Supplementary information
Creating custom synthetic genomes in
Escherichia coli with REXER and GENESIS
In the format provided by the
authors and unedited
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https://doi.org/10.1038
/s41596-020-00464-3
REXER2
REXER4
Target region for replacement
Target region for replacement
gSpacer 1
30 nt total
5’
3’
gSpacer 2
30 nt total
5’
3’
HR3
HR4
GGN
NNN
CCN
NNN
ACT
AGG
TGA
TCC
sacB
cat
HR3
HR4
locus
1
locus
2
locus
1
locus
2
loc1
geno_F
loc1
geno_R
loc2
geno_F
loc2
geno_R
loc1
geno_R
loc2
geno_R
loc1
geno_F
loc2
geno_F
HR3
PAM
bSpacer 1
30 nt total
5’
3’
PAM
HR4
YAC ori
pSC101
Amp
R
BsaI
BsaI
iii) BAC vector
CCT
TCC
AGG
GGA
CCT
TCC
AGG
GGA
AGG
rK_scF
rK_scR
bb2_scF
bb2_scR
BAC template
BAC ori
URA3
OriT
BAC2_R
BAC_F
HR3
CCT
TCC
AGG
GGA
bSpacer 2
30 nt total
HR4
ii) pre-cloned selection fragment
YAC ori
rpsL
kanR
sacB
CCT
TCC
AGG
GGA
AGG
3’
5’
j1_F
j3_R
rpsL
kanR
sacB
ACT
AGG
TGA
TCC
sacB
cat
HR2
HR2
a
b
sc_seq1_F
sc_seq1_R
i) synthetic stretches
j2_R
j
n
_F
jn_R
j
n
_F
j
3_
F
jn_R