IGEM:MIT/2005/Sequences of primers specified: Difference between revisions
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>Aanniev |
>Ymk |
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==<b>FecA-scFv fusion</b>== | ==<b>FecA-scFv fusion</b>== | ||
*Final: Type1s | *Final: Type1s | ||
Rev138: | Rev138: TGTTTGAGTCATAACGACGTC GTCACGCGCGCCAGCATGTTC | ||
Fwd138: | Fwd138: accagcagcgcgccgggcagc gtgatccgccgtgaggatttc | ||
Rev155: | Rev155: TGTTTGAGTCATAACGACGTC ctcacgcatggtggttgcgcc | ||
Fwd155: | Fwd155: accagcagcgcgccgggcagc gtacttaaccgcatccctggc | ||
Rev176: | Rev176: TGTTTGAGTCATAACGACGTC caggtcgtggctgccggtgcc T_m,fec=80.6 | ||
Fwd176: | Fwd176: accagcagcgcgccgggcagc gcgatgaactttggcatccggggc T_m,fec=79 | ||
Rev178: | Rev178: TGTTTGAGTCATAACGACGTC catcgccaggtcgtggctgcc | ||
Fwd178: | Fwd178: accagcagcgcgccgggcagc aactttggcatccggggcctg | ||
Rev180: | Rev180: TGTTTGAGTCATAACGACGTC aaagttcatcgccaggtcgtggctg T_m,fec=77.5 | ||
Fwd180: | Fwd180: accagcagcgcgccgggcagc ggcatccggggcctgaacccg T_m,fec=80 | ||
Rev527: | Rev527: TGTTTGAGTCATAACGACGTC cgccggaagcggtgcgttata | ||
Fwd527: | Fwd527: accagcagcgcgccgggcagc ttgaacgtgctctatcacctg | ||
Rev571: | Rev571: TGTTTGAGTCATAACGACGTC tcgcgctttttccggttcaac | ||
Fwd571: | Fwd571: accagcagcgcgccgggcagc acctgggaactcggtacccgc | ||
*Final: Type2s | *Final: Type2s | ||
Rev138-141: | Rev138-141: TGTTTGAGTCATAACGACGTC acgcgcgccagcatgttcaaa | ||
Fwd138-141: | Fwd138-141: accagcagcgcgccgggcagc cgtgaggatttcgccaaaacc | ||
Rev155-158: | Rev155-158: TGTTTGAGTCATAACGACGTC acgcatggtggttgcgccggt | ||
Fwd155-158: | Fwd155-158: accagcagcgcgccgggcagc cgcatccctggcgtcagcgcg | ||
Rev176-180: | Rev176-180: TGTTTGAGTCATAACGACGTC gtcgtggctgccggtgccgtt | ||
Fwd176-180: | Fwd176-180: accagcagcgcgccgggcagc ggcatccggggcctgaacccg | ||
Rev178-182: | Rev178-182: TGTTTGAGTCATAACGACGTC cgccaggtcgtggctgccggt | ||
Fwd178-182: | Fwd178-182: accagcagcgcgccgggcagc cggggcctgaacccgcgcctc | ||
Rev180-184: | Rev180-184: TGTTTGAGTCATAACGACGTC gttcatcgccaggtcgtggct | ||
Fwd180-184: | Fwd180-184: accagcagcgcgccgggcagc ctgaacccgcgcctcgccagc | ||
Rev521-529: | Rev521-529: TGTTTGAGTCATAACGACGTC gctcacttcttcgtgcgtgcc | ||
Fwd521-529: | Fwd521-529: accagcagcgcgccgggcagc gtgctctatcacctgactgac | ||
Rev523-531: | Rev523-531: TGTTTGAGTCATAACGACGTC gttatagctcacttcttcgtg | ||
Fwd523-531: | Fwd523-531: accagcagcgcgccgggcagc tatcacctgactgacagctgg | ||
Rev527-531: | Rev527-531: TGTTTGAGTCATAACGACGTC cggaagcggtgcgttatagct | ||
Fwd527-531: | Fwd527-531: accagcagcgcgccgggcagc tatcacctgactgacagctgg | ||
Rev567-576: | Rev567-576: TGTTTGAGTCATAACGACGTC ttcaacattgccgctttgcacagc T_m,fec=73.9 | ||
Fwd567-576: | Fwd567-576: accagcagcgcgccgggcagc acccgctacgacgacggcgcg T_m,fec=80 | ||
Rev571-574: | Rev571-574: TGTTTGAGTCATAACGACGTC cgctttttccggttcaacattgccgc T_m,fec=77.6 | ||
Fwd571-574: | Fwd571-574: accagcagcgcgccgggcagc ctcggtacccgctacgacgac T_m,fec=76 |
Revision as of 13:09, 5 August 2005
The melting temperatures were calculated mostly by the following site: Oligonucleotide Primer Check
Fur knockout with Chloramphenicol resistance marker
Fwd: 5' atgactgataacaataccgccctaaagaaagctggcctgaa gctcgaggcttggattctcac 3' T_m,i=72.8 T_m,f= 90.5 Rev: 5' ttatttgccttcgtgcgcatgttcatcttcgcggcaatcgc ctcgagttgatcgggcacgta 3' t_m,i=72.8 T_m,f=92.4
FecA knockout with Tetracycline resistance marker
Fwd: 5' atgacgccgttacgcgtttttcgtaaaacaacacctttggt gagattctcatgtttgacagc 3' T_m,i=66.9 T_m,f=88.5 Rev: 5' tcagaacttcaacgacccctgcatatacagcgtgcgcggct gcattggtaactcgagttagg 3' T_m,i=68.9 T_m,f=92.4
ToxR (ATCC)
Fwd: tctagatgttcggattaggacacaa
Rev: ctgcaggcggccgctactagtcgttaggggtttaaagctgga
scFv
- (pCT-4-4-20, pCT302 S101A, pCT-4M5.3 anti-fluorescein plasmids)
FWD: 5' CATCAT GAATTC GCGGCCGC T TCTAGA GACGTCGTTATGACTCAAACACC 3' T_m,i=71.8 T_m,f=88.5 rand EcoRI NotI XbaI Primer
REV: 5' GCTCAT ACTAGT TTATTA CTTATCATCATCATCCTTATAATC GGAGACGGTGACTGAGGTTCC 3' T_m,i=74.7 T_m,f=87.1 rand SpeI Stop FLAG Tag Primer
FWD-flag-test: CATCAT GAATTC GCGGCCGC T TCTAGA TG GACGTCGTTATGACTCAAACACC -rand- -EcoRI --NotI-- -XbaI[---] Primer start
FecA
fecA-Fwd 5’CATCAT GAATTC GCGGCCGC TCTAGA TGACGCCGTTACGCGTTTTTCG 3’ T_m,i=73.2 T_m,f=89.6 rand EcoRI NotI XbaI primer fecA-Rev 5’GCCATA ACTAGT TTATTA TCAGAACTTCAACGACCCCT 3’ T_m,i=68.2 T_m,f=79.9 rand SpeI stop primer
FecA'
Middle Fwd- 5'GGACGGCAGCGGACTGCAAGTAAAACCGCTGGGAAATAAC 3' T_m=84.7 Middle Rev- 5’GTTATTTCCCAGCGGTTTTACTTGCAGTCCGCTGCCGTCC 3' T_m=84.7
FecA Promoter
- registry specified 5'-3'
fecA Promoter Fwd- 5’CATCAT GAATTC GCGGCCGC T TCTAGA TGGATAAACATTTCACCACTG 3' T_m,i=65.0 T_m,f=86.2 rand EcoRI NotI XbaI primer fecA Promoter Rev- 5'GCCATA CTGCAG CGGCCGC T ACTAGT GTGTTCAGCTATGAGTCGAAC 3' T_m,i= 68.9 T_m,f=89.8 rand PstI NotI SpeI primer
- registry specified 3'-5'
fecA Promoter Fwd- 5’CATCAT GAATTC GCGGCCGC T TCTAGA CACAAGTCGATACTCAGCTTGCTC 3' T_m,i=73.9 T_m,f=87.9 rand EcoRI NotI XbaI primer fecA Promoter Rev- 5'GCCATA CTGCAG CGGCCGC T ACTAGT ACCTATTTGTAAAGTGGTGACATTCC 3' T_m,i=71.3 T_m,f=89 rand PstI NotI SpeI primer
FecR
fecR-Fwd 5' CATCAT GAATTC GCGGCCGC TCTAGA tgaatcctttgttaaccgattccc 3'T_m 66 rand EcoRI NotI XbaI primer fecR-Rev 5' GCCATA ACTAGT TTATTA ttacagtggtgaaatgtttatccag 3' T_m 68 rand SpeI stop primer
FecR'
Middle Fwd 5'gtgaaagcctacagttcagcgcctcag3' T_m 84 Middle Rev 5'ctgaggcgctgaactgtaggctttcac3' T_m 84
FecI
fecI-Fwd 5'CATCAT GAATTC GCGGCCGC T TCTAGA tgtctgaccgcgccactacc3' T_m 68 rand EcoRI NotI XbaI primer fecI-Rev 5'GCCATA CTGCAG CGGCCGC T ACTAGT A TTATTA tcataacccatactccagacggaa3' T_m 70 rand PstI NotI SpeI stop primer
==MalE== (updated/fixed)
fwd: 5' CCGTTA GAATTC GCGGCCGC T TCTAGA atgaaaataaaaacaggtgcacgc3' T_m,i=68.8 T_m,f=85.5 junk EcoRI NotI XbaI Primer rev: 5' ACTGAG CTGCAG GCGGCCGC T ACTAGT TTATTA TTACTTGGTGATACGAGTCTGC 3' T_m,i=69.5 T_m,f=89.4 junk PstI NotI SpeI Stop Primer
PhoA
fwd: 5' CATGAG GAATTC GCGGCCGC T TCTAGA ATGAAACAGAGCACCATTGCG 3' T_m,i=68.9 T_m,f=88.8 junk EcoRI NotI XbaI Primer rev: 5' AAGCTA CTGCAG GCGGCCGC T ACTAGT TTATTA TTATTTCAGGCCCAGCGCC 3' T_m,i=69.7 T_m,f=89.8 junk PstI NotI SpeI Stop Primer
Middle(1) fwd: 5' caccgcggaactgcaagatgcgaccccggcg 3' T_m=88.8 * rev: 5' CGCCGGGGTCGCATCTTGCAGTTCCGCGGTG 3' T_m=88.8 *
Middle(2) fwd: 5' aggcttttttctgcaagtggaaggcgcgagc 3' T_m=82.2 * rev: 5' GCTCGCGCCTTCCACTTGCAGAAAAAAGCCT 3' T_m=82.2 *
Adding Terminal Linker in scFv
linker fwd1: 5'GACGTCGTTATGACTCAAACACC 3' T_m,i= 71.8 primer linker rev1': 5' CGCGCTGCCGCTATGGCTCGCGCTGTTCGGGGAGACGGTGACTGAGGTTC 3' T_m,i=74.7 T_m,f=95.9 primer linker fwd2: 5' CATCAT GAATTC GCGGCCGC T TCTAGA GACGTCGTTATGACTCAAACACC 3' T_m,i=71.8 T_m,f=88.5 rand EcoRI NotI XbaI primer linker rev2': 5'GCTGCCCGGCGCGCTGCTGGTGTTCGGCGCGCTGCCGCTATGGCTCG 3' T_m,i=74.7 T_m,f=98.5 primer linker rev3: 5' GCTCAT ACTAGT CTTATCATCATCATCCTTATAATC TCCGGACCGTCGTCTAGTTCT 3' T_m,i=72.8 T_m,f=86.9 rand SpeI FLAG Tag Primer
FecA-scFv fusion
- Final: Type1s
Rev138: TGTTTGAGTCATAACGACGTC GTCACGCGCGCCAGCATGTTC Fwd138: accagcagcgcgccgggcagc gtgatccgccgtgaggatttc
Rev155: TGTTTGAGTCATAACGACGTC ctcacgcatggtggttgcgcc Fwd155: accagcagcgcgccgggcagc gtacttaaccgcatccctggc
Rev176: TGTTTGAGTCATAACGACGTC caggtcgtggctgccggtgcc T_m,fec=80.6 Fwd176: accagcagcgcgccgggcagc gcgatgaactttggcatccggggc T_m,fec=79
Rev178: TGTTTGAGTCATAACGACGTC catcgccaggtcgtggctgcc Fwd178: accagcagcgcgccgggcagc aactttggcatccggggcctg
Rev180: TGTTTGAGTCATAACGACGTC aaagttcatcgccaggtcgtggctg T_m,fec=77.5 Fwd180: accagcagcgcgccgggcagc ggcatccggggcctgaacccg T_m,fec=80
Rev527: TGTTTGAGTCATAACGACGTC cgccggaagcggtgcgttata Fwd527: accagcagcgcgccgggcagc ttgaacgtgctctatcacctg
Rev571: TGTTTGAGTCATAACGACGTC tcgcgctttttccggttcaac Fwd571: accagcagcgcgccgggcagc acctgggaactcggtacccgc
- Final: Type2s
Rev138-141: TGTTTGAGTCATAACGACGTC acgcgcgccagcatgttcaaa Fwd138-141: accagcagcgcgccgggcagc cgtgaggatttcgccaaaacc
Rev155-158: TGTTTGAGTCATAACGACGTC acgcatggtggttgcgccggt Fwd155-158: accagcagcgcgccgggcagc cgcatccctggcgtcagcgcg
Rev176-180: TGTTTGAGTCATAACGACGTC gtcgtggctgccggtgccgtt Fwd176-180: accagcagcgcgccgggcagc ggcatccggggcctgaacccg
Rev178-182: TGTTTGAGTCATAACGACGTC cgccaggtcgtggctgccggt Fwd178-182: accagcagcgcgccgggcagc cggggcctgaacccgcgcctc
Rev180-184: TGTTTGAGTCATAACGACGTC gttcatcgccaggtcgtggct Fwd180-184: accagcagcgcgccgggcagc ctgaacccgcgcctcgccagc
Rev521-529: TGTTTGAGTCATAACGACGTC gctcacttcttcgtgcgtgcc Fwd521-529: accagcagcgcgccgggcagc gtgctctatcacctgactgac
Rev523-531: TGTTTGAGTCATAACGACGTC gttatagctcacttcttcgtg Fwd523-531: accagcagcgcgccgggcagc tatcacctgactgacagctgg
Rev527-531: TGTTTGAGTCATAACGACGTC cggaagcggtgcgttatagct Fwd527-531: accagcagcgcgccgggcagc tatcacctgactgacagctgg
Rev567-576: TGTTTGAGTCATAACGACGTC ttcaacattgccgctttgcacagc T_m,fec=73.9 Fwd567-576: accagcagcgcgccgggcagc acccgctacgacgacggcgcg T_m,fec=80
Rev571-574: TGTTTGAGTCATAACGACGTC cgctttttccggttcaacattgccgc T_m,fec=77.6 Fwd571-574: accagcagcgcgccgggcagc ctcggtacccgctacgacgac T_m,fec=76