# US 12,600,985 B2 · Compositions and methods for controlling production of polypeptides in cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,600,985 B2 - Document ID: US12600985B2 - Status: Granted - Kind: B2 - Application number: 17/796,839 - Filed: 2021-02-03 - Published: 2023-03-02 - Granted: 2026-04-14 - Earliest claimed: 2020-02-04 - Assignees: The Board of Trustees of the Leland Stanford Junior University - Applicants: The Board of Trustees of the Leland Stanford Junior University - Inventors: Karl A. Deisseroth; Charu Ramakrishnan; Yoon Seok Kim; Lief E. Fenno - Web page: https://patentgrep.com/patent/US12600985B2 ## 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 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease - C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT - C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present disclosure provides recombinant expression vectors for modulating production of polypeptides of interest in a target cell or target cell population. Aspects of the disclosure include recombinant expression vectors having coding sequences encoding portions of a polypeptide of interest, where the coding sequences are flanked by recombinase recognition sites. Also provided are methods for using the recombinant expression vectors as well as a device for monitoring expression of the polypeptide of interest. ## Claims ### Claim 1 A recombinant expression vector comprising: a) a first coding sequence encoding a portion of a polypeptide of interest, wherein a first recombinase recognition site is positioned 5′ to the first coding sequence; b) a second coding sequence positioned 3′ to the first coding sequence, the second coding sequence encoding a portion of the polypeptide of interest; c) a first non-coding sequence comprising a first recombinase recognition site and a second recombinase recognition site positioned between the first coding sequence and the second coding sequence; d) a third coding sequence positioned 3′ to the second coding sequence, the third coding sequence encoding a portion of the polypeptide of interest, wherein a third recombinase recognition site is positioned 3′ to the third coding sequence; and e) a second non-coding sequence comprising a second recombinase recognition site and a third recombinase recognition site positioned between the second coding sequence and the third coding sequence. ### Claim 2 (depends on 1) The recombinant expression vector of claim 1, wherein the first coding sequence is in reverse complement orientation. ### Claim 3 (depends on 1, 2) The recombinant expression vector of claim 1 or claim 2, wherein the second coding sequence is in reverse complement orientation. ### Claim 4 (depends on 1) The recombinant expression vector of claim 1, wherein the third coding sequence is in reverse complement orientation. ### Claim 5 (depends on 1) The recombinant expression vector of claim 1, wherein the first coding sequence, the second coding sequence, and the third coding sequence are in reverse complement orientation. ### Claim 6 (depends on 1) The recombinant expression vector of claim 1, wherein the polypeptide of interest comprises any one of a fluorescent polypeptide, a calcium indicator, an excitatory opsin, and an inhibitory opsin. ### Claim 7 (depends on 1) The recombinant expression vector of claim 1, wherein the first recombinase recognition site is a Cre recombinase recognition site. ### Claim 8 (depends on 7) The recombinant expression vector of claim 7, wherein the Cre recombinase recognition site comprises a loxP sequence, lox2722 sequence, loxN sequence, vloxP sequence, or vlox2722 sequence. ### Claim 9 (depends on 1) The recombinant expression vector of claim 1, wherein the second recombinase recognition site is a Flp recombinase recognition site. ### Claim 10 (depends on 9) The recombinant expression vector of claim 9, wherein the Flp recombinase recognition site comprises a F3 sequence, F5 sequence, FRT sequence, variant FRT sequence, or F72 sequence. ### Claim 11 (depends on 1) The recombinant expression vector of claim 1, wherein the third recombinase recognition site is a vCre recombinase recognition site. ### Claim 12 A method for modulating production of a polypeptide of interest in a target cell or a target cell population, the method comprising: introducing a recombinant expression vector comprising a) a first coding sequence encoding a portion of a polypeptide of interest, wherein a first recombinase recognition site is positioned 5′ to the first coding sequence; b) a second coding sequence positioned 3′ to the first coding sequence, the second coding sequence encoding a portion of the polypeptide of interest; c) a first non-coding sequence comprising a first recombinase recognition site and a second recombinase recognition site positioned between the first coding sequence and the second coding sequence; d) a third coding sequence positioned 3′ to the second coding sequence, the third coding sequence encoding a portion of the polypeptide of interest, wherein a third recombinase recognition site is positioned 3′ to the third coding sequence; and e) a second non-coding sequence comprising a second recombinase recognition site and a third recombinase recognition site positioned between the second coding sequence and the third coding sequence into the target cell or the target cell population. ### Claim 13 (depends on 12) The method of claim 12, wherein the target cell or the target cell population expresses one or more of Cre recombinase, Flp recombinase, and vCre recombinase. ### Claim 14 (depends on 13) The method of claim 13, wherein the method further comprises introducing one or more recombinant expression vectors encoding one or more of Cre recombinase, Flp recombinase, and vCre recombinase into the target cell or the target cell population. ### Claim 15 (depends on 14) The method of claim 14, wherein the method further comprises modulating the amount of Cre recombinase, Flp recombinase, vCre recombinase, or any combination thereof expressed by the target cell or the target cell population. ### Claim 16 (depends on 12) The method of claim 12, wherein the first coding sequence is in reverse complement orientation. ### Claim 17 (depends on 12) The method of claim 12, wherein the second coding sequence is in reverse complement orientation. ### Claim 18 (depends on 12) The method of claim 12, wherein the third coding sequence is in reverse complement orientation. ### Claim 19 (depends on 12) The method of claim 12, wherein the first coding sequence, the second coding sequence, and the third coding sequence are in reverse complement orientation. ### Claim 20 (depends on 12) The method of claim 12, wherein the polypeptide of interest comprises any one of a fluorescent protein, a calcium indicator, an excitatory opsin, and an inhibitory opsin. ### Claim 21 (depends on 12) The method of claim 12, wherein the first recombinase recognition site is a Cre recombinase recognition site. ### Claim 22 (depends on 21) The method of claim 21, wherein the Cre recombinase recognition site comprises a loxP sequence, lox2722 sequence, loxN sequence, vloxP sequence, or vlox2722 sequence. ### Claim 23 (depends on 12) The method of claim 12, wherein the second recombinase recognition site is a Flp recombinase recognition site. ### Claim 24 (depends on 23) The method of claim 23, wherein the Flp recombinase recognition site comprises a F3 sequence, F5 sequence, FRT sequence, variant FRT sequence, or F72 sequence. ### Claim 25 (depends on 12) The method of claim 12, wherein the third recombinase recognition site is a vCre recombinase recognition site.