Primer Design for the eCDM8 and RFP
PCR primers for insertion of Genes between Junctions C and D
1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for.
2. Conduct PCR of eCDM8 gene and RFP gene templates with primers below.
3. Conduct J-GGA with products from 1 and 2 above.
Gene primer considerations.
1. Four extra bases on 5’ end (eg. GCAT)
2. BsaI site directed toward the gene
3. Sticky end of 5’ CATC 3’ for Junction C on For primer
4. Sticky end of 5’ TCCG 3’ for Junction D on Rev primer
5. 18 nt of gene coding sequence on each primer
Primer designs for the eCDM8 gene
Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg
Rev ECDM8 ccagggtctcatccgttattaacctgcccaaac
5’ gttTgggcaggttaataa 3’
3’ caaacccgtccaattatt 5’
>BBa_J100100 Part-only sequence (3204 bp) gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg
ataaaccggttcatgcactgatgaaaattgcagatgaactgggcgagatctttaaattcgaagcaccgggtagcgttacccgttatctgagcagccagcg
tctgattaaagaagcatgcgacgaaagccgctttgataaaaatctgattcagtggctgaaattcatccgcgattttctgggtgatggtctgagcaccagc
tggacccatgaaaaaaactggaaaaaagcccataatattctgctgccgagctttagccagcaggcaatgaaaggttatcatgcaatgatggttgatattg
ccgttcagctggttcagaaatgggaacgtctgaatgcagatgaacatattgaagttccggaagatatgacccgtctgaccctggataccattggtctgtg
tggttttaactatcgcttcaacagcttttatcgtgatcagccgcatccgtttgttaccagcatggttcgtgcactggatgaagcagtgaataaatggcag
cgtgcaaatccggatgatccggcatatgatgaaaataaacgtcagttccaagaggatattaaagtgatgaatgacctggtggacaaaattatcgcagatc
gtaaagcaagcggtgaacagagtgatgatctgctgacccatatgctgaatggtaaagatccggaaaccggtgaaccgctggatgatgaaaacattcgtta
tcagattattaccttcctgattgccggtcatgaaaccaccagtggtctgctgagctttgcactgtattttctggttaaaaatccgcacgttctgcaaaaa
gcagccgaagaagcagcacgcgttctggttgatccggttccgagctataaacaggttaaacagctgaaatatgtgggcatggttctgaatgaagcactgc
gtctgtggccgacctttccgtggtttagcctgtatgcaaaagaagataccgttctgggaggtgaatatccgctggaaaaaggtgatgaactgatggttct
gattccgcagctgcatcgtgataaaaccatttggggtgatgatgtggaagaatttcgtccggaacgttttgaaaatccgagcgcaattccgcagcatgcc
tttaaaccgtttggtaatggtcagcgtgcatgtattggtcagcagtttgcactgcatgaagccaccctggttctgggtatgatgctgaaacattttgatt
ttgaggaccacaccaactatgagctggatattaaaggcaccctgaccctgaaaccggaaggttttgttgttaaagccaaaagcaaaaaaatcccgctggg
tggtattccgagcccgagcaccgaacagagcgcaaaaaaagttcgtaaaaaagccgaaaacgcccataatacaccgctgctggttctgtatggtagcaat
atgggtacagccgaaggcaccgcacgtgatctggcagatattgcaatgagcaaaggttttgcaccgcaggttgcaaccctggatagccatgcaggtaatc
tgcctcgtgaaggtgcagttctgattgttaccgcaagctataatggtcatccgcctgataatgcaaaacagtttgttgattggctggatcaggcaagcgc
agatgaagttaaaggtgttcgttatagcgtttttggttgcggtgataaaaactgggcaaccacctatcagaaagttccggcatttattgatgaaaccctg
gcagcaaaaggtgcagaaaatattgcagatcgtggtgaaaccgatgccagtgatgattttgaaggcacctatgaagaatggcgtgaacatatgtggtcag
atgttgcagcctattttaacctggatatcgaaaacagcgaggacaataaaagcaccctgagcctgcaatttgtggatagcgcagcagatatgccgctggc
aaaaatgcatggtgcatttagcaccaatgttgttgcaagcaaagaactgcaacagcctggtagcgcacgtagcacccgtcatctggaaattgaactgccg
aaagaagcaagctatcaagagggtgatcatctgggtgttattccgcgtaattatgaaggtattgttaatcgtgttaccgcacgttttggtctggatgcaa
gccagcagattcgtctggaagcagaagaagaaaaactggcacatctgccgctggccaaaaccgttagcgttgaagaactgctgcaatatgttgaactgca
agatccggttacccgtacccagctgcgtgcaatggcagcaaaaaccgtttgtccgcctcataaagttgaactggaagcactgctggagaaacaggcatat
aaagaacaggttctggcaaaacgtctgaccatgctggaactgctggaaaaatatccggcatgcgaaatgaaattcagcgaatttattgcactgctgccga
gtattcgtccgcgttattatagcattagcagcagtccgcgtgttgatgaaaaacaggccagcattaccgttagcgtggttagcggtgaagcatggtcagg
ttatggtgaatataaaggtattgccagcaattatctggccgaactgcaagaaggtgataccattacctgttttattagcacaccgcagagcgaatttacc
ctgccgaaagatcctgaaacaccgctgattatggttggtccgggtacaggtgttgcaccgtttcgtggttttgttcaggcacgtaaacaactgaaagaac
agggtcagagcctgggtgaagcacatctgtattttggttgtcgtagtccgcatgaggattatctgtatcaagaagaactggaaaacgcacagagcgaagg
tattattaccctgcataccgcatttagccgtatgccgaatcagccgaaaacatatgttcagcatgttatggaacaggatggcaaaaaactgattgaactg
ctggatcagggtgcccatttctatatttgtggtgatggtagccagatggcaccggcagttgaagcaaccctgatgaaaagctatgcagatgttcatcagg
ttagcgaagcagatgcacgtctgtggctgcaacagctggaagaaaaaggtcgttatgcaaaagatgtttgggcaggttaataatactagtagcggccgct gcag
Primer designs for the RFP gene
RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt
RFP Rev ccagggtctcatccgttattaagcaccggtgga
5’ Tccaccggtgcttaataa 3’
3’ Aggtggccacgaattatt 5’
RFP Gene Sequence
>BBa_E1010 Part-only sequence (706 bp)
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg
aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta
cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa
gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc
cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact
gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac
atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa
