Table 2.

Sequence rewriting of C. eth-1.0 into C. eth-2.0 leads to massive reduction of genetic features

Type	C. eth-1.0	C. eth-2.0	Fraction (%)
Sequence rewriting
Base substitutions	None	133,313	17.0
Rewritten codons*	None	123,562	56.1
Codons
TTG	1,154	0	100
TTA	46	0	100
TAG	173	10	94.2
Alternative genetic features
ORFs†	3,229	407	87.4
TSS‡	1,730	82	95.3
RBS§	1,331	310	76.7
Remaining genetic features¶	6,290	799
DNA synthesis constraints
High GC regions#	4,342	0	100
Direct repeats $\ge$8 bp	880	113	87.2
Hairpins $\ge$8 bp	606	140	76.9
Homopolymers	139	46	66.9
Restriction sites‖	1,047	2	99.8
Synthesis constraints**	7,014	301

^*Number of synonymous codon substitutions introduced on sequence rewriting.

^†Number of alternative ORFs residing within the 676 CDSs of C. eth-1.0 and C. eth-2.0.

^‡Number of TSSs internal to CDSs.

^§Number of ribosome binding sites (RBSs) internal to CDSs.

^¶Number of remaining genetic features within CDS of C. eth-1.0 and C. eth-2.0.

^#Regions of high GC content > 0.8 within a 100-bp window.

^‖Total number of type IIS restriction sites that were removed (AarI, BsaI, BspQI, PacI, PmeI, I-CeuI, I-SceI). Note that the two unique PmeI and PacI sites remained within the pMR10Y backbone to facilitate linearization of the final assembled chromosome for subsequent analysis by pulsed field gel electrophoresis.

^**Number of DNA synthesis constraints of C. eth-1.0 and C. eth-2.0.