name	type	start_1based	end_1based	strand	length_bp	sequence_5to3	source	justification
T7 promoter	promoter	1	19	1	19	TAATACGACTCACTATAGG	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Inducible expression with host-supplied T7 RNA polymerase.
lac operator	protein_bind	20	44	1	25	GGAATTGTGAGCGGATAACAATTCC	pET-28a(+); MilliporeSigma/Novagen via SnapGene	LacI-mediated control of basal expression.
RBS	RBS	75	80	1	6	AAGGAG	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Preserve existing bacterial translation-initiation context and spacing.
His6-GSG-TG2_candidate	CDS	88	390	1	303	ATGCATCATCATCATCATCATGGCAGCGGCGATGAAAAAGCGAAAGAACTGCTGAAAAAAGCGAAAGAAGCGGGCAAAAAAGCGCTGGAAGAAGTGAAAAAAGCGAAAGAAAAAGGCGATCTGGGCGCGGCGAGCAAAGCGGCGGCGGAAGGCACCGAAAAAATGGGCGAACTGGTGCAGGAAGCGGTGGATCTGGGCCTGCCGCAGGAAGCGATTGATGAACTGAAAAAACTGCTGTTTGAAACCGGCCGCGAAATGATGAAACTGGTGGAAGAAACCCGCGAAGCGCGCGAAAAAGCGTAA	designed from local candidate; see candidate-source.json	Full expressed product: 100 aa plus stop. Tag not cleavable in this design.
start_ATG	misc_feature	88	90	1	3	ATG	designed from local candidate; see candidate-source.json	Translation starts here.
His6_tag	misc_feature	91	108	1	18	CATCATCATCATCATCAT	designed from local candidate; see candidate-source.json	Six histidines for purification; not a secretion signal.
GSG_linker	misc_feature	109	117	1	9	GGCAGCGGC	designed from local candidate; see candidate-source.json	Three residue spacer; empirical effect on binding unknown.
TG2_binder_90aa	misc_feature	118	387	1	270	GATGAAAAAGCGAAAGAACTGCTGAAAAAAGCGAAAGAAGCGGGCAAAAAAGCGCTGGAAGAAGTGAAAAAAGCGAAAGAAAAAGGCGATCTGGGCGCGGCGAGCAAAGCGGCGGCGGAAGGCACCGAAAAAATGGGCGAACTGGTGCAGGAAGCGGTGGATCTGGGCCTGCCGCAGGAAGCGATTGATGAACTGAAAAAACTGCTGTTTGAAACCGGCCGCGAAATGATGAAACTGGTGGAAGAAACCCGCGAAGCGCGCGAAAAAGCG	designed from local candidate; see candidate-source.json	Exact original amino-acid candidate preserved.
stop_TAA	misc_feature	388	390	1	3	TAA	designed from local candidate; see candidate-source.json	Translation stop, distinct from transcription terminator.
T7 terminator	terminator	454	501	1	48	CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Retain the backbone transcription terminator.
f1 ori	rep_origin	538	993	1	456	ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Retained legacy backbone feature; not required for the proposed expression use.
KanR	CDS	1086	1901	-1	816	ATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Select bacteria retaining the plasmid.
ori	rep_origin	2023	2611	1	589	TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Maintain plasmid replication in E. coli.
rop	CDS	3041	3232	-1	192	GTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Retained backbone replication-control context.
lacI	CDS	4041	5123	-1	1083	GTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Supply lac repressor for expression control.
lacI promoter	promoter	5124	5201	-1	78	GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAAT	pET-28a(+); MilliporeSigma/Novagen via SnapGene	Express the lac repressor.