Team:MIT/sequences

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Contents

Primers

  • All BioBrick parts are Phillips-Silver to enable fusion peptide construction


P1: Forward Primer for Promoter

P1-2.jpg

    • foward sequence: CCG CTT CTA GAG TAA TAC GAC TCA CTA TAG GGA ATA CAA GCT ACT TGT TCT TTT TGC ATA CTA GAG ATT AAA GAG GAG AAA TAC TAG ATG CAG AAG AAA AAA TCC GC
    • length = 107 bp
    • GC content = 37.4 %
    • melting temp = 68.9 ºC
    • concentration:
      • original:
      • dilute:


P2: Reverse Primer for Peptide

P2.jpg

    • reverse sequence: GTT CTT CTC CTT TAC GCA TGC TGC CCC CGC CGC TGC CGC C
    • length = 40 bp
    • GC content = 67.5 %
    • melting temp = 74.7 ºC
    • concentration:
      • original:
      • dilute:


P3: Forward Primer for GFP

P3.jpg

    • forward sequence: GGC GGC AGC GGC GGG GGC AGC ATG CGT AAA GGA GAA GAA C
    • length = 40 bp
    • GC content = 67.5 %
    • melting temp = 74.7 ºC
    • concentration:
      • original:
      • dilute: 5μM?


P4: Reverse Primer for GFP

P4-5.jpg

    • sequence: CTG CAG CGG CCG CTA CTA GTA AGA GAA TAT AAA AAG CCA GAT TAT TAA TCC GGC TTT TTT ATT ATT TTT ATT AGT GGT GAT GGT GAT GAT GTT TGT ATA GTT CAT CCA TGC
    • length = 111 bp
    • GC content % = 36.0 %
    • melting temp = 69.6 ºC
    • concentration =


P5: Reverse Primer for GFP without Terminator

P5.jpg

    • sequence: CTG CAG CGG CCG CTA CTA GTA TTA TTA GTG GTG ATG GTG ATG ATG TTT GTA TAG TTC ATC CAT GC
    • length = 65bp
    • GC content = 44.6 %
    • melting temp = 68.5 ºC
    • concentration:


P6: Forward Primer for BioBrick FLAG tag

P6.png

    • sequence: GAATTCGCGGCCGCTTCTAGAGACTACAAAGACGACGACGACAAAACTAGTAGCGGCCGCTGCAG
    • length = 65bp
    • GC content = 55.4%
    • melting temp = 72.1 ºC
    • concentration =


P7: Reverse Primer for BioBrick FLAG tag

P7.png

    • sequence: CTGCAGCGGCCGCTACTAGTTTTGTCGTCGTCGTCTTTGTAGTCTCTAGAAGCGGCCGCGAATTC
    • length = 65bp
    • GC content = 55.4%
    • melting temp = 72.1 ºC
    • concentration =


P8: Forward Primer for BioBrick HA tag

P8.png

    • sequence: GAATTCGCGGCCGCTTCTAGATACCCGTACGACGTTCCGGACTACGCTACTAGTAGCGGCCGCTGCAG
    • length = 68bp
    • GC content = 60.3%
    • melting temp = 73.4 ºC
    • concentration =


P9: Reverse Primer for BioBrick HA tag

P9.png

    • sequence:CTGCAGCGGCCGCTACTAGTAGCGTAGTCCGGAACGTCGTACGGGTATCTAGAAGCGGCCGCGAATTC
    • length = 68bp
    • GC content = 60.3%
    • melting temp = 73.4 ºC
    • concentration =


P10: Forward Primer for BioBrick His tag

P10.png

    • sequence: GAATTCGCGGCCGCTTCTAGACATCATCACCATCACCACACTAGTAGCGGCCGCTGCAG
    • length = 59bp
    • GC content = 57.6%
    • melting temp = 72.7 ºC
    • concentration =


P11: Reverse Primer for BioBrick His tag

P11.png

    • sequence: CTGCAGCGGCCGCTACTAGTGTGGTGATGGTGATGATGTCTAGAAGCGGCCGCGAATTC
    • length = 59bp
    • GC content = 57.6%
    • melting temp = 72.7 ºC
    • concentration =


P12: Forward Primer for BioBrick TEV protease cut site

P12.png

    • sequence: GAATTCGCGGCCGCTTCTAGAGAAAACCTGTACTTCCAGGGTACTAGTAGCGGCCGCTGCAG
    • length = 62bp
    • GC content = 56.5%
    • melting temp = 72.2 ºC
    • concentration =


P13: Reverse Primer for BioBrick TEV protease cut site

P13.png

    • sequence: CTGCAGCGGCCGCTACTAGTACCCTGGAAGTACAGGTTTTCTCTAGAAGCGGCCGCGAATTC
    • length = 62bp
    • GC content = 56.5%
    • melting temp = 72.2 ºC
    • concentration =


P14: Forward Primer for BioBrick pRT B l.bulgaricus signal sequence

P14.png

    • sequence = GAATTCGCGGCCGCTTCTAGAATGCAGAAGAAAAAATCCGC
    • length = 41bp
    • GC content = 48.8%
    • melting temp = 67.1 ºC
    • concentration =


P15: Reverse Primer for BioBrick prt B l.bulgaricus signal sequence

P15.png

    • sequence = CTGCAGCGGCCGCTACTAGTGGTAACCGGAGCCGTTTCTTG
    • length = 41bp
    • GC content = 61%
    • melting temp = 71.3 ºC
    • concentration =


P16: Forward Primer for BioBrick L. Bulgaricus lacS promoter

    • sequence = GAATTCGCGGCCGCTTCTAGAGAAGAGGCTATATCGC

bold = biobrick prefix

    • length = 37 bp
    • GC content = 54.1%
    • melting temp = 66.6 ºC

P17: Reverse Primer for BioBrick L. Bulgaricus lacS promoter

    • sequence = CTGCAGCGGCCGCTACTAGTAGAAATTCTCCTTTAGGTGTG

bold = biobrick suffix

    • length = 41 bp
    • GC content = 51.2%
    • melting temp = 66.7 ºC

Plasmids

Include sequence, BioBrick #, functional features, digestion map.


PCR Products

1-1 GFP

GFP with linker @ 3' and [His, Terminator, Suffix] @ 5'

  • Template: GFP from Felix
  • Primers: 3 and 4

1-2 GFP

GFP with linker @ 3' and [His, Suffix] @ 5'

  • Template: GFP from Felix
  • Primers: 3 and 5

2-1 p1025

p1025 construct with [prefix, promoter, rbs] @ 3' and linker @ 5'

  • Template: p1025 construct
  • Primers: 1 and 2

2-2

L. bulgaricus signal peptide with Silver modified BioBricks prefix and suffix

  • Template: p1025 cnstruct
  • Primers: 14 and 15

2-3

Flag tag with Silver modified BioBricks prefix and suffix

  • Oligo annealing
  • Primers 6 and 7

2-4

HA tag with Silver modified BioBricks prefix and suffix

  • Oligo annealing
  • Primers 8 and 9

2-5

His tag with Silver modified BioBricks prefix and suffix

  • Oligo annealing
  • Primers 10 and 11

2-6

Tev cleavage site with Silver modified BioBricks prefix and suffix

  • Oligo annealing
  • primers 12 and 13

Other

TEV Protease cut site

  • Amino Acid Sequence : Glu-Asn-Leu-Tyr-Phe-Gln-Gly (Cuts between Gln-Gly)
  • Base Pair Sequence: GAAAACCTGTACTTCCAGGGT


DNA sequence to synthesize - epitope/p1025/linker (6/17/08)

  • The signal peptide sequence (141 bp) can be found at [http://www.ncbi.nlm.nih.gov/sites/entrez?dispmax=1&db=nucleotide&doptcmdl=graph&term=104773257 the NCBI genome database]
  • 6/17, lengthened signal peptide so that it will cleave properly
  • silent mutations in linkers to reduce repetition and to ensure specificity of primers (ex. GGC/GGA/GGG instead of GGT coding for Gly)

P1025-epitope.png

sequence: ATGCAGAAGAAAAAATCCGCACGCCATTTGAACAAAGTGGCTGAATTAGCCGCAGCACTGCTCC TATCAGCGAGTCCACTGGCGGGAACTTTCCAGTCAGCCGCTTTTGTCCAAGCTGCCAGTCAAGA AACGgctccggttaccGACTACAAAGACGACGACGACAAAGGaGGTGGcCAGCTGAAAACCGCT GACCTGCCGGCTGGTCGTGACGAAACCACCTCTTTCGTTCTGGTTGGTGGaGGgTACCCGTACG ACGTTCCGGACTACGCTGGaGGtGGgTCTGGcGGtGGaTCaGGgGGtGGaTCgGAAAACCTGTA CTTCCAGGGTGGTGGaGGcTCcGGTGGcGGCagcGGcGGgGGCagc

Whole Sequence

CCGCTTCTAGAGTAATACGACTCACTATAGGGAATACAAG CTACTTGTTCTTTTTGCATACTAGAGATTAAAGAGGAGAA ATACTAGATGCAGAAGAAAAAATCCGCACGCCATTTGAAC AAAGTGGCTGAATTAGCCGCAGCACTGCTCCTATCAGCGA GTCCACTGGCGGGAACTTTCCAGTCAGCCGCTTTTGTCCA AGCTGCCAGTCAAGAAACGGCTCCGGTTACCGACTACAAA GACGACGACGACAAAGGaGGTGGcCAGCTGAAAACCGCTG ACCTGCCGGCTGGTCGTGACGAAACCACCTCTTTCGTTCT GGTTGGTGGaGGgTACCCGTACGACGTTCCGGACTACGCT GGaGGtGGgTCTGGcGGtGGaTCaGGgGGtGGaTCgGAAA ACCTGTACTTCCAGGGTGGTGGaGGcTCcGGTGGcGGCag cGGcGGgGGCagcATGCGTAAAGGAGAAGAACTTTTCACT GGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTA ATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGA TGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACT ACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTA CTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGA TCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCC GAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATG ACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGG TGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGAT TTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAAT ACAACTATAACTCACACAATGTATACATCATGGCAGACAA ACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACAC AACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATC AACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACC AGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAA GATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGT TTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACT ATACAAACATCATCACCATCACCACTAATAAAAATAATAA AAAAGCCGGATTAATAATCTGGCTTTTTATATTCTCTTAC TAGTAGCGGCCGCTGCAGGATTA

Wholesequence.png


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