IGEM:MIT/2006/Notebook/2006-6-8

SAMT from A. Majus

checked by Reshma

<bbpart>BBa_J45001</bbpart>

PCR primers

Forward Strand

• Sequence to order: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA G ATG ACA AAA CAA ACA CAA AAG C
• Length of matching: 22 bp
• GC content: 31.8%
• Melt Temp. 51.0 C
• Whole sequence analysis
  • Lowest secondary structure deltaG: -.93 kcal/mol
  • No significant homodimers or heterodimers

Reverse Strand

• Sequence to order: 5'- GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAT TA GTT TCT TTT TGC TTG TCA CTC
• Length of matching: 21 bp
• GC content: 38.1.0%
• Melt Temp. 50.4 C
• Whole sequence analysis
  • Lowest secondary structure deltaG: -1.68 kcal/mol
  • No significant homodimers or heterodimers

BAMT from A. majus

PCR primers

checked by Reshma

<bbpart>BBa_J45002</bbpart> - note, this part needs to be fixed up to remove noncoding region on 3' end and replace original stop codon with the TAATAA stop codon in the primers

Forward primers

• Forward primer (5' -> 3'): GTT TCT TCG AAT TCG CGG CCG CTT CTA G ATG AAA GTG ATG AAG AAA C
  • Length: 47
  • GC Content: 31.6%
  • Melting Temp: 45.2

Reverse primers

• Reverse primer (5' -> 3'): GTT TCT TCC TGC AGC GGC CGC TAC TAG TA TTA TTA TCT CCT ACT TAG AGA AAC TAC
  • Length: 56
  • GC Content: 38.1%
  • Melting Temp: 47.7

Mutagenesis primers

mutation OK, checking primer now

Primer pair 1

                              *
   Forward: 5' GCTTGCAAAAACACTGGTTGATATGGTGGC 3'
   Reverse: 5' GCCACCATATCAACCAGTGTTTTTGCAAGC 3'
                             *
    GC content: 46.67%           Location: 687-716
    Melting temp: 75.3°C         Mismatched bases: 1
    Length: 30 bp                Mutation: Substitution
    5' flanking region: 15 bp    Forward primer MW: 9286.14 Da
    3' flanking region: 14 bp    Reverse primer MW: 9126.06 Da

Primer pair 2

                              *
   Forward: 5' GCTTGCAAAAACACTGGTTGATATGGTGGCTG 3'
   Reverse: 5' CAGCCACCATATCAACCAGTGTTTTTGCAAGC 3'
                               *
    GC content: 46.88%           Location: 687-718
    Melting temp: 76.7°C         Mismatched bases: 1
    Length: 32 bp                Mutation: Substitution
    5' flanking region: 15 bp    Forward primer MW: 9919.55 Da
    3' flanking region: 16 bp    Reverse primer MW: 9728.46 Da

Primer pair 3

                                  *
   Forward: 5' CATTGCTTGCAAAAACACTGGTTGATATGGTGGCTGAG 3'
   Reverse: 5' CTCAGCCACCATATCAACCAGTGTTTTTGCAAGCAATG 3'
                                 *
    GC content: 44.74%           Location: 683-720
    Melting temp: 79.2°C         Mismatched bases: 1
    Length: 38 bp                Mutation: Substitution
    5' flanking region: 19 bp    Forward primer MW: 11772.77 Da
    3' flanking region: 18 bp    Reverse primer MW: 11581.68 Da

BSMT from P. x hybrida

Part <bbpart>BBa_J45004</bbpart> checked by Austin:

• Sequence matches that of Genbank sequence AY233465
• No double TAA stop codon in the part but primer does include it
• Forward primer ok
• Reverse primer ok

PCR primers

Forward primer

Designed Forward Strand (1 and 2): 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA G ATG GAA GTT GTT GAA GTT C -3'

• LENGTH: 19
• GC CONTENT: 36.8 %
• MELT TEMP: 47.8 °C

Reverse primer

Designed Reverse Strand (phBSMT1): 5'- GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAT TAA TTT ATT TTG GTC AAG GAG -3'

• LENGTH: 22
• GC CONTENT: 27.3 %
• MELT TEMP: 46.6 °C

ATF1 from S. Cerevisiae

Part <bbpart>BBa_J45006</bbpart>

checked by Austin:

• EcoRI mutation is silent mutation GAA->GAG
• Sequence matches that of Genbank sequence Z75285
• Double TAA stop codon
• Forward primer ok
• Reverse primer
  • The anneal length is only 16 bp not 19 as listed below. melt temp is even lower at 45.9
  • Also 3'-ends with sticky GG, I would add 2 more bases (AG) to 3' end
• Mutation primers look ok

PCR primers

Forward primer

Designed forward primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA G ATG AAT GAA ATC GAT GAG -3'

• Length: 46 bp
• Length only anneal: 18
• GC content: 33.3%
• Melt temp: 44.2 deg. C
• DeltaG hairpin: -1.52 kcal/mol
• DeltaG homodimer: -91.97 kcal/mol

Reverse primer

Designed reverse primer: 5'- GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAT TAA GGG CCT AAA AGG -3'

• Length: 48 bp
• Length only anneal: 19
• GC content: 36.8%
• Melt temp: 46.5 deg. C
• DeltaG hairpin: -2.73 kcal/mol
• DeltaG homodimer: -96.09 kcal/mol

DeltaG heterodimer: -96.09 kcal/mol

Mutagenesis Primers

Primer pair 4

                                 *
   Forward: 5' GAAGCAAATATTAGAAGAGTTCAAAAATAGTAAGGG 3'
   Reverse: 5' CCCTTACTATTTTTGAACTCTTCTAATATTTGCTTC 3'
                                *
    GC content: 30.56%           Location: 396-431
    Melting temp: 72.5°C         Mismatched bases: 1
    Length: 36 bp                Mutation: Substitution
    5' flanking region: 18 bp    Forward primer MW: 11246.47 Da
    3' flanking region: 17 bp    Reverse primer MW: 10867.22 Da