DNA

This repository contains sequence files for the Nucleus Distribution DNA library. It is the companion DNA registry to the Nucleus Distribution documentation, which describes the protocols and modules that use these parts.

All files are in GenBank (.gb) format and can be opened in Benchling, Snapgene, Benchling, or any other sequence editor that supports the GenBank format.

Naming convention

Most parts in this library use the pOpen backbone — a modular cloning (MoClo) compatible entry vector (medium copy, ampicilin resistance). File names follow the pattern pOpen-[part-name].gb, where the part name describes the genetic element cloned into the backbone. Expression constructs for PURE system proteins use the pET28a backbone (medium copy, kanamycin resistance, lac inducible T7 expression) and follow the pattern pET28a-[protein-name].gb.

Repository structure

DNA/
├── PURE/
│   ├── cloning/         # pOpen entry vectors for all PURE system proteins
│   └── expression/      # pET28a expression vectors for PURE system proteins
├── assembly/            # MoClo backbone (pOpen-pOpenv3-MCL0)
├── promoters/           # Level-matched T7 promoter library 
├── RBS/                 # Ribosome binding site and UTR parts
├── terminators/         # T7 terminator variants
├── reporters/           # Protein reporter constructs 
├── pores/               # passive membrane transport
├── emitters/            # signal emission modules
└── detectors/
    ├── quorum-sensing/  # Quorum sensing circuit components
    └── ...              # LacI/TetR-based repressor and operator constructs

---

Part types

PURE/ — Protein components of PURE system

The PURE/ directory contains genes encoding the 36 proteins included in the PURE cell-free translation system. These genes include all 20 canonical aminoacyl-tRNA synthetases (AlaRS, ArgRS, AsnRS, etc) and methionyl-tRNA formyltransferase (MTF); E. coli translation initiation factors (IF1, IF2, IF3), elongation factors (EF-G, EF-Ts, EF-Tu), and release factors (RF1, RF2, RF3, RRF); energy regeneration enzymes (AK, CK, NDK, PPiase), and T7 phage RNA polymerase (T7RNAP).

This distribution includes several variants of a few genes:

• adenylate kinase (AK) from E. coli, S. cerevisiae, chicken (G. gallus) and rabbit (O. cuniculus)
• creatine kinase (CK) from chicken (G. gallus) and rabbit (O. cuniculus)
• glyRS (heterodimer) as individual monomers (glyS, glyQ) and as integrated transcription units (glyQS, glySQ, glyQS-DualHis)
• pheRS (heterodimer) as individual monomers (pheS, pheT) and as integrated transcription units (pheST, pheTS, pheST-DualHis)

PURE/cloning/ — PURE protein cloning vectors

The PURE/cloning/ directory contains pOpen entry vectors encoding each protein component of the PURE cell-free transcription/translation system. These plasmids are meant to be used for stable maintenance of these genes in vivo and downstream assembly into appropriate expression plasmids. They are NOT appropriate to use directly as expression plasmids.

---

PURE/expression/ — PURE protein expression vectors

The PURE/expression/ directory contains pET28a-based expression vectors for producing each PURE system protein in E. coli. These are the constructs used for protein production to make the protein components of the PURE system using a lac inducible E. coli expression system.

---

assembly/ — MoClo backbone

The assembly/ directory contains the pOpen MoClo backbone vector:

• pOpen-pOpenv3-MCL0.gb — MCL0 destination backbone for MoClo Level 0 assembly

---

promoters/ — T7 promoter library

The promoters/ directory contains ten T7 promoter variants (pOpen-PURET7-1.gb through pOpen-PURET7-10.gb) designed for use in PURE cell-free expression. The variants are level-matched: expression is calibrated relative to PURET7-10, which is defined as 1.0. PURET7-1 has a relative expression of ~0.35, providing approximately a 3× dynamic range across the series.

---

RBS/ — Ribosome binding sites and UTRs

The RBS/ directory contains translation initiation parts:

• pOpen-RBS.gb — Elowitz (1999) reference E. coli ribosome binding site
• pOpen-UTR1.gb — 5′ UTR element

---

terminators/ — T7 terminator variants

The terminators/ directory contains three T7 terminator variants originally developed in Calvopina-Chavez, Gardner, and Griffitts, 2022:

• pOpen-tT7.gb — native T7 terminator
• pOpen-tT7hyb6.gb — hybrid T7 terminator variant 6
• pOpen-tT7hyb10.gb — hybrid T7 terminator variant 10

---

reporters/ — Protein reporters

The reporters/ directory contains fluorescent protein and chromoprotein reporter constructs. Includes cjBlue, eforRed, plamGFP, amajLime, gfasPurple, meleRFP, and mmilCFP. Several reporters include a lacO operator insert for lac-regulated expression, and some include a C-terminal His6 tag for downstream purification.

---

pores/ — Passive transport through the membrane

The pores/ directory contains passive transport membrane channel proteins for use in synthetic cells. Includes Cx43 and Cx43-eGFP in pOpen.

---

emitters/ — Signal emission modules

The emitters/ directory contains constructs for signal emission modules, typically small molecule generators. Includses bjaI (tet regulation), which produces the quorum sensing module IV-HSL.

---

detectors/ — Regulatory and sensing circuits

The detectors/ directory contains constructs for building gene circuits that respond to small molecule inputs.

Root level — LacI/TetR-based regulation:

• pOpen-lacI.gb, pOpen-tetR.gb — repressor expression constructs
• pOpen-pT7-lacO.gb, pOpen-pT7-tetO.gb — T7 promoters with single operator sites
• pOpen-pT7-lacO-tetO.gb, pOpen-pT7-tetO-lacO.gb — dual-operator promoters for AND-gate logic

detectors/quorum-sensing/ — BjaI/BjaR quorum sensing system from Bradyrhizobium japonicum:

• pOpen-pT7-bjaI.gb — BjaI synthase (signal production)
• pOpen-bjaR-GFP-native.gb — BjaR receptor fused to GFP reporter under native promoter. Used in an E. coli strain as a reporter for signal emitted by a syncell with bjaI.

---

Relationship to Nucleus Distribution docs

Protocol pages in the Nucleus Distribution documentation reference specific constructs from this repository by filename. When a protocol calls for a specific plasmid, the corresponding .gb file can be found here.