Reagent/Resource	Reference or source	Identifier or catalog number
Experimental models
Escherichia coli strain MG1655∆lac	Sarkar et al (2020)	Addgene cat. #164844
One Shot TOP10 Electrocomp E. coli	Invitrogen	Cat. #C404050
Recombinant DNA
Plasmid pTY1	This study	Addgene cat. #164831
Plasmid pVER	This study	Addgene cat. #164830
plasmid pUC19	New England Biolabs	Cat. #N3041
Oligonucleotides and sequence‐based reagents
PCR primers for library construction	This study	Appendix Table S7
Barcode PCR amplification primers	This study	Appendix Tables S8 and S9
Paired‐end adapter PCR amplification primers	This study	Appendix Table S10
Chemicals, enzymes, and other reagents
Sera‐mag SpeedBeads	GE Healthcare	Cat. #65152105050250
GeneMorph II Random Mutagenesis Kit	Agilent	Cat. #200550
Phusion Flash High‐Fidelity PCR Master Mix	Thermo Scientific	Cat. #548L
T4 DNA Ligase	Thermo Scientific	Cat. #EL0011
Calf Intestinal Phosphatase	New England Biolabs	Cat. #M0525L
FastDigest Buffer	Thermo Scientific	Cat. #B64
FastDigest SgsI	Thermo Scientific	Cat. #FD1894
FastDigest NheI	Thermo Scientific	Cat. #FD0974
FastDigest XhoI	Thermo Scientific	Cat. #FD0695
FastDigest ApaI	Thermo Scientific	Cat. #FD1414
FastDigest DpnI	Thermo Scientific	Cat. #FD1703
BspOI	Thermo Scientific	Cat. #ER2041
XmaI	New England Biolabs	Cat. #R0180S
FseI	New England Biolabs	Cat. #R0588S
Gibson Assembly Master Mix	New England Biolabs	Cat. #E2611S
Rainbow Calibration Particles, 8 peaks	Spherotech	Cat. #RCP‐30‐20A
LB	BD Biosciences	Cat. #244620
Casamino Acids	Fisher Bioreagents	Cat. #BP1424
Bacto Tryptone	Gibco	Cat. #211705
Bacto Yeast Extract	Thermo Scientific	Cat. #212750
Glucose	Gibco	Cat. #2494001
Glycerol	Thermo Scientific	Cat. #15514029
Chloramphenicol	Fisher Bioreagents	Cat. #BP904‐100
Kanamycin	Thermo Scientific	Cat. #J1792406
IPTG	Thermo Scientific	Cat. #R0393
Tetracycline	Alfa Aesar	Cat. #B21408
Neutralization Buffer	Qiagen	Cat. #19064
Binding Buffer	Qiagen	Cat. #19066
Rnase A	Qiagen	Cat. #19101
Nuclease‐free water	Thermo Scientific	Cat. #AM9938
M9 Salts	BD Biosciences	Cat. #248510
CaCl2	Fisher Bioreagents	Cat. #BP210‐100
MgSO4	Fisher Bioreagents	Cat. #BP213‐1
NaCl	Thermo Scientific	Cat. #AM9759
KCl	Thermo Scientific	Cat. #AM9640G
MgCl2	Thermo Scientific	Cat. #AM9530G
NaOH	Millipore Sigma	Cat. #106462
Tris–Cl, pH 7.5	Fisher Bioreagents	Cat. #BP1757‐100
Tris–Cl, pH 8.0	Invitrogen	Cat. #15‐568‐025
EDTA	Fisher Bioreagents	Cat. #1311‐200
SDS	Millipore Sigma	Cat. #24802350
Absolute Ethanol	Fisher Bioreagents	Cat. #BP2818500
Tris–Cl, pH 8.5	VWR	Cat. #MB‐027‐1000
PEG‐8000	Sigma Aldrich	Cat. #89510
Focusing fluid	Invitrogen	Cat. #4488621
PBS	Invitrogen	Cat. #AM9625
Tween‐20	Fisher Bioreagents	BP337‐100
1× TE Buffer	Thermo Scientific	Cat. #12090015
96‐deep well plate	Eppendorf	Cat. #951033405
96‐well plate	Abgene	Cat. #AB‐1127
96‐deep well plate	Eppendorf	Cat. #951033588
96‐well DNA elution plate	Eppendorf	Cat. #30603303
96‐well DNA binding plate	Nunc	Cat. #278010
96‐well midi plate	Abgene	Cat. #AB‐0765
96‐well growth plate	4titude	Cat. #4ti‐0255
Gas‐permeable membrane	4titude	Cat. #4ti‐0598
Software
Data analysis software	This study, Zhao et al (2018), and Schlecht et al (2017)	github.com/djross22/nist_lacI_landscape_analysis
Other
Qubit 1× dsDNA HS Assay Kit	Thermo Fisher Scientific	Cat. # Q33231
QIAquick PCR Purification Kit	Qiagen	Cat. #28106
QIAquick Gel Extraction Kit	Qiagen	Cat. #28115
QIAprep Spin Miniprep Kit	Qiagen	Cat. #27106
Laboratory automation system	Peak Analysis and Automation, S‐Cell	Integrated components marked with (**)
Star liquid handler**	Hamilton
a4S plate sealer**	4titude
Xpeel plate desealer**	Brooks
Neo2SM plate reader**	BioTek
Rotanta 460 Robotic Centrifuge**	Hettich Zentrifugen
NGS Star liquid handler	Hamilton
Illumina HiSeqX 300‐cycle paired‐end service	Novogene
PacBio Sequel II	University of Maryland Institute of Genome Sciences
Qubit 3	Thermo Fisher Scientific
Attune NxT Flow Cytometer	Thermo Fisher Scientific
SH800S Cell Sorter	Sony