Team:MIT/sequences

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Revision as of 19:25, 7 July 2008

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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 = 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

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

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

gel image of parts

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

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)

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

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@@ Line 155: / Line 155: @@
 ====P16: Forward Primer for BioBrick L. Bulgaricus lacS promoter====
+[[Image:P16.png]]
 **sequence = '''GAATTCGCGGCCGCTTCTAGAG'''AAGAGGCTATATCGC
@@ Line 165: / Line 165: @@
 ====P17: Reverse Primer for BioBrick L. Bulgaricus lacS promoter====
+[[Image:P17.png]]
 **sequence = '''CTGCAGCGGCCGCTACTAGTA'''GAAATTCTCCTTTAGGTGTG
 bold = biobrick suffix
@@ Line 175: / Line 175: @@
 ====P18: Forward Primer for BioBrick p1025====
+[[Image:P18.png]]
 **sequence = GAATTCGCGGCCGCTTCTAGACAGCTGAAAACCGCTGACCTG
 **length = 42bp
@@ Line 182: / Line 182: @@
 ====P19: Reverse Primer for BioBrick p1025====
+[[Image:P19.png]]
 **sequence = CTGCAGCGGCCGCTACTAGTAACCAGAACGAAAGAGGTGG
 **length = 40 bp
@@ Line 189: / Line 189: @@
 ====P20: Forward Primer for BioBrick GFP====
+[[Image:P20.png]]
 **sequence = GAATTCGCGGCCGCTTCTAGACGTAAAGGAGAAGAACTTTTC
 **length = 42 bp
@@ Line 196: / Line 196: @@
 ====P21: Reverse Primer for BIoBrick  GFP====
+[[Image:P21.png]]
 **sequence = CTGCAGCGGCCGCTACTAGTTTTGTATAGTTCATCCATGC
 **length = 40bp