# US 2025/0146014 A1 · Plasmid for production of recombinant protein in mammalian cell

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- Number: US 2025/0146014 A1
- Document ID: US20250146014A1
- Status: Published application
- Kind: A1
- Application number: 18/867,841
- Filed: 2023-05-30
- Published: 2025-05-08
- Earliest claimed: 2022-06-03
- Assignees: Izmir Biyotip Ve Genom Merkezi
- Applicants: Izmir Biyotip Ve Genom Merkezi
- Inventors: Mehmet OZTURK; Aybike ERDOGAN
- Web page: https://patentgrep.com/patent/US20250146014A1

## CPC

- C12N 15/85: for animal cells
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 2800/107: for mammalian
- C12N 2830/15: chimeric enhancer/promoter combination
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal
- C12N 2830/60: from viruses
- C12N 2840/105: enhancing translation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A plasmid developed for the production of recombinant protein in mammalian cells is provided. The plasmid vector includes the nucleic acid sequence with at least 80% similarity to SEQ ID NO: 1. The plasmid vector includes a CMV promoter, a CMV enhancer, CMV intron A, SV40 small t antigen intron, SV40 poly(A) signal, Syn21, and an hTERT-SV40 enhancer.

## Claims

### Claim 1

A plasmid vector, comprising a nucleic acid sequence with at least 80% similarity to SEQ ID NO: 1.

### Claim 2 (depends on 1)

The plasmid vector according to claim 1, comprising:
a cytomegalovirus (CMV) promoter containing the sequence specified at nucleotides 836 to 1040 in SEQ ID NO: 1,
a CMV enhancer containing the sequence specified at nucleotides 456 to 835 in SEQ ID NO: 1,
CMV intron A containing the sequence specified at nucleotides 1176 to 1997 in SEQ ID NO: 1,
simian vacuolating virus 40 (SV40) small t antigen intron containing the sequence specified at nucleotides 2247 to 2312 in SEQ ID NO: 1,
an SV40 poly(A) signal containing the sequence specified at nucleotides 2471 to 2592 in SEQ ID NO: 1,
a synthetic AT-rich 21-bp sequence (Syn21) containing the sequence specified at nucleotides 2106 to 2130 in SEQ ID NO: 1,
a human telomerase reverse transcriptase (hTERT)-SV40 enhancer containing the sequence specified at nucleotides 2604 to 3111 in SEQ ID NO: 1.

### Claim 3 (depends on 2)

The plasmid vector according to claim 2, further comprising an ampicillin resistance gene containing the sequence specified at nucleotides 6527 to 7387 in SEQ ID NO: 1, or kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, puromycin, erythromycin, polymyxin B, tetracycline, hygromycin chloramphenicol, or an equivalent resistance gene playing a same role as the ampicillin resistance gene in a plasmid selection in prokaryotic cells.

### Claim 4 (depends on 2)

The plasmid vector according to claim 2, further comprising a puromycin resistance gene containing the sequence specified at nucleotides 4633 to 5232 in SEQ ID NO: 1, or blasticidin, G418/geneticin, hygromycin B, zeocin, neomycin, or an equivalent gene playing a same role as the puromycin resistance gene in selecting cells containing the plasmid vector in mammalian cells.

### Claim 5 (depends on 2)

The plasmid vector according to claim 2, further comprising a dihydrofolate reductase (DHFR) gene containing the sequence specified at nucleotides 3729 to 4292 in SEQ ID NO: 1, or a glutamine synthetase (GS) gene, or an equivalent gene playing a same role as the DHFR gene in cell survival depending on a host cell.

### Claim 6 (depends on 2)

The plasmid vector according to claim 2, further comprising a multiple cloning site (MCS) at nucleotides 2132 to 2240 in SEQ ID NO: 1, wherein the MCS contains a plurality of restriction enzyme sites, the plurality of restriction enzyme sites allow an addition of a gene of interest into the plasmid vector.