primerdesigner

name	primerdesigner
description	Design PCR primers for molecular cloning. Use when the user asks about designing primers, LIC cloning primers, sequencing primers, Gibson assembly primers, or working with NCBI accession codes for cloning.
allowed-tools	Bash(primer_cli:*) Bash(primerdesigner:*)

Primer Designer Tool

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

CLI Location

scripts/primer_cli.py

Quick Start

LIC Cloning Primers (default v1)

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

LIC Cloning with Specific Tag Version

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

Gibson/HiFi Assembly Primers

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

List Available Options

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:

# 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:

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:

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:

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

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

LICv3 for C-terminal Tags

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

Gibson/HiFi with Custom Overlap Tm

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

JSON Output for Parsing

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)