCTTCGCCAATAACGATATGACCACGGCACAAAACATTTACCGCGATAAATACGCGCCGACCTATGGTGAAATGCGTAAACAAGCCAACCAGATCCTCGATACGCTTTTGCAGCAAGCGGAGCAGCAAAACCATGCCAGTGTGGAAAGCTTCGAAGCAGGACGCACCAAGCAAATGGTGATCATTGCAGCAGGCTTGATCATTTCATTCATCACTTCACTGGTTATCATAACGAACTTACGTAGCCGAGTGGCTTACCTGAAAGATCGTATGAGTTCTGCGGCGGCGAATCTTTCACTGCGTACTCGATTGGAGTTGGATGGTAACGATGAACTGTGTGACATCGGTAAAAGCTTCAATGCGTTCATTGATAAAGTGCATCACTCGATTGAAGAAGTGGCAGAAAACTCAAAAGAGCTGGCGACGATGGCCTCTAGTGTGTCGCAGCGCGCGCACATGACGCAATCTAACTGTGCTTCGCAGCGAGATAGAACAGTGCAAGTTGCGACGGCGATTCATGAGCTTGGTGCCACCGTATCCGAAATCGCTTCCAATGCGGCCATGGCTGCCGATGTCGCGAAGCAAGCGACGCTGCATTCTGGTGAAGGGAAAAAAGTGGTAGGCGAAGTGCAAAATCGGATCCAAACACTGGTCAATGAACTCGATAATGCCACTCAAGTTGTCTCATCACTGGCGACCCAAATTAACGGTATTAGCTCAACACTTGATACCATTCGCAGTATTTCTGAGCAAACGAACCTATTGGCGCTCAACGCTGCGATTGAAGCTGCGCGAGCGGGTGAACAAGGTCGTGGTTTTGCGGTGGTGGCGGATGAAGTTCGCACATTAGCAAGTCGTTCAGCGGCATCGACGGAAGAGATCCAGCAAGTCATTAATCGCCTTCAAACGGAGTCAACTCGCGCAGTAGAAGCAATGGAAAAAGGTCGCTCGCAAAGTGATGTGGTGGTTGAGTTTTCCGCTAAAGCGAACCAATCTCTCACAGAGATCAACAGCCAAATTGATCAGATTAATGATCAAAATATTCAAGTTGCGACCGCGACAGAGGAACAATCAACCGTGGTGGAAGACATTAATCGCAACGTTGAAGACATCAACCAACTGACGACAGAAACCTCGCATGTTGCGGATGAGTTAAGCCGAGCCAGTGCAAGCTTGCAACGTCTCTCTTCGCAACTGGATAAACTGGTGGGCAGTTTTGAACTTTAA http://www.ncbi.nlm.nih.gov/nuccore/NC_012583.1/?from=274978&to=276624&report=fasta NCBI Reference Sequence: NC_012583.1 from Vibrio cholerae O395 chromosome II, complete genome

• HylD

ATGGCTGCGTTCGGACAAGACGTCTTCAGGCGCTTCGGCACGGTCACCGAACGCTTCTCCGGCACCGGCGGCAAACTCTTCGGCGGCCTGCTCGCGCGCCTGTCGAGCGTGGAGCGCGACGACAGCCTGGACTGGGCGAGCGACGCCGACTGGGCGCGCCTGCAGCAGGAGCCGCTGCGCGCGCGCGCGGTACTCAAGGTCAGCGCGATCGCGCTGCTGGTGCTGCTCGGCTGGGCCGCGGTGGCGCATGTGGACGAGGTGACCAAGGGCGACGGCAAGGTGATCCCGTCCAGCCAGCTGCAGATCATCCAGTCGGTCGATGGCGGCGTGGTGGAGTCGATCGCGGTGCGCGAGGGCGAGGTGGTCGAGGCCGGGCAGCTGCTGCTCAAGGTCGATCCCACCCGTTTCATGTCCTCGCTGCTGGAAAACCGCGCCGAGTTCCTCGCGCTGGAAATCAAGCGCCGGCGGCTCGAGGCGCTGACCCAGGGGACGCCGTTCTCGGTGCCGGCCGAGCTGGAGAAGGAGGCGGCGGACATCGCCGCCCACGAGCGCCGGCTGTATGAATCCAGCCGCGCCGAGATGGACGCGCAGCAGTCGATCGCGCGCGACCAGCTGCGCCAGCGCGAGCAGGAACTCAACGAGGTGCGCGCCCGCAGTGAGAACGCCGCCCGCGCCTACGAACTGGTGCAGCAGGAACTCAAGGTCACCAAGCCGCTCGCGGCCTCCGGCGCGGTATCCGAGGTCGAGCTGCTGCGTCTGGAGCGCGACGTGTCGCGCTTGCGCGGCGAGCGCGAGCAGAGCGCGGCGCAGATTTCGCGCATCCAGTCGGCCATCGCCGAATCCACGCGCAAGATCCAGGAGATCGAGCTGACGGTGCGCAACCAGCTGCGCAACGAGCTTTCGGACACCATGGCGCGGCTGGGCAGCCTGACCCAGGGCAGCCGCATGCTGGCCGACCGCGTCAAGCACGCCGAAATCCGCGCGCCGATGCGCGGCACGGTCAAGCGCATCCTCGTCAACACCGTGGGTGGCGTGGTCCAGCCGGGCAAGGAAGTGGCCGAGATCGTGCCGCTCGACGACGCGCTGATCCTCGAAGCGCAGGTCAAGCCGAGCGACATCGCCTTCCTGCGCCCGGGTCAGTCGGCGCTGGTCAAGTTCTCCGCGTACGACTTCTCGATCTACGGCGGCCTGGAGGCGGTCGTGGACCAGATTTCCGCCGACACCGTGGTCGATGCAAAGGGTTCCGCGTTCTACGTGGTGCGGGTGCGCACGCTGCATTCCTCTCTGGGTGAAAACCTGCCGATCATTCCGGGCATGGTGGCCGAAGTGGACATCCTGACCGGCAAGAAGACCATCCTCTCCTACCTGCTGAAGCCCGTGCTGCGGGCCAAGGCCAACGCACTGTCCGAACGATGA http://www.ncbi.nlm.nih.gov/nuccore/NC_008702.1/?from=674498&to=675913&report=fasta NCBI Reference Sequence: NC_008702.1 from Azoarcus sp. BH72, complete genome

• TolC

ATGAAGAAATTGCTCCCCATTCTTATCGGCCTGAGCCTTTCTGGGTTCAGTTCGTTGAGCCAGGCCGAGAACCTGATGCAAGTTTATCAGCAAGCACGCCTTAGTAACCCGGAATTGCGTAAGTCTGCCGCCGATCGTGATGCTGCCTTTGAAAAAATTAATGAAGCGCGCAGTCCATTACTGCCACAGCTAGGTTTAGGTGCAGATTACACCTATAGCAACGGCTACCGCGACGCGAACGGCATCAACTCTAACGCGACCAGTGCGTCCTTGCAGTTAACTCAATCCATTTTTGATATGTCGAAATGGCGTGCGTTAACGCTGCAGGAAAAAGCAGCAGGGATTCAGGACGTCACGTATCAGACCGATCAGCAAACCTTGATCCTCAACACCGCGACCGCTTATTTCAACGTGTTGAATGCTATTGACGTTCTTTCCTATACACAGGCACAAAAAGAAGCGATCTACCGTCAATTAGATCAAACCACCCAACGTTTTAACGTGGGCCTGGTAGCGATCACCGACGTGCAGAACGCCCGCGCACAGTACGATACCGTGCTGGCGAACGAAGTGACCGCACGTAATAACCTTGATAACGCGGTAGAGCAGCTGCGCCAGATCACCGGTAACTACTATCCGGAACTGGCTGCGCTGAATGTCGAAAACTTTAAAACCGACAAACCACAGCCGGTTAACGCGCTGCTGAAAGAAGCCGAAAAACGCAACCTGTCGCTGTTACAGGCACGCTTGAGCCAGGACCTGGCGCGCGAGCAAATTCGCCAGGCGCAGGATGGTCACTTACCGACTCTGGATTTAACGGCTTCTACCGGGATTTCTGACACCTCTTATAGCGGTTCGAAAACCCGTGGTGCCGCTGGTACCCAGTATGACGATAGCAATATGGGCCAGAACAAAGTTGGCCTGAGCTTCTCGCTGCCGATTTATCAGGGCGGAATGGTTAACTCGCAGGTGAAACAGGCACAGTACAACTTTGTCGGTGCCAGCGAGCAACTGGAAAGTGCCCATCGTAGCGTCGTGCAGACCGTGCGTTCCTCCTTCAACAACATTAATGCATCTATCAGTAGCATTAACGCCTACAAACAAGCCGTAGTTTCCGCTCAAAGCTCATTAGACGCGATGGAAGCGGGCTACTCGGTCGGTACGCGTACCATTGTTGATGTGTTGGATGCGACCACCACGTTGTACAACGCCAAGCAAGAGCTGGCGAATGCGCGTTATAACTACCTGATTAATCAGCTGAATATTAAGTCAGCTCTGGGTACGTTGAACGAGCAGGATCTGCTGGCACTGAACAATGCGCTGAGCAAACCGGTTTCCACTAATCCGGAAAACGTTGCACCGCAAACGCCGGAACAGAATGCTATTGCTGATGGTTATGCGCCTGATAGCCCGGCACCAGTCGTTCAGCAAACATCCGCACGCACTACCACCAGTAACGGTCATAACCCTTTCCGTAACTGA http://www.ncbi.nlm.nih.gov/nuccore/NC_000913.2/?from=3176137&to=3177618&report=fasta NCBI Reference Sequence: NC_000913.2 from Escherichia coli str. K-12 substr. MG1655, complete genome

• IL-6 fusion gene

• Protease-binding sequence?

Anti-Inflammatory Device

Primers:

SoxR (Escherichia coli E24377A)

Forward: TATAgaattcgcggccgcttctagATGGAAAAGAAATTACCCCGC

Reverse: TATAtactagtagcggccgctgcagTTAGTTTTGTTCATCTTCCAGCAAGCGTG

Added 7/1/09 - Anusuya

CrtB (Erwinia Uredovora)

Forward: TATAgaattcgcggccgcttctagATGGCAGTTGGCTCGAAAAG

Reverse: TATAtactagtagcggccgctgcagCTAGAGCGGGCGCTGCCAG

CrtE (Erwinia Uredovora)

Forward: TATAgaattcgcggccgcttctagATGACGGTCTGCGCAAAAAAAC

Reverse: TATAtactagtagcggccgctgcagTTAACTGACGGCAGCGAGTTTTTTG

CrtI (Erwinia Uredovora)

Forward: TATAgaattcgcggccgcttctag ATGAAACCAACTACGGTAATTGGTGC

Reverse: TATAtactagtagcggccgctgcag TGATTGAGTAACGACGG

CrtY (Erwinia Uredovora)

Forward: TATAgaattcgcggccgcttctag ATGCAACCGCATTATGATCTG

Reverse: TATAtactagtagcggccgctgcag TTAACGATGAGTCGTCATAATGGC

Soxs: primer dimers

Blh (Halobacterium sp. NRC1)

Forward: TATAgaattcgcggccgcttctagATGCCACACGGCGCGATCG

Reverse: TATAtactagtagcggccgctgcagTCAGAGGACGCCCTGCAC

RalDH(II) (Rattus Norvegicus)

Forward: TATAgaattcgcggccgcttctagATGCCCGGCGAGGTGAAGGC

Reverse: TATAtactagtagcggccgctgcagTTAGGAGTTCTTCTGGGGGATC

Gene sequences: SoxR ATGGAAAAGAAATTACCCCGCATTAAAGCGCTGCTAACCCCCGGCGAAGTGGCGAAACGCAGCGGTGTGGCGGTATCGGCGCTGCATTTCTATGAAAGTAAAGGGTTGATTACCAGTATCCGTAACAGCGGCAATCAGCGGCGATATAAACGTGATGTGTTGCGATATGTTGCAATTATCAAAATTGCTCAGCGTATTGGCATTCCGCTGGCGACCATTGGTGAAGCGTTTGGCGTGTTGCCCGAAGGGCATACGTTAAGTGCGAAAGAGTGGAAACAGCTTTCGTCCCAATGGCGAGAAGAGTTGGATCGGCGCATTCATACCTTAGTGGCGCTGCGTGACGAACTGGACGGATGTATTGGTTGTGGCTGCCTTTCGCGCAGTGATTGCCCGTTGCGTAACCCGGGCGACCGCTTAGGAGAAGAAGGTACCGGCGCACGCTTGCTGGAAGATGAACAAAACTAA http://www.ncbi.nlm.nih.gov/nuccore/NC_009801.1/?from=4616553&to=4617017&report=fasta (NCBI Reference Sequence: NC_009801.1 from Escherichia coli E24377A, complete genome)

Crt genes GenBank accession no. D90087—should we do as the entire cluster (in this case how should we design primers) or in parts?

Blh ATGGGTCTGATGCTGATTGATTGGTGTGCACTGGCTCTGGTTGTTTTCATTGGCCTGCCGCACGGCGCGCTGGATGCTGCCATTTCTTTTTCTATGATCTCTTCTGCAAAACGCATTGCTCGTCTGGCTGGTATTCTGCTGATCTATCTGCTGCTGGCGACCGCGTTCTTCCTGATCTGGTATCAGCTGCCAGCGTTTAGCCTGCTGATCTTCCTGCTGATCTCCATTATCCACTTTGGTATGGCAGACTTCAACGCGTCCCCAAGCAAACTGAAATGGCCGCATATCATCGCCCACGGCGGTGTTGTTACTGTTTGGCTGCCGCTGATCCAGAAAAACGAAGTAACTAAACTGTTTAGCATCCTGACTAACGGTCCGACTCCGATCCTGTGGGACATCCTGCTGATTTTCTTCCTGTGTTGGTCTATTGGCGTGTGTCTGCACACGTACGAAACCCTGCGCTCTAAACATTACAACATCGCCTTTGAACTGATCGGTCTGATTTTCCTGGCGTGGTATGCGCCGCCTCTGGTTACGTTTGCCACTTACTTCTGCTTCATTCATTCCCGTCGCCACTTCTCCTTTGTGTGGAAGCAGCTGCAACACATGTCTTCCAAAAAGATGATGATTGGCAGCGCGATTATCCTGTCCTGTACCTCTTGGCTGATCGGCGGTGGTATCTATTTCTTCCTGAACTCCAAAATGATCGCCTCTGAGGCTGCGCTGCAGACTGTGTTCATCGGTCTGGCGGCACTGACCGTGCCGCACATGATTCTGATCGACTTCATCTTCCGTCCGCACTCTTCCCGTATCAAAATCAAAAACTGA

Kim et al. In vitro characterization of a recombinant Blh protein from an uncultured marine bacterium as a beta-carotene 15,15'-dioxygenase. J Biol Chem. 2009 Jun 5;284(23):15781-93. (supplementary material; 15,15'-Dioxygenase, product of Blh gene, codon-optimized and expressed in E. coli, forms a 64k protein of two 32 kDa dimers)

RALDH?