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GoldenBraid 2.0 E. coli Kit
(Kit # 1000000284 )

Depositing Lab:   Christophe Herman

A modular cloning toolkit adhering to the GoldenBraid 2.0 standard. This toolkit is designed for use in E. coli and related enteric bacteria.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

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$774 USD + shipping
Available to academics and nonprofits only.

Original Publication

GoldenBraid2.0 E. coli: a comprehensive and characterized toolkit for enterics. Cooke MB, Welch KT, Ramirez LD, Wen AX, Marciano DC, Herman C. Synth Biol (Oxf). 2025 Aug 14;10(1):ysaf015. doi: 10.1093/synbio/ysaf015. eCollection 2025. PubMed (Link opens in a new window) Article (Link opens in a new window)

Description

The GoldenBraid 2.0 E. coli Toolkit is a comprehensive modular cloning resource designed to facilitate genetic engineering in Escherichia coli. Built on the GoldenBraid 2.0 Type IIS assembly platform, the toolkit enables rapid and standardized construction of plasmid-borne and chromosomally integrated genetic circuits through the reuse of interchangeable DNA parts. The collection includes 192 pre-assembled genetic parts for gene expression, protein tagging, and strain engineering, along with specialized cloning strains that streamline construct assembly and genome modification.

Schematic overview of the GoldenBraid 2.0 E. coli modular cloning toolkit. The figure illustrates the system's hierarchical assembly logic across three stages. A conceptual diagram shows how individual genetic parts (Golden, Braid, 2.0, E., coli), each flanked by red directional arrows representing Type IIS restriction sites, are assembled into a composite construct within an E. coli cell. GoldenBraid 2.0 E. coli Grammar: A hierarchical part taxonomy defines the allowed structure of a Transcription Unit (TU). The 5-prime UTR (flanked by overhangs GGAG and AATG) accommodates a Full Promoter or a Partial Promoter combined with an RBS. The coding region may consist of TL 1-5 (AATG and GCTT overhangs); TL 1-3, Link TL4, and Tag TL5 (AATG, TCGGGC, GGCTCG, and GCTT overhangs); Tag TL1, Link TL2, and TL 3-5 (AATG, AAGTGGC, GGTAGC, and GCTT overhangs); or Tag TL1, Link TL2, TL3, Link TL4, and Tag TL5 (AATG, AGTGGC, GGTAGC, TCGGGC, GGCTCG, and GCTT overhangs). The 3-prime UTR may consist of a Full Terminator (GCTT and CGCT overhangs); RTag and RTag Terminator (GCTT, TGGC, and CGCT overhangs); DTag Terminator and DTag (GCTT, CAGT, and CGCT overhangs); or RTag, RD Term, and DTag (GCTT, TGGC, CAGT, and CGCT overhangs).  The three-stage assembly workflow is domestic parts, assemble TUs, and combine TUs. First a PCR-amplified insert flanked by Esp3I sites is cloned into a domestication vector (e.g., pUPD3) carrying a screening insert between Esp3I and BsaI sites. Esp3I digestion–ligation removes the screening insert and produces a domesticated part (e.g., pUPD3-Insert) flanked by BsaI sites. Next multiple domesticated parts (e.g., Promoter, Coding Sequence, Terminator), each in BsaI-flanked vectors, are combined with an alpha-level assembly vector (e.g., pBSKalpha1) containing a screening insert between BsaI and Esp3I sites. BsaI digestion–ligation produces an assembled Transcription Unit flanked by Esp3I sites. And then two or more alpha-level assembled TUs are combined with an omega-level assembly vector (e.g., pBSKomega1) bearing a screening insert flanked by Esp3I and BsaI sites. Esp3I digestion–ligation produces a multi-TU construct flanked by BsaI sites. The last two steps can repeat indefinitely to build increasingly complex multigenic constructs. A legend illustrates the dual-enzyme vector logic. Each assembled construct is flanked by two enzyme sites: an inner site (Enz1) that cleaves the vector to insert a new sequence (vector serves as assembly output), and an outer site (Enz2) that cleaves to liberate the sequence (vector serves as assembly input). This alternating enzyme architecture enables iterative, scarless hierarchical assembly.

Figure 1: The GoldenBraid 2.0 E. coli toolkit is a modular cloning toolkit designed for use in E. coli and related enteric bacteria. The toolkit is designed to enable the “domestication” of genetic parts into the GoldenBraid 2.0 assembly standard, the assembly of these parts into transcription units which are functional in these species, and the combination of these units together into genetic constructs of arbitrary size. This toolkit is composed of a set of standardized E. coli vectors and genetic parts arranged in a “grammatical” standard optimized for the generation of E. coli genetic constructs.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

"The GoldenBraid 2.0 E. coli Kit was a gift from Christophe Herman (Addgene kit #1000000284)."

For your Reference section:

GoldenBraid2.0 E. coli: a comprehensive and characterized toolkit for enterics. Cooke MB, Welch KT, Ramirez LD, Wen AX, Marciano DC, Herman C. Synth Biol (Oxf). 2025 Aug 14;10(1):ysaf015. doi: 10.1093/synbio/ysaf015. eCollection 2025. PubMed (Link opens in a new window) Article (Link opens in a new window)

GoldenBraid 2.0 E. coli Kit - #1000000284

Resistance Color Key

Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.

Inventory

Searchable and sortable table of all plasmids in kit. The Well column links to its kit plate map on the right, as well as lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.

Kit Plate Map

96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.

Resistance Color Key

Kanamycin
Ampicillin
Chloramphenicol

Inventory

Well Plasmid Resistance
Plate 1 / A / 1 pBSKa1_Red
Kanamycin
Plate 1 / A / 2 pBSKa2_Red
Kanamycin
Plate 1 / A / 3 pR6Ka1_Red
Kanamycin
Plate 1 / A / 4 pR6Ka2_Red
Kanamycin
Plate 1 / A / 5 pBSKa1_FRTampFRT
Kanamycin
Plate 1 / A / 6 pBSKa1_FRTkanFRT
Kanamycin
Plate 1 / A / 7 pBSKa1_FRTcamFRT
Kanamycin
Plate 1 / A / 8 pBSKa1_FRTtetFRT
Kanamycin
Plate 1 / A / 9 pBSKa1_FRTspcFRT
Kanamycin
Plate 1 / A / 10 pBSKa2_FRTampFRT
Kanamycin
Plate 1 / A / 11 pBSKa2_FRTkanFRT
Kanamycin
Plate 1 / A / 12 pBSKa2_FRTcamFRT
Kanamycin
Plate 1 / B / 1 pBSKa2_FRTtetFRT
Kanamycin
Plate 1 / B / 2 pBSKa2_FRTspcFRT
Kanamycin
Plate 1 / B / 3 pBSKa1_Stuffer1
Kanamycin
Plate 1 / B / 4 pBSKa2_Stuffer1
Kanamycin
Plate 1 / B / 5 pBSKa1_Stuffer2
Kanamycin
Plate 1 / B / 6 pBSKa2_Stuffer2
Kanamycin
Plate 1 / B / 7 p15Aa1_Blue
Ampicillin
Plate 1 / B / 8 p15Aa2_Blue
Ampicillin
Plate 1 / B / 9 p15Ao1_Blue
Ampicillin
Plate 1 / B / 10 p15Ao2_Blue
Ampicillin
Plate 1 / B / 11 p15Aa1_Red
Ampicillin
Plate 1 / B / 12 p15Aa2_Red
Ampicillin
Plate 1 / C / 1 p15Ao1_Red
Ampicillin
Plate 1 / C / 2 p15Ao2_Red
Ampicillin
Plate 1 / C / 3 pMB1a1_Blue
Ampicillin
Plate 1 / C / 4 pMB1a2_Blue
Ampicillin
Plate 1 / C / 5 pMB1o1_Blue
Ampicillin
Plate 1 / C / 6 pMB1o2_Blue
Ampicillin
Plate 1 / C / 7 pMB1a1_Red
Ampicillin
Plate 1 / C / 8 pMB1a2_Red
Ampicillin
Plate 1 / C / 9 pMB1o1_Red
Ampicillin
Plate 1 / C / 10 pMB1o2_Red
Ampicillin
Plate 1 / C / 11 pSC101TSa1_Blue
Ampicillin
Plate 1 / C / 12 pSC101TSa2_Blue
Ampicillin
Plate 1 / D / 1 pSC101TSo1_Blue
Ampicillin
Plate 1 / D / 2 pSC101TSo2_Blue
Ampicillin
Plate 1 / D / 3 pSC101TSa1_Red
Ampicillin
Plate 1 / D / 4 pSC101TSa2_Red
Ampicillin
Plate 1 / D / 5 pSC101TSo1_Red
Ampicillin
Plate 1 / D / 6 pSC101TSo2_Red
Ampicillin
Plate 1 / D / 7 ptetgRNA-FRT
Ampicillin
Plate 1 / D / 8 pUPD3_Red
Chloramphenicol
Plate 1 / D / 9 pBSKo1_Red
Chloramphenicol
Plate 1 / D / 10 pBSKo2_Red
Chloramphenicol
Plate 1 / D / 11 pR6Ko1_Red
Chloramphenicol
Plate 1 / D / 12 pR6Ko2_Red
Chloramphenicol
Plate 1 / E / 1 pUPD3_FULLUNIT_Stuffer1
Chloramphenicol
Plate 1 / E / 2 pUPD3_FULLUNIT_Stuffer2
Chloramphenicol
Plate 1 / E / 3 pUPD3_FULLPRM_Stuffer3
Chloramphenicol
Plate 1 / E / 4 pUPD3_FULLPRM_Stuffer4
Chloramphenicol
Plate 1 / E / 5 pUPD3_PARTPRM_Stuffer5
Chloramphenicol
Plate 1 / E / 6 pUPD3_PARTPRM_Stuffer6
Chloramphenicol
Plate 1 / E / 7 pUPD3_RBS_Stuffer7
Chloramphenicol
Plate 1 / E / 8 pUPD3_RBS_Stuffer8
Chloramphenicol
Plate 1 / E / 9 pUPD3_CDS_Stuffer9
Chloramphenicol
Plate 1 / E / 10 pUPD3_CDS_Stuffer10
Chloramphenicol
Plate 1 / E / 11 pUPD3_FULLTRM_Stuffer11
Chloramphenicol
Plate 1 / E / 12 pUPD3_FULLTRM_Stuffer12
Chloramphenicol
Plate 1 / F / 1 pUPD3_RTAGTRM_Stuffer13
Chloramphenicol
Plate 1 / F / 2 pUPD3_RTAGTRM_Stuffer14
Chloramphenicol
Plate 1 / F / 3 pUPD3_DTAGTRM_Stuffer15
Chloramphenicol
Plate 1 / F / 4 pUPD3_DTAGTRM_Stuffer16
Chloramphenicol
Plate 1 / F / 5 pUPD3_RDTAGTRM_Stuffer17
Chloramphenicol
Plate 1 / F / 6 pUPD3_RDTAGTRM_Stuffer18
Chloramphenicol
Plate 1 / F / 7 pBSKo1_Stuffer1_Stuffer2
Chloramphenicol
Plate 1 / F / 8 pBSKo2_Stuffer2_Stuffer1
Chloramphenicol
Plate 1 / F / 9 pR6Ko1_FRTampFRT
Chloramphenicol
Plate 1 / F / 10 pR6Ko1_FRTkanFRT
Chloramphenicol
Plate 1 / F / 11 pR6Ko1_FRTcamFRT
Chloramphenicol
Plate 1 / F / 12 pR6Ko1_FRTtetFRT
Chloramphenicol
Plate 1 / G / 1 pR6Ko1_FRTspcFRT
Chloramphenicol
Plate 1 / G / 2 pR6Ko2_FRTtetFRT
Chloramphenicol
Plate 1 / G / 3 pR6Ko2_FRTcamFRT
Chloramphenicol
Plate 1 / G / 4 pR6Ko2_FRTkanFRT
Chloramphenicol
Plate 1 / G / 5 pR6Ko2_FRTampFRT
Chloramphenicol
Plate 1 / G / 6 pR6Ko2_FRTspcFRT
Chloramphenicol
Plate 1 / G / 7 pUPD3_FULLPRM_Pem7
Chloramphenicol
Plate 1 / G / 8 pUPD3_FULLPRM_lacI_Plac
Chloramphenicol
Plate 1 / G / 9 pUPD3_FULLPRM_Plac
Chloramphenicol
Plate 1 / G / 10 pUPD3_FULLPRM_araC_Para
Chloramphenicol
Plate 1 / G / 11 pUPD3_FULLPRM_Para
Chloramphenicol
Plate 1 / G / 12 pUPD3_FULLPRM_tetR_Ptet
Chloramphenicol
Plate 1 / H / 1 pUPD3_FULLPRM_Ptet
Chloramphenicol
Plate 1 / H / 2 pUPD3_FULLPRM_cI857_PR
Chloramphenicol
Plate 1 / H / 3 pUPD3_FULLPRM_PR
Chloramphenicol
Plate 1 / H / 4 pUPD3_FULLPRM_PT7
Chloramphenicol
Plate 1 / H / 5 pUPD3_FULLPRM_PT7lac
Chloramphenicol
Plate 1 / H / 6 pUPD3_PARTPRM_Pem7
Chloramphenicol
Plate 1 / H / 7 pUPD3_PARTPRM_lacI_Plac
Chloramphenicol
Plate 1 / H / 8 pUPD3_PARTPRM_Plac
Chloramphenicol
Plate 1 / H / 9 pUPD3_PARTPRM_araC_Para
Chloramphenicol
Plate 1 / H / 10 pUPD3_PARTPRM_Para
Chloramphenicol
Plate 1 / H / 11 pUPD3_PARTPRM_tetR_Ptet
Chloramphenicol
Plate 1 / H / 12 pUPD3_PARTPRM_Ptet
Chloramphenicol
Plate 2 / A / 1 pUPD3_PARTPRM_cI857_PR
Chloramphenicol
Plate 2 / A / 2 pUPD3_PARTPRM_PR
Chloramphenicol
Plate 2 / A / 3 pUPD3_PARTPRM_PT7
Chloramphenicol
Plate 2 / A / 4 pUPD3_PARTPRM_PT7lac
Chloramphenicol
Plate 2 / A / 5 pUPD3_PARTPRM_J23100
Chloramphenicol
Plate 2 / A / 6 pUPD3_PARTPRM_J23101
Chloramphenicol
Plate 2 / A / 7 pUPD3_PARTPRM_J23102
Chloramphenicol
Plate 2 / A / 8 pUPD3_PARTPRM_J23103
Chloramphenicol
Plate 2 / A / 9 pUPD3_PARTPRM_J23104
Chloramphenicol
Plate 2 / A / 10 pUPD3_PARTPRM_J23105
Chloramphenicol
Plate 2 / A / 11 pUPD3_PARTPRM_J23106
Chloramphenicol
Plate 2 / A / 12 pUPD3_PARTPRM_J23107
Chloramphenicol
Plate 2 / B / 1 pUPD3_PARTPRM_J23108
Chloramphenicol
Plate 2 / B / 2 pUPD3_PARTPRM_J23109
Chloramphenicol
Plate 2 / B / 3 pUPD3_PARTPRM_J23110
Chloramphenicol
Plate 2 / B / 4 pUPD3_PARTPRM_J23111
Chloramphenicol
Plate 2 / B / 5 pUPD3_PARTPRM_J23113
Chloramphenicol
Plate 2 / B / 6 pUPD3_PARTPRM_J23114
Chloramphenicol
Plate 2 / B / 7 pUPD3_PARTPRM_J23115
Chloramphenicol
Plate 2 / B / 8 pUPD3_PARTPRM_J23116
Chloramphenicol
Plate 2 / B / 9 pUPD3_PARTPRM_J23117
Chloramphenicol
Plate 2 / B / 10 pUPD3_PARTPRM_J23118
Chloramphenicol
Plate 2 / B / 11 pUPD3_PARTPRM_J23119
Chloramphenicol
Plate 2 / B / 12 pUPD3_RBS_J61100
Chloramphenicol
Plate 2 / C / 1 pUPD3_RBS_J61101
Chloramphenicol
Plate 2 / C / 2 pUPD3_RBS_J61102
Chloramphenicol
Plate 2 / C / 3 pUPD3_RBS_J61103
Chloramphenicol
Plate 2 / C / 4 pUPD3_RBS_J61104
Chloramphenicol
Plate 2 / C / 5 pUPD3_RBS_J61105
Chloramphenicol
Plate 2 / C / 6 pUPD3_RBS_J61106
Chloramphenicol
Plate 2 / C / 7 pUPD3_RBS_J61107
Chloramphenicol
Plate 2 / C / 8 pUPD3_RBS_J61108
Chloramphenicol
Plate 2 / C / 9 pUPD3_RBS_J61109
Chloramphenicol
Plate 2 / C / 10 pUPD3_RBS_J61110
Chloramphenicol
Plate 2 / C / 11 pUPD3_RBS_J61111
Chloramphenicol
Plate 2 / C / 12 pUPD3_RBS_J61112
Chloramphenicol
Plate 2 / D / 1 pUPD3_RBS_J61113
Chloramphenicol
Plate 2 / D / 2 pUPD3_RBS_J61114
Chloramphenicol
Plate 2 / D / 3 pUPD3_RBS_J61115
Chloramphenicol
Plate 2 / D / 4 pUPD3_RBS_J61116
Chloramphenicol
Plate 2 / D / 5 pUPD3_RBS_J61117
Chloramphenicol
Plate 2 / D / 6 pUPD3_RBS_J61118
Chloramphenicol
Plate 2 / D / 7 pUPD3_RBS_J61119
Chloramphenicol
Plate 2 / D / 8 pUPD3_RBS_J61120
Chloramphenicol
Plate 2 / D / 9 pUPD3_RBS_J61121
Chloramphenicol
Plate 2 / D / 10 pUPD3_RBS_J61122
Chloramphenicol
Plate 2 / D / 11 pUPD3_RBS_J61123
Chloramphenicol
Plate 2 / D / 12 pUPD3_RBS_J61124
Chloramphenicol
Plate 2 / E / 1 pUPD3_RBS_J61125
Chloramphenicol
Plate 2 / E / 2 pUPD3_RBS_J61126
Chloramphenicol
Plate 2 / E / 3 pUPD3_RBS_J61127
Chloramphenicol
Plate 2 / E / 4 pUPD3_RBS_J61128
Chloramphenicol
Plate 2 / E / 5 pUPD3_RBS_J61129
Chloramphenicol
Plate 2 / E / 6 pUPD3_RBS_J61130
Chloramphenicol
Plate 2 / E / 7 pUPD3_RBS_J61131
Chloramphenicol
Plate 2 / E / 8 pUPD3_RBS_J61132
Chloramphenicol
Plate 2 / E / 9 pUPD3_RBS_J61133
Chloramphenicol
Plate 2 / E / 10 pUPD3_RBS_J61134
Chloramphenicol
Plate 2 / E / 11 pUPD3_RBS_J61135
Chloramphenicol
Plate 2 / E / 12 pUPD3_RBS_J61136
Chloramphenicol
Plate 2 / F / 1 pUPD3_RBS_J61137
Chloramphenicol
Plate 2 / F / 2 pUPD3_RBS_J61138
Chloramphenicol
Plate 2 / F / 3 pUPD3_RBS_J61139
Chloramphenicol
Plate 2 / F / 4 pUPD3_RBS_RBS1
Chloramphenicol
Plate 2 / F / 5 pUPD3_RBS_RBS2
Chloramphenicol
Plate 2 / F / 6 pUPD3_RBS_RBS3
Chloramphenicol
Plate 2 / F / 7 pUPD3_RBS_RBS4
Chloramphenicol
Plate 2 / F / 8 pUPD3_RBS_RBS5
Chloramphenicol
Plate 2 / F / 9 pUPD3_RBS_RBS6
Chloramphenicol
Plate 2 / F / 10 pUPD3_RBS_RBS7
Chloramphenicol
Plate 2 / F / 11 pUPD3_RBS_RBS8
Chloramphenicol
Plate 2 / F / 12 pUPD3_RBS_RBS9
Chloramphenicol
Plate 2 / G / 1 pUPD3_RBS_RBS10
Chloramphenicol
Plate 2 / G / 2 pUPD3_RBS_RBS11
Chloramphenicol
Plate 2 / G / 3 pUPD3_RBS_RBS12
Chloramphenicol
Plate 2 / G / 4 pUPD3_RBS_RBS13
Chloramphenicol
Plate 2 / G / 5 pUPD3_RBS_RBS14
Chloramphenicol
Plate 2 / G / 6 pUPD3_RBS_RBS15
Chloramphenicol
Plate 2 / G / 7 pUPD3_RBS_RBS16
Chloramphenicol
Plate 2 / G / 8 pUPD3_RBS_RBS17
Chloramphenicol
Plate 2 / G / 9 pUPD3_RBS_RBS18
Chloramphenicol
Plate 2 / G / 10 pUPD3_RBS_RBS19
Chloramphenicol
Plate 2 / G / 11 pUPD3_RBS_RBS20
Chloramphenicol
Plate 2 / G / 12 pUPD3_RBS_RBS21
Chloramphenicol
Plate 2 / H / 1 pUPD3_RBS_RBS22
Chloramphenicol
Plate 2 / H / 2 pUPD3_RBS_RBS23
Chloramphenicol
Plate 2 / H / 3 pUPD3_RBS_RBS24
Chloramphenicol
Plate 2 / H / 4 pUPD3_RBS_RBS25
Chloramphenicol
Plate 2 / H / 5 pUPD3_RBS_RBS26
Chloramphenicol
Plate 2 / H / 6 pUPD3_RBS_RBS27
Chloramphenicol
Plate 2 / H / 7 pUPD3_RBS_RBS28
Chloramphenicol
Plate 2 / H / 8 pUPD3_CDS_lacI
Chloramphenicol
Plate 2 / H / 9 pUPD3_CDS_araC
Chloramphenicol
Plate 2 / H / 10 pUPD3_CDS_tetR
Chloramphenicol
Plate 2 / H / 11 pUPD3_CDS_cI857
Chloramphenicol
Plate 2 / H / 12 pUPD3_CDS_T7pol
Chloramphenicol
Data calculated @ 2026-09-13

Kit Plate Map - #1000000284

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