Sph-E
Nco-E
Ack-Gen-F
Ack-E-R
Ack-F
Gen-F
Ack-catF
Ack-catR
TetA-F
Ack-R
EG-pkd-E
EG-pkd-G
Pkd-cx
Gen-R
PigA-EGF
PigA-EGR
Pig-520F
pigA-ssF
PigA-T7-R1
AraB-T7-F
Pig-1690R
T7-F
Ack-EGSD
Arab-EGSDF
Arab-EGR
Arab-EGF
Arab-F
Arab-R
E-R
Hyp-EGSDF
Hyp-EGSDR
Mar-EGSDF
Mar-EGSDR
Rcs-EGSDF
Rcs-EGSDR
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Sph-E
Nco-E
Ack-Gen-F
Ack-E-R
Ack-F
Gen-F
Ack-catF
Ack-catR
TetA-F
Ack-R
EG-pkd-E
EG-pkd-G
Pkd-cx
Gen-R
PigA-EGF
PigA-EGR
Pig-520F
pigA-ssF
PigA-T7-R1
AraB-T7-F
Pig-1690R
T7-F
Ack-EGSD
Arab-EGSDF
Arab-EGR
Arab-EGF
Arab-F
Arab-R
E-R
Hyp-EGSDF
Hyp-EGSDR
Mar-EGSDF
Mar-EGSDR
Rcs-EGSDF
Rcs-EGSDR
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CCAGCATGCATGGTACGCTGGACTTTGT
ACTCCATGGTCACTCCTTCCGCACGTAA
CTGACGTTTTTTTAGCCACGTATCAATTATAGGTACTTCCTTAGGTGGCGGTACTTGGG
ATTAATACTGAAACTGAGATCAAGCAAAAGCATTCACTAATTGACATAAGCCTGTTCGG
TGCAGTGCATGATGTTAATC
TTGACATAAGCCTGTTCGG
TGACGTTTTTTTAGCCACGTATCAATTATAGGTACTTCCAGCTACCAGATACCATCGTC
TTACAAAACAGCACCGCCAGCTGAGCTGGCGGTGTGAAATAAGAGCGCATGATACGGAC
CAAGAGGGTCATTATATTTCG
GGGATCAGCATAATAATACG
GTTTTTTTGGGAATTCGAGCTCTAAGGAGGTTATAAAAAATGGTACGCTGGACTTTGT
AAGTGATTGCGCCTACCCGGATATTATCGTGAGGATGCGTTAGGTGGCGGTACTTGGG
GCTCTTCTCGCTAACCAAAC
TTAGGTGGCGGTACTTGGG
ACTGATCGCGGCGAATGCGGCGACGGAGCCGGACGCCGGTTAGGTGGCGGTACTTGGG
CAGGGTTGAGCGGTTGATTTCATGGCATAAATATCGGAATTATGACAACTTGACGGCTA
AACGAATCGTCTTGCTGGTT
ACTGATCGCGGCGAATGCGGCGACGGAGCCGGACGCCGG//TTCCGATATTTATGCCATGAAATCAACCGCTCAACCCTG
ACTGATCGCGGCGAATGCGGCGACGGAGCCGGACGCCGGTACGTTGGCGACCGCTAGG
CTGTTTCTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGAACACGATTAACATCG
CCAGATCATTGAATCGTGGA
ATGAACACGATTAACATCG
AATACTGAAACTGAGATCAAGCAAAAGCATTCACTAAGAAGGAGATATACCATGGTACGCTGGACTTTG
CTGTTTCTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGTGCCAATCGCGGGGGGT
CACACTTCATAATTATCAAAAATCGTCATTATCGTGTCCTTAGGTGGCGGTACTTGGGT
CTGTTTCTCCATACCCGTTTTTTTGGATGGAGTGAAACGATGGTACGCTGGACTTTGTG
TCCATAAGATTAGCGGATC
ATGTTGGGTGACCTGACGCA
TCACTCCTTCCGCACGTAAT
AGATGCTAGTGATATGCTTATTAACATTTCTGAATACTTTTATGACAACTTGACGGCTA
ATAGTAATCTCCCATTTCACTTTCGCCATCATAACCAACTTAGGTGGCGGTACTTGGGT
AGAGTTATCTTGGAATAAGTTAGGGCGAGGCGGAGAAATTTATGACAACTTGACGGCTA
ATAATCTGTACCGGTTGAAAACGTGGGCAAAAACCGGTGTTAGGTGGCGGTACTTGGGT
ATATTACCGCAGTAAAACGGATTTAAGTGGGGGTGCGATTTATGACAACTTGACGGCTA
TAACGGCTGGTAAAACAGAGCGGGGGCGTTCTCTGTGGTTTAGGTGGCGGTACTTGGGT
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Construction of plasmid pBBR1-E
Replacement of kil by E-GmR
Identification of strain CWE-1
Replacement of ack by partial fragment of catenin gene
Identification of strain CWE-2
Insertion of E-GmR after promoter
PBAD in plasmid pKD46
Identification of plasmid pKD-EG
Insertion of PBAD-E-GmR double
selection cassette at pigA locus
Paired with Gen-F to identify strain GY4
Reparation of PBAD-E-GmR
insertion mutation using ssDNA
Replacement of PBAD-E-GmR by
partial fragment of T7 RNA polymerase gene
Paired with Pig-520F to identify
PBAD-E-GmR deletion by ssDNA
Paired with Pig-520F to identify GY4-1
Paired with Ack-Gen-F to replace kil at ack locus
Replacement of araB by PBAD-kil-sd-E-
GmR cassette
Paired with Arab-EGR to Replacement of araB by
PBAD-E- GmR cassette
Identification of gene Replacement at araB locus
Paired with Arab-F to identify MG-4A/4B
Replacement of hyp-1 by PBAD-kil-sd-E-
GmR cassette
Replacement of marR-1 by PBAD-kil-sd-E-
GmR cassette
Replacement of rcsA by PBAD-kil-sd-E-
GmR cassette
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