Provided is a recombinant Herpes Simplex Virus type 1 (HSV-1) strain H129-derived anterograde multi-synaptic transneuronal viral tracer for mutli-synaptic neural circuit mapping, which comprises two or more fluorescence expression cassettes being integrated into the H129 genome at different locations; wherein each fluorescence expression cassette contains at least two copies of fluorescent protein-encoding sequence that are arranged in tandem, and at least one linker-encoding sequence, where at least one linker-encoding sequence is disposed between two fluorescent protein-encoding sequences, allowing transcription of fluorescent protein-encoding sequences and linker-encoding sequence as a single transcript; and wherein the linker-encoding sequence encodes a linker peptide containing at least two adjacent amino acids that are highly inefficient in forming a peptide bond between them; thereby, when the single transcript is translated, at least two fluorescent proteins are stoichiometrically generated due to the impedence of peptide bond formation by the linker peptide.
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