IGEM:MIT/2005/Sequences of primers specified

The melting temperatures were calculated mostly by the following site:Oligonucleotide Primer Check Reverse Complement Codon Table Translation Tools

Check KO
gacgttcccgctgaaaaata, FecAKO check left1
 * FecA/TetR

gcgactcctgcattaggaag, FecAKO check right1

gcttgcggtattcggaatcttg, FecAKO check left2

TGCGGGACTTAAGCAGCAGTAC, FecAKO check right2

Primers for Sequencing
VR1 5' attaccgcctttgagtgagc 3' T_m=68.2 VF2 5' tgccacctgacgtctaagaa 3' T_m=68.2 FecR'seq.fwd: 5' ctcactgctttagggacag 3' goes w/ VR1 T_m=67.6 FecR'seq.rev: 5' CTGGCCCTGACGGGTCAGG 3' goes w/ VF2 T_m=70.3
 * FecR'

VR1 VF2 FecA'seq.fwd1: 5' cttacggtcagccgcagc 3' goes w/ FecA'seq.rev2 T_m=71.3 FecA'seq.rev1: 5' ACGGGGATGCCGTCCATC 3' goes w/ VF2 T_m=71.3 FecA'seq.fwd2: 5' cgagcctgggctaccag 3' goes w/ FecA'seq.rev3 T_m=70.7 FecA'seq.rev2: 5' CATACGGGCGGGTGGATTG 3' goes w/ FecA'seq.fwd1 T_m=71.9 FecA'seq.fwd3: 5' gtacagtacagccagattggc 3' goes w/ VR1 T_m=70.8 FecA'seq.rev3: 5' GAACGAGCCTTCAGTGTTTGC 3' goes w/ FecA'seq.fwd2 T_m=70.8
 * FecA'

VR1 VF2 PhoAseq.fwd: 5' ggcaaccgctggtgaatggcag 3' goes w/ PhoAseq.rev2 T_m=76.9 PhoA''seq.rev: 5' CAGCAAAGGTTTTTGCGCCGCCG 3' goes w/ VF2 T_m=77.1
 * PhoA''

scFv-L fwd: 5' gacgtcgttatgactcaaacaccac 3' T_m=74.2 scFv-L rev: 5' GCTGCCCGGCGCGCTGCT 3' T_m=78.2
 * scFv-linker

Fur knockout with Chloramphenicol resistance marker
Cm fwd: 5' atgactgataacaataccgccctaaagaaagctggcctgaa ctggatctatcaacaggagtcc 3'T_m,i=71.3 T_m,f=89.7 Cm rev: 5' ttatttgccttcgtgcgcatgttcatcttcgcggcaatcgc GTTGATCGGGCACGTAAGAGGTT 3'T_m,i=73.6 T_m,f=92.1 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
 * NEW: 41bp homologus with Fur gene + 20-25bp primer to PCR Chloramphenicol gene
 * OLD:

FecA KO with Chloramphenicol resistance marker

 * homologous to FecA gene + 20-25bp primer designed by Primer3

gtctctcgttttccgcttttgctgcacaggttaatatcgc ctggatctatcaacaggagtcc, FecA-Cm fwd

aggcatctatgcaggccagccgcgcacgctgtatatgcag GTTGATCGGGCACGTAAGAGGTT, FecA-Cm rev

ctggatctatcaacaggagtcc, Cm-left

GTTGATCGGGCACGTAAGAGGTT, Cm-right

FecA knockout with Tetracycline resistance marker

 * homologous to FecA gene + 20-25bp primer designed by Primer3

gtctctcgttttccgcttttgctgcacaggttaatatcgc tgagtaaacttggtctgacagctc, TetR_fwd1

aggcatctatgcaggccagccgcgcacgctgtatatgcag cgctgagataggtgcctcac, TetR_rev1

gtctctcgttttccgcttttgctgcacaggttaatatcgc tgagtaaacttggtctgacagc, TetR_fwd2

aggcatctatgcaggccagccgcgcacgctgtatatgcag cgctgagataggtgcctcac, TetR_rev2

gtctctcgttttccgcttttgctgcacaggttaatatcgc tgagtaaacttggtctgacagc, TetR_fwd3

aggcatctatgcaggccagccgcgcacgctgtatatgcag gctgagataggtgcctcactg, TetR_rev3

gtctctcgttttccgcttttgctgcacaggttaatatcgc atgagtaaacttggtctgacagctc, TetR_fwd4

aggcatctatgcaggccagccgcgcacgctgtatatgcag cgctgagataggtgcctcac, TetR_rev4


 * TetR primers designed by Primer3 (NO FecA homology)

left1 tgagtaaacttggtctgacagctc

right1 cgctgagataggtgcctcac

left2 tgagtaaacttggtctgacagc

right2 cgctgagataggtgcctcac

left3 tgagtaaacttggtctgacagc

right3 gctgagataggtgcctcactg

left4 atgagtaaacttggtctgacagctc

right4 cgctgagataggtgcctcac

Tet fwd: 5' gtctctcgttttccgcttttgctgcacaggttaatatcgc gagattctcatgtttgacagc 3' T_m,i=66.9 T_m,f=89.9 Tet rev: 5' aggcatctatgcaggccagccgcgcacgctgtatatgcag gcattggtaactcgagttagg 3' T_m,i=68.9 T_m,f=93.3 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
 * NEW 41bp homologous w/ FecA gene + 20-25bp primer to PCR Tetracycline resistance
 * OLD

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
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 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
 * registry specified 5'-3'
 * registry specified 3'-5'

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
fwd2: 5' CATGAG GAATTC GCGGCCGC T TCTAGA gtgaaacaaagcactattgcactggc 3' T_m,i=74.5 T_m,f=89.7 rev2: 5' AAGCTA CTGCAG GCGGCCGC T ACTAGT TTATTA TTATTTCAGCCCCAGAGCGGCTTTC 3' T_m,i=75.8 T_m,f=90.4 E1fwd: 5' cgatgctgcctcactgaactcggtgacggaagcgaatcag 3' T_m=88.2 E1rev: 5' CTGATTCGCTTCCGTCACCGAGTTCAGTGAGGCAGCATCG 3' T_m=88.2 E2fwd: 5' cgtacaacgggcgctggagttcgctaaaaaggagggtaac 3' T_m=87.2 E2rev: 5' GTTACCCTCCTTTTTAGCGAACTCCAGCGCCCGTTGTACG 3' T_m=87.2
 * NEW:

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
 * OLD:

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

scFv-L fwd: 5' gacgtcgttatgactcaaacaccac 3' T_m=74.2 scFv-L rev1: 5' GCTGCCCGGCGCGCTGCT 3' T_m=78.2 scFv-L rev2: 5' CTATGGCTCGCGCTGTTCGG 3' T_m=74.7
 * Verifying addition:

FecA-scFv fusion
Note: scFv_rev_Tm=76.6 (44.8%); scFv_fwd_Tm=82.5 (81.0%)

Rev_primer=first_29_bp_of_scFv(reversecomplemented) + 21_bp_of_FecA(reversecomplemented)

Fwd_primer=last_21_bp_of_scFv + 21_bp_of_FecA

more documentation in http://web.mit.edu/ymk/Public/igem/feca_scfv_fusions2.txt

Rev138: gatAGTGGTGTTTGAGTCATAACGACGTC GTCACGCGCGCCAGCATGTTC T_m=76.7 Fwd138: accagcagcgcgccgggcagc gtgatccgccgtgaggatttc T_m=72.8
 * Final: Type1s

Rev155: gatAGTGGTGTTTGAGTCATAACGACGTC ctcacgcatggtggttgcgcc T_m=76.7 Fwd155: accagcagcgcgccgggcagc gtacttaaccgcatccctggc T_m=72.8

Rev176: gatAGTGGTGTTTGAGTCATAACGACGTC caggtcgtggctgccggtgcc T_m,fec=80.6 Fwd176: accagcagcgcgccgggcagc gcgatgaactttggcatccggggc T_m,fec=79

Rev178: gatAGTGGTGTTTGAGTCATAACGACGTC catcgccaggtcgtggctgcc T_m=78.6 Fwd178: accagcagcgcgccgggcagc aactttggcatccggggcctg T_m=74.7

Rev180: gatAGTGGTGTTTGAGTCATAACGACGTC aaagttcatcgccaggtcgtggctg T_m,fec=77.5 Fwd180: accagcagcgcgccgggcagc ggcatccggggcctgaacccg T_m,fec=80.6

Rev527: gatAGTGGTGTTTGAGTCATAACGACGTC cgccggaagcggtgcgttata T_m=74.7 Fwd527: accagcagcgcgccgggcagc ttgaacgtgctctatcacctg T_m=68.9

Rev571: gatAGTGGTGTTTGAGTCATAACGACGTC tcgcgctttttccggttcaac T_m=70.8 Fwd571: accagcagcgcgccgggcagc acctgggaactcggtacccgc T_m=76.7


 * Final: Type2s

Rev138-141: gatAGTGGTGTTTGAGTCATAACGACGTC acgcgcgccagcatgttcaaa T_m=72.8 Fwd138-141: accagcagcgcgccgggcagc cgtgaggatttcgccaaaacc T_m=70.8

Rev155-158: gatAGTGGTGTTTGAGTCATAACGACGTC acgcatggtggttgcgccggt T_m=76.7 Fwd155-158: accagcagcgcgccgggcagc cgcatccctggcgtcagcgcg T_m=80.6

Rev176-180: gatAGTGGTGTTTGAGTCATAACGACGTC gtcgtggctgccggtgccgtt T_m=78.6 Fwd176-180: accagcagcgcgccgggcagc ggcatccggggcctgaacccg T_m=80.6

Rev178-182: gatAGTGGTGTTTGAGTCATAACGACGTC cgccaggtcgtggctgccggt T_m=80.6 Fwd178-182: accagcagcgcgccgggcagc cggggcctgaacccgcgcctc T_m=82.5

Rev180-184: gatAGTGGTGTTTGAGTCATAACGACGTC gttcatcgccaggtcgtggct T_m=74.7 Fwd180-184: accagcagcgcgccgggcagc ctgaacccgcgcctcgccagc T_m=80.6

Rev521-529: gatAGTGGTGTTTGAGTCATAACGACGTC gctcacttcttcgtgcgtgcc T_m=74.7 Fwd521-529: accagcagcgcgccgggcagc gtgctctatcacctgactgac T_m=70.8

Rev523-531: gatAGTGGTGTTTGAGTCATAACGACGTC gttatagctcacttcttcgtg T_m=66.9 Fwd523-531: accagcagcgcgccgggcagc tatcacctgactgacagctgg T_m=70.8

Rev527-531: gatAGTGGTGTTTGAGTCATAACGACGTC cggaagcggtgcgttatagct T_m=72.8 Fwd527-531: accagcagcgcgccgggcagc tatcacctgactgacagctgg T_m=70.8

Rev567-576: gatAGTGGTGTTTGAGTCATAACGACGTC ttcaacattgccgctttgcacagc T_m,fec=73.9 Fwd567-576: accagcagcgcgccgggcagc acccgctacgacgacggcgcg T_m,fec=80.6

Rev571-574: gatAGTGGTGTTTGAGTCATAACGACGTC cgctttttccggttcaacattgccgc T_m,fec=77.6 Fwd571-574: accagcagcgcgccgggcagc ctcggtacccgctacgacgac T_m,fec=76.7