Team:MIT/sequences

Primers

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

P1: Forward Primer for Promoter

 * 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: 5uM

P2: Reverse Primer for Peptide

 * 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: 5uM

P3: Forward Primer for GFP

 * 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

 * 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

 * 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

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

P7: Reverse Primer for BioBrick FLAG tag

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

P8: Forward Primer for BioBrick HA tag

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

P9: Reverse Primer for BioBrick HA tag

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

P10: Forward Primer for BioBrick His tag

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

P11: Reverse Primer for BioBrick His tag

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

P12: Forward Primer for BioBrick TEV protease cut site

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

P13: Reverse Primer for BioBrick TEV protease cut site

 * 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

 * 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

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

P16: Forward Primer for BioBrick L. Bulgaricus lacS promoter

 * sequence =

GAATTCGCGGCCGCTTCTAGAGAAGAGGCTATATCGCCATCATTAGCAGCTT

bold = biobrick prefix


 * length = 57 bp
 * GC content = 47.4%
 * melting temp = 68.7 ºC

P17: Reverse Primer for BioBrick L. Bulgaricus lacS promoter

 * sequence =

CTGCAGCGGCCGCTACTAGTAGAAATTCTCCTTTAGGTGTGTTAACTTG

bold = biobrick suffix


 * length = 49 bp
 * GC content = 46.9%
 * melting temp = 67 ºC

P18: Forward Primer for BioBrick p1025

 * sequence = GAATTCGCGGCCGCTTCTAGACAGCTGAAAACCGCTGACCTG
 * length = 42bp
 * GC content = 57.1%
 * melting temp = 70.2ºC

P19: Reverse Primer for BioBrick p1025

 * sequence = CTGCAGCGGCCGCTACTAGTAACCAGAACGAAAGAGGTGG
 * length = 40 bp
 * GC content = 57.5%
 * melting temp = 69.2ºC

P20: Forward Primer for BioBrick GFP

 * sequence = GAATTCGCGGCCGCTTCTAGACGTAAAGGAGAAGAACTTTTC
 * length = 42 bp
 * GC content = 47.6%
 * melting temp = 65.9ºC

P21: Reverse Primer for BIoBrick GFP

 * sequence = CTGCAGCGGCCGCTACTAGTTTTGTATAGTTCATCCATGC
 * length = 40bp
 * GC content = 50%
 * melting temp = 66.5 ºC

P22: Forward Primer for BBa_I13521

 * Will basically remove the promoter from this RFP device


 * sequence = gaattcgcggccgcttctagagtactagagaaagaggagaatac
 * length = 41 bp
 * GC content = 48.8%
 * melting temp = 65.9 C

P23: Reverse Primer for BBa_I13521

 * sequence = ctgcagcggccgctactagtatataaacgcagaaaggcccac
 * length = 41 bp
 * GC content = 53.7%
 * melting temp = 68.9 C

P24: bbRBSfw: attach RBS upstream of p1025 part, with BB end

 * sequence = GGAATTCGCGGCCGCTTCTAGAGTC...?
 * length = ? bp
 * GC content = ?%
 * melting temp = 69.4 C

P25: expfw: attach NdeI end to beginning of p1025 part

 * sequence = GGAATTCCATATGCAGAAGAAAAA...?
 * length = ? bp
 * GC content = ?%
 * melting temp = 61.3 C

P26: exprev: attach XhoI end after p1025 part

 * sequence = CCGCTCGAGTTATTAGTGGTGATG...?
 * length = ? bp
 * GC content = ?%
 * melting temp = 63.5 C

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

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'

gel image of parts
 * Template: p1025 construct
 * Primers: 1 and 2

2-2 signal
L. bulgaricus signal peptide with Silver modified BioBricks prefix and suffix


 * Template: p1025 cnstruct
 * Primers: 14 and 15

2-3 Flag
Flag tag with Silver modified BioBricks prefix and suffix


 * Oligo annealing
 * Primers 6 and 7

2-4 HA
HA tag with Silver modified BioBricks prefix and suffix


 * Oligo annealing
 * Primers 8 and 9

2-5 His
His tag with Silver modified BioBricks prefix and suffix


 * Oligo annealing
 * Primers 10 and 11

2-6 Tev
Tev cleavage site with Silver modified BioBricks prefix and suffix


 * Oligo annealing
 * primers 12 and 13

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 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)



sequence: ATGCAGAAGAAAAAATCCGCACGCCATTTGAACAAAGTGGCTGAATTAGCCGCAGCACTGCTCC TATCAGCGAGTCCACTGGCGGGAACTTTCCAGTCAGCCGCTTTTGTCCAAGCTGCCAGTCAAGA AACGgctccggttaccGACTACAAAGACGACGACGACAAAGGaGGTGGcCAGCTGAAAACCGCT GACCTGCCGGCTGGTCGTGACGAAACCACCTCTTTCGTTCTGGTTGGTGGaGGgTACCCGTACG ACGTTCCGGACTACGCTGGaGGtGGgTCTGGcGGtGGaTCaGGgGGtGGaTCgGAAAACCTGTA CTTCCAGGGTGGTGGaGGcTCcGGTGGcGGCagcGGcGGgGGCagc

L. bulgaricus lacS promoter to synthesize (7/11/08)


sequence:

GAATTCGCGGCCGCTTCTAGAGAAGAGGCTATATCGCCATCATTAGCAGCTTAATTGA ATATTTACTGGCTAAACTATTGAGTTTTCAAGGCTTCATAGTTCTTTTTGGTGTGGGA AGTTTAAATTACTAAAAATATTTTAGTAAAACATCTTGGTTTATTTAGTAAACAAGTC TATACTGTAATTATAAACAAGTTAACACACCTAAAGGAGAATTTCTACTAGTAGCGGC CGCTGCAG

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