Primer Designer Tool

Design PCR primers for LIC cloning, sequencing, and Gibson/HiFi assembly using Primer3.

CLI Location

bash

scripts/primer_cli.py

Quick Start

LIC Cloning Primers (default v1)

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192

LIC Cloning with Specific Tag Version

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192 --lic-tag v2

Gibson/HiFi Assembly Primers

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192 --hifi --vector 438-A

List Available Options

bash

python3 scripts/primer_cli.py --list-lic-tags
python3 scripts/primer_cli.py --list-vectors

Options

Option	Description
`--index, -i`	Starting index number for primer naming (required)
`--accession, -a`	NCBI accession code (NM_ or XM_ prefix)
`--sequence, -s`	Raw DNA sequence (ATCG only)
`--gene, -g`	Gene name (required with --sequence)
`--lic-tag, -t`	LIC tag version: v1, v2, v3, vKoz, vBac, vGFP1, vGFP2, vHRV
`--list-lic-tags`	List available LIC tag versions and exit
`--hifi, --gibson`	Design HiFi/Gibson assembly primers
`--vector, -v`	Vector name (e.g., 438-A) - also auto-selects LIC tag
`--overlap-tm`	Target Tm for overlap regions (default: 60°C)
`--list-vectors`	List available vectors and exit
`--lic-only`	Only generate LIC cloning primers
`--seq-only`	Only generate sequencing primers
`--json`	Output results as JSON

LIC Tag Versions (MacroLab Vectors v8)

Tag	Forward Overhang	Reverse Overhang	ORF Requirement
v1	`TACTTCCAATCCAATGCA`	`TTATCCACTTCCAATGTTATTA`	No ATG in ORF
v2	`TTTAAGAAGGAGATATAGATC`	`TTATGGAGTTGGGATCTTATTA`	ORF needs ATG
v3	`TTTAAGAAGGAGATATAGTTC`	`GGATTGGAAGTAGAGGTTCTC`	ORF needs ATG
vKoz	`TACTTCCAATCCAATGCCACC`	`TTATCCACTTCCAATGTTATTA`	ORF needs ATG
vBac	`TACTTCCAATCCAATCG`	`TTATCCACTTCCAATGTTATTA`	ORF needs ATG
vGFP1	`TACTTCCAATCCAATGCA`	`CTCCCACTACCAATGCC`	No stop codon
vGFP2	`TTTAAGAAGGAGATATAGATC`	`GTTGGAGGATGAGAGGATCCC`	ORF needs ATG
vHRV	`GTGCTGTTCCAGGGTCCGAAT`	`TGGTGGTGGTGGTGCTCGATTA`	HRV 3C SLIC

When to Use Each Tag

Tag	Use Case	Example Vectors
v1	N-terminal tagged constructs (His6-MBP, His6-GST, etc.)	1B, 1C, 1M, 2BT, 2CT, 4B, 4C, 438-B, 438-C
v2	Untagged expression	2AT
v3	C-terminal tagged constructs	2Bc-T, 2Cc-T, 2Oc-T, 2Tc-T
vKoz	Eukaryotic expression with Kozak sequence	-
vBac	Baculovirus/insect cell untagged	4A, 5A, 438-A
vGFP1	C-terminal fluorescent protein fusion (N-term tagged)	MBP-mCherry, H6-msfGFP
vGFP2	C-terminal fluorescent protein fusion (untagged)	u-mCherry, u-msfGFP
vHRV	HRV 3C protease site SLIC cloning	-

Auto-Selection from Vector

When using --vector, the appropriate LIC tag is auto-selected:

bash

# Auto-selects vBac for 438-A (untagged insect)
python3 scripts/primer_cli.py --index 1 --accession NM_007192 --vector 438-A

# Auto-selects v1 for 438-B (His6-MBP)
python3 scripts/primer_cli.py --index 1 --accession NM_007192 --vector 438-B

# Auto-selects v2 for 2AT (untagged E. coli)
python3 scripts/primer_cli.py --index 1 --accession NM_007192 --vector 2AT

You can override auto-selection with explicit --lic-tag:

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192 --vector 438-B --lic-tag v2

Primer Types

1. LIC Primers (Ligation Independent Cloning)

Overhangs determined by tag version:

code

LF1_GeneName_LICv1_F    TACTTCCAATCCAATGCA{binding_region}
LF2_GeneName_LICv1_R    TTATCCACTTCCAATGTTATTA{binding_region}

2. HiFi/Gibson Assembly Primers

Dynamic overlaps calculated to achieve target Tm (default 60°C):

•Forward: [vector upstream overlap] + [gene binding region]
•Reverse: [vector downstream overlap RC] + [gene binding region]

Output includes detailed Tm information:

code

LF1_SUPT16H_HiFi_F    ACACCTCCCCCTGAACCTGATGGCTGTGACTCTGGACAAAGACGCTTAT
  # Overlap: ACACCTCCCCCTGAACCTG (Tm: 61.5°C, 19 bp)
  # Binding: ATGGCTGTGACTCTGGACAAAGACGCTTAT (Tm: 67.8°C)

3. Sequencing Primers

Spaced ~750bp apart for full-length sequencing coverage.

Available Vectors

Vectors are loaded from ./vectors/ directory:

Vector	Tag	LIC Tag	Host
438-A	- (untagged)	vBac	Insect
438-B	His6-TEV	v1	Insect
438-C	His6-MBP-TEV	v1	Insect
438-D	His6-GST-TEV	v1	Insect
438-G	His6-GB1-TEV	v1	Insect

Examples

LICv2 for Untagged Expression

bash

python3 scripts/primer_cli.py --index 1 --sequence ATGCATGC... \
  --gene MyGene --lic-tag v2 --lic-only

LICv3 for C-terminal Tags

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192 \
  --lic-tag v3 --lic-only

Gibson/HiFi with Custom Overlap Tm

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192 \
  --hifi --vector 438-B --overlap-tm 65

JSON Output for Parsing

bash

python3 scripts/primer_cli.py --index 1 --accession NM_007192 \
  --lic-only --json

Restriction Site Check

Automatically warns about internal SwaI and PmeI sites that could interfere with cloning.

Configuration

Settings in scripts/settings.py:

•Email: configured in scripts/settings.py
•Initials: configured in scripts/settings.py (used in primer names)