EcoFlex MoClo Extension, Volume I
(Kit # 1000000253 )

Depositing Lab:   Mikhail Shapiro

The EcoFlex MoClo system is a collection of plasmid parts that can be easily combined to form new plasmids with functional expression units (and multi-gene expression systems) using Golden Gate assembly with BsaI and BsmBI. While MoClo is used across a variety of systems and organisms, this kit contains parts designed for E. coli expression.

This kit contains 96 plasmids to quickly and efficiently build a wide variety of gene expression constructs using the EcoFlex-standard MoClo system. This kit can be used as a stand-alone product, but is intended to be used with the EcoFlex MoClo Kit from the Paul Freemont Lab, which contains a number of useful parts not included in this kit.

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

Description

EcoFlex MoClo Extension, Volume I extends the EcoFlex kit by adding 11 chemically inducible promoters of varying strengths, 16 ribosome-binding sites (RBSs) of varying strengths, 8 tag-compatible RBSs of varying strengths, 2 N-terminal secretion tags, 6 CDSs, 9 strong terminators, 34 backbones with ORIs of varying copy number, 6 destination vectors for cloning additional parts, and 4 nonfunctional “dummy” parts that are intended for use in certain multi-transcription unit (TU) assemblies. These parts are cross-compatible with parts from the original EcoFlex kit.

There are 10 types of vectors used for parts:

• The backbones used for assemply (100 µg/mL kanamycin, NEB Stable):
• pTU1-A will hold 1 TU, assembled using BsaI from Level 0 parts.
• pTU2-a will hold 2 TUs, assembled using BsmBI from Level 1 plasmids.
• pTU2-b will hold 3 TUs, assembled using BsmBI from Level 1 plasmids.

• The pBP vectors (25 µg/mL chloramphenicol, NEB Stable):
• pBP-P (holds Level 0 promoter parts; overhangs CTAT and GTAC)
• pBP-R (holds Level 0 RBS parts that cannot be assembled with N-terminal tag parts; overhangs GTAC and CATA)
• pBP-TagR (holds Level 0 RBS parts that must be assembled with N-terminal tag parts; overhangs GTAC and TAAA)
• pBP-Tag (holds Level 0 N-terminal tag parts; overhangs TAAA and CATA)
• pBP-C (holds Level 0 CDS parts; overhangs CATA and TCGA)
• pBP-T (holds Level 0 terminator parts; overhangs TCGA and TGTT)

• The entry vectors for making your own parts (100 µg/mL kanamycin and 25 µg/mL Zeocin®, DH10B-M.Osp807II):
• pMetBP
• The nomenclature is the same as above, with the addition that “RC” is a combined RBS-CDS part (overhangs GTAC and TCGA) used when the RBS is contained in the CDS part rather than a separate RBS part. These entry vectors are based on the MetClo system, and must be propagated in the DH10B-M.Osp807II strain. To create a new part, perform a BsaI assembly with the pMetBP vector and a dsDNA part fragment (usually a PCR product or duplex oligo) flanked by the appropriate overhangs.

In cases where a promoter requires a repressor or activator for induction, that protein is included in the promoter part, rather than in the backbone part. “Dummy” parts are random sequences without any activity flanked by the 4-bp overhangs corresponding to that part type, and are intended for use in assemblies where a part of that type is not desired for the function of the assembled construct but is needed for the assembly to correctly circularize (e.g., a negative control plasmid where no promoter is driving the gene of interest, or a multi-TU construct without a promoter and terminator between the TUs).

Two types of Level 1 assemblies can be performed in the base EcoFlex system. These always include a backbone, promoter, RBS, CDS, and terminator, and may include an N-terminal tag (in which case a TagR part must be used instead of an R part). If you don’t want to include one of these parts, you must use the “dummy” version of that part for the assembly to work. Level 1 assemblies are always performed using BsaI. During successful Level 1 assemblies, the dropout chromophore (amilCP or cRed) is lost and replaced with the desired TU. After transforming the assembly, only pick colonies that do not produce a blue or orange color. For additional information on these and other types of assemblies, consult the EcoFlex User Guide (DOCX, 50 KB) .

Figure 1. The two types of Level 1 assemblies that can be performed using the EcoFlex system. Note that the type of RBS part used differs between the two types of assemblies.

Kit Documentation

EcoFlex User Guide (DOCX, 50 KB)

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 EcoFlex MoClo Extension, Volume I kit was a gift from Mikhail Shapiro (Addgene kit #1000000253).”

EcoFlex MoClo Extension, Volume I - #1000000253

Inventory

Well	Plasmid	Resistance
A / 1	pTU1-A_Sv7K_amilCP	Kanamycin
A / 2	pTU1-A_Sv6K_amilCP	Kanamycin
A / 3	pTU1-A_Sv5K_amilCP	Kanamycin
A / 4	pTU1-A_Sv4K_amilCP	Kanamycin
A / 5	pTU1-A_Sv3K_amilCP	Kanamycin
A / 6	pTU1-A_Sv2K_amilCP	Kanamycin
A / 7	pTU1-A_Sv1K_amilCP	Kanamycin
A / 8	pTU1-A_SK_amilCP	Kanamycin
A / 9	pTU1-A_UK_amilCP	Kanamycin
A / 10	pTU1-A_UrK_amilCP	Kanamycin
A / 11	pTU1-A_CK_amilCP	Kanamycin
A / 12	pTU1-A_CrK_amilCP	Kanamycin
B / 1	pTU1-A_PK_amilCP	Kanamycin
B / 2	pTU2-a_Sv7KA_amilCP	Kanamycin
B / 3	pTU2-a_Sv6KA_amilCP	Kanamycin
B / 4	pTU2-a_Sv5KA_amilCP	Kanamycin
B / 5	pTU2-a_Sv4KA_amilCP	Kanamycin
B / 6	pTU2-a_Sv3KA_amilCP	Kanamycin
B / 7	pTU2-a_Sv2KA_amilCP	Kanamycin
B / 8	pTU2-a_Sv1KA_amilCP	Kanamycin
B / 9	pTU2-a_SKA_amilCP	Kanamycin
B / 10	pTU2-b_Sv7K_amilCP	Kanamycin
B / 11	pTU2-b_Sv6K_amilCP	Kanamycin
B / 12	pTU2-b_Sv5K_amilCP	Kanamycin
C / 1	pTU2-b_Sv4K_amilCP	Kanamycin
C / 2	pTU2-b_Sv3K_amilCP	Kanamycin
C / 3	pTU2-b_Sv2K_amilCP	Kanamycin
C / 4	pTU2-b_Sv1K_amilCP	Kanamycin
C / 5	pTU2-b_SK_amilCP	Kanamycin
C / 6	pTU2-b_UK_amilCP	Kanamycin
C / 7	pTU2-b_UrK_amilCP	Kanamycin
C / 8	pTU2-b_CK_amilCP	Kanamycin
C / 9	pTU2-b_CrK_amilCP	Kanamycin
C / 10	pTU2-b_PK_amilCP	Kanamycin
C / 11	pBP-P_dummy-promoter	Chloramphenicol
C / 12	pBP-P_araC-PBAD	Chloramphenicol
D / 1	pBP-P_cymR-PCymRC	Chloramphenicol
D / 2	pBP-P_lacI-Ptac-lacO	Chloramphenicol
D / 3	pBP-P_luxR-PLuxB	Chloramphenicol
D / 4	pBP-P_nahR-PSalTTC	Chloramphenicol
D / 5	pBP-P_phlF-PPhlF	Chloramphenicol
D / 6	pBP-P_rhaS-PRhaB	Chloramphenicol
D / 7	pBP-P_tetR-PTet*	Chloramphenicol
D / 8	pBP-P_vanR-PVanCC	Chloramphenicol
D / 9	pBP-P_TALEsp1_PUPsp1-lacI_Ptac-lacO	Chloramphenicol
D / 10	pBP-P_TALEsp1_PUPsp1-phlF_PPhlF	Chloramphenicol
D / 11	pBP-R_dummy-RBS	Chloramphenicol
D / 12	pBP-R_RiboJ-MCD2	Chloramphenicol
E / 1	pBP-R_SarJ-MCD2	Chloramphenicol
E / 2	pBP-R_PlmJ-MCD2	Chloramphenicol
E / 3	pBP-R_BydvJ-MCD2	Chloramphenicol
E / 4	pBP-R_ElvJ-MCD2	Chloramphenicol
E / 5	pBP-R_CchJ-MCD2	Chloramphenicol
E / 6	pBP-R_AraJ-MCD2	Chloramphenicol
E / 7	pBP-R_RiboJ57-MCD2	Chloramphenicol
E / 8	pBP-R_MCD2	Chloramphenicol
E / 9	pBP-R_MCD1	Chloramphenicol
E / 10	pBP-R_MCD17	Chloramphenicol
E / 11	pBP-R_MCD10	Chloramphenicol
E / 12	pBP-R_MCD7	Chloramphenicol
F / 1	pBP-R_MCD15	Chloramphenicol
F / 2	pBP-R_MCD22	Chloramphenicol
F / 3	pBP-R_MCD8	Chloramphenicol
F / 4	pBP-TagR_MCD2	Chloramphenicol
F / 5	pBP-TagR_MCD1	Chloramphenicol
F / 6	pBP-TagR_MCD17	Chloramphenicol
F / 7	pBP-TagR_MCD10	Chloramphenicol
F / 8	pBP-TagR_MCD7	Chloramphenicol
F / 9	pBP-TagR_MCD15	Chloramphenicol
F / 10	pBP-TagR_MCD22	Chloramphenicol
F / 11	pBP-TagR_MCD8	Chloramphenicol
F / 12	pBP-Tag_dsbA	Chloramphenicol
G / 1	pBP-Tag_phoA	Chloramphenicol
G / 2	pBP-C_dummy-CDS	Chloramphenicol
G / 3	pBP-C_mTagBFP2	Chloramphenicol
G / 4	pBP-C_mScarlet-I	Chloramphenicol
G / 5	pBP-C_sfGFP	Chloramphenicol
G / 6	pBP-C_NanoLuc	Chloramphenicol
G / 7	pBP-C_sacB	Chloramphenicol
G / 8	pBP-C_ccdB	Chloramphenicol
G / 9	pBP-T_dummy-term	Chloramphenicol
G / 10	pBP-T_L3S2P21	Chloramphenicol
G / 11	pBP-T_ECK120029600	Chloramphenicol
G / 12	pBP-T_ECK120033737	Chloramphenicol
H / 1	pBP-T_L3S1P13	Chloramphenicol
H / 2	pBP-T_ECK120033736	Chloramphenicol
H / 3	pBP-T_L3S1P47	Chloramphenicol
H / 4	pBP-T_BBa-B1006-U10	Chloramphenicol
H / 5	pBP-T_his-min-S	Chloramphenicol
H / 6	pBP-T_T7-term	Chloramphenicol
H / 7	pMetBP-P_cRed_BsaI	Chloramphenicol and Bleocin (Zeocin)
H / 8	pMetBP-R_cRed_BsaI	Chloramphenicol and Bleocin (Zeocin)
H / 9	pMetBP-Tag_cRed_BsaI	Chloramphenicol and Bleocin (Zeocin)
H / 10	pMetBP-C_cRed_BsaI	Chloramphenicol and Bleocin (Zeocin)
H / 11	pMetBP-RC_cRed_BsaI	Chloramphenicol and Bleocin (Zeocin)
H / 12	pMetBP-T_cRed_BsaI	Chloramphenicol and Bleocin (Zeocin)

Data calculated @ 2025-04-03