Digital tuning of selectable markers

Abstract

The present invention provides host cells and vector systems that allow precise tuning of expression of selectable marker expression and increased integrated copy number of product genes so that host cell growth and expression of desired proteins or nucleic acid products from the host cell can be optimized.

Description

Atorney Docket No. CATA-43085.601DIGITAL TUNING OF SELECTABLE MARKERSCROSS REFERENCE TO RELATED APPLICATIONThe present application claims priority to U.S. Provisional Application No. 63 / 689,183, filed August 30, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTINGThe text of the computer readable sequence listing filed herewith, titled “CATA_43085_601_SequenceListing.xml”, created August 28, 2025, having a file size of 69,312 bytes, is hereby incorporated by reference in its entirety.FIELD OF THE INVENTIONThe present invention provides host cells and vector systems that allow enhanced integration and expression of desired proteins or nucleic acid products through independent and tunable control of selection marker copy number.BACKGROUND OF THE INVENTIONTherapeutic recombinant products are extensively used for human medical purposes. Recombinant proteins can be expressed in various types of living organisms. CHO and HEK cells are some of the most commonly used host cells for the manufacture of bi opharmaceuti cal s .Biopharmaceuticals are manufactured in living cells and require specialized infrastructure. The cost of building and maintaining facilities containing commercial scale bioreactors is very high. Moreover, the costs of goods for runs in commercial bioreactors is high due to the use of expensive components such as cell culture media and additives and the need to maintain the cultures in a sterile environment. For these reasons, there is a need to produce high titer cell lines so that production from bioreactors can be maximized.SUMMARY OF THE INVENTIONThe present invention provides host cells and vector systems that allow enhanced integration and expression of desired proteins or nucleic acid products through independent and tunable control of selection marker copy number.Accordingly, in one aspect, the present invention provides a host cell comprising: a plurality of docking sites integrated into the genome of the host cell, each docking siteAtorney Docket No. CATA-43085.601 comprising at least one dock site insertion element; at least integrated first nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a first protein or nucleic acid of interest; and at least integrated second nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion and encoding a first selectable marker; wherein the at least integrated first nucleic acid constructs and the at least second integrated nucleic acid constructs are integrated at the plurality of docking sites at a ratio of first nucleic acid constructs to second nucleic acid constructs of from 500: 1 to 1 :500.In some embodiments, the first nucleic acid constructs do not comprise a selectable marker. In some embodiments, the second nucleic acid constructs do not comprise an additional protein or product of interest in addition to the selectable marker. In some embodiments, the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs.The present invention is not limited to the use of any particular selectable marker(s). In some embodiments, the selectable marker is GS. In some embodiments, the selectable marker is DHFR.In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200:1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 10: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 20: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 30: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 40: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 50: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 10: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 20: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 30: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 40: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 50: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 20: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to secondAtorney Docket No. CATA-43085.601 nucleic acid constructs is from 15: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 12: 1 to 7: 1.In some embodiments, the host cell further comprises at least integrated third nucleic acid constructs (or fourth, fifth, sixth, seventh, or eight constructs, etc.) comprising at least one insertion element compatible with the dock site insertion element and encoding a second protein or nucleic acid of interest. In some embodiments, the ratio of third nucleic acid constructs (or fourth, fifth, sixth, seventh, or eight constructs, etc.) to second nucleic acid constructs is selected from the group consisting of 200: 1 to 5:l, 200: 1 to 10: 1, 200: 1 to 20:1, 200: 1 to 30: 1, 200:1 to 40:1, 200: 1 to 50: 1, 100:1 to 5: 1, 100: 1 to 10:1, 100: 1 to 20: 1, 100:1 to 30: 1, 100: l to 40: l, 100: l to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1, and 12: 1 to 7: 1.In some embodiments, the first protein of interest is selected from the group consisting of an antigen binding protein or portion thereof, an enzyme, and a growth factor receptor or agonist or antagonist thereof. In some embodiments, the first and second proteins of interest are subunits of a multi-subunit protein. In some embodiments, one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain. In some embodiments, the first and / or second proteins of interest are subunits of a viral particle or the first and / or second nucleic acids of interest are part of a viral genome.In some embodiments, the at least first nucleic acid constructs further comprise at least the following elements in operable association in 5’ to 3?order: an internal promoter sequence; a nucleic acid sequence encoding the first protein of interest operably linked to the internal promoter; and a poly A signal sequence.In some embodiments, the at least second nucleic acid constructs further comprise at least the following elements in operable association in 5' to 3’ order: an internal promoter sequence; a nucleic acid sequence encoding the selectable marker operably linked to the internal promoter; and a poly A signal sequence.In some embodiments, the at least second nucleic acid constructs comprise at least the following elements in operable association in 5 ‘ to 3’ order: the nucleic acid sequence encoding the selectable marker; and a poly A signal sequence; wherein the nucleic acid sequence encoding the selectable marker is not operably linked to a promoter.In some embodiments, the at least second nucleic acid constructs do not include a poly A tail operably linked to the nucleic acid sequence encoding the selectable marker and / or do not include a promoter sequence operably linked to the nucleic acid sequence encoding the selectable marker.Atorney Docket No. CATA-43085.601In some embodiments, the nucleic acid expression constructs are provided in a vector. In some embodiments, the vector is a plasmid vector.In some embodiments, the host cell comprises a nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the dock site. In some embodiments, the nucleic acid construct encoding an enzy me that facilitates insertion of the nucleic acid expression construct at the dock site is provided in a vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the enzyme is selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.In some embodiments, the host cell genome comprises from 5 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 5 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 5 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 25 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 25 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 25 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the integrated docking sites are independently positioned throughout the host cell genome. In some embodiments, the host cell genome comprises from 50 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 50 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 50 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element.In some embodiments, the host cell comprises from 20 to 10,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 1,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 500 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 250 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 200 copies of theAtorney Docket No. CATA-43085.601 at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 10,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 1,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 500 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 250 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 200 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 10.000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 1,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 500 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 250 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 200 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios.In some embodiments, the dock site insertion element is targeted by enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase. In some embodiments, the dock site insertion element is selected from the group consisting of a recombinase dock site insertion element and a HDR dock site insertion element. In some embodiments, the dock site insertion element is a recombinase dock site insertion element. In some embodiments, the recombinase dock site insertion element comprises an atachment site (at). In some embodiments, the atachment site (at) is selected from the group consisting of attB and atP and atR and atL. In some embodiments, the recombinase dock site insertion element comprises a LoxP sequence. In some embodiments, the recombinase dock site insertion element is a Flp Recombination Target (FRT) site. In some embodiments, the dock site insertion element is a HDR dock site insertion element. In some embodiments, the HDR dock site insertion element comprises one or two dock site homology' arms. In some embodiments, the HDR dock site insertion element further comprises one or more sequences homologous to a guide RNA sequence. In some embodiments, the dock site homology arms are from about 30 to 1000 bases in length. In some embodiments, the integrase dock siteAtorney Docket No. CATA-43085.601 insertion element comprises an AAVS1 safe harbor locus sequence. In some embodiments, each docking site is flanked by exogenous integrating vector sequences. In some embodiments, the exogenous integrating vector sequences are selected from the group consisting of viral vector sequences and transposon vector sequences.In some embodiments, the host cell further comprises an expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase. In some embodiments, the expression construct encoding an exogenous enzy me selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is provided in an episomal expression vector. In some embodiments, the expression construct encoding an exogenous enzy me selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is integrated into the host cell genome.In some embodiments, the dock site insertion element is positioned to facilitate cassete exchange. In some embodiments, each docking site comprises two dock site insertion elements. In some embodiments, the two dock site insertion elements are positioned to facilitate cassete exchange. In some embodiments, the two dock site insertion elements flank sequences encoding a selectable marker, an enzyme, or a combination thereof.In some embodiments, the host cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells. HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 line transformed by SV40. baby hamster kidney cells, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, VERO cells, NSO cells, and human hepatoma line cells. In some embodiments, the host cell is selected from the group consisting of a Chinese Hamster Ovary (CHO) cells, a HEK 293 cells and a CAP cells. In some embodiments, the host cell is not a CHO cell. In some embodiments, the host cell is a GS knockout cell line. In some embodiments, the host cell is a DHFR knockout cell line.In a second aspect, the present invention provides a cell culture comprising host cells as described above.In a third aspect, the present invention provides processes for producing a protein of interest comprising culturing host cells as described above (or a cell culture comprising the host cells) under conditions that the protein(s) of interest are expressed and purifying the protein(s) of interest from the host cell culture. In some embodiments, the host cells areAtorney Docket No. CATA-43085.601 grown in a medium comprising an inhibitor of the selectable marker. In some embodiments, the selectable marker is GS and the inhibitor is phosphinothricin or methionine sulphoximine (Msx). In some embodiments, the selectable marker is DHFR and the inhibitor is methotrexate.In a fourth aspect, the present invention provides methods comprising introducing at least first nucleic acid constructs encoding a first protein or nucleic acid of interest and second nucleic acid constructs encoding a selectable marker at a ratio of first nucleic acid constructs to second nucleic acid constructs selected from the group consisting of 5000: 1 to 5:1, 5000:1 to 10:1, 5000:1 to 20:1, 5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to 50:1, 4000:1 to5:1, 4000:1 to 10:1, 4000:1 to 20:1, 4000:1 to 30:1, 4000:1 to 40:1, 4000:1 to 50:1.3000:1 to5:1, 3000: 1 to 10: 1, 3000: 1 to 20: 1, 3000: 1 to 30: 1, 3000: 1 to 40: 1.3000: 1 to 50: 1.2000: 1 to5:1, 2000:1 to 10:1, 2000:1 to 20:1, 2000:1 to 30:1, 2000:1 to 40:1, 2000:1 to 50:1, 1000:1 to5:1, 1000:1 to 10:1, 1000:1 to 20:1, 1000: 1 to 30:1, 1000:1 to 40:1, 1000:1 to 50:1, 500:1 to 5:1, 500:1 to 10:1, 500:1 to 20:1, 500:1 to 30:1, 500:1 to 40:1, 500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1,200:1 to 20:1.200:1 to 30:1,200:1 to 40:1.200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1, 20:1 to 5:1, 15:1 to 5:1, and 12:1 to 7:1 into ahost cell having genome comprising from 1, 5, 10, 20, 50 or 100 to 100, 200, 250, 500, 1000 or 10,000 integrated docking sites, each docking site comprising at least one dock site insertion element and the nucleic acid constructs each comprising at least one insertion element compatible with the at least one dock site insertion element in the integrated docking sites.In some embodiments, the first nucleic acid constructs do not comprise a selectable marker. In some embodiments, the second nucleic acid constructs do not comprise an additional protein or product of interest in addition to the selectable marker. In some embodiments, the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs.In some embodiments, the methods further comprise introducing at least a third nucleic acid constructs (or fourth, fifth, sixth, seventh, or eighth constructs, etc.) into the host cell, wherein the ratio of third nucleic acid constructs (or fourth, fifth, sixth, seventh, or eighth constructs, etc.) to second nucleic acid constructs is selected from the group consisting of 5000:1 to 5:1, 5000:1 to 10:1, 5000:1 to 20:1, 5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to50:1,4000:1 to 5:1, 4000:1 to 10:1,4000:1 to 20:1,4000:1 to 30:1, 4000:1 to 40:1.4000:1 to50:1, 3000:1 to 5:1, 3000:1 to 10:1, 3000:1 to 20:1, 3000:1 to 30:1.3000:1 to 40:1.3000:1 to50:1, 2000:1 to 5:1, 2000:1 to 10:1, 2000:1 to 20:1, 2000:1 to 30:1, 2000:1 to 40:1, 2000:1 toAtorney Docket No. CATA-43085.60150: 1, 1000:1 to 5: 1, 1000: 1 to 10: 1, 1000: 1 to 20: 1, 1000:1 to 30: 1, 1000: 1 to 40:1, 1000: 1 to 50: 1, 500: 1 to 5: 1, 500: 1 to 10: 1, 500: 1 to 20: 1, 500: 1 to 30: 1, 500: 1 to 40: 1, 500: 1 to 50: 1, 200: 1 to 5: 1, 200: 1 to 10: 1, 200: 1 to 20: 1, 200: 1 to 30: 1, 200: 1 to 40: 1, 200: 1 to 50: 1, 100: 1 to 5: 1, 100:1 to 10:1, 100: 1 to 20: 1, 100: 1 to 30: 1, 100: 1 to 40: 1, 100: 1 to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1, and 12: 1 to 7: 1.In some embodiments, the first protein of interest is selected from the group consisting of an antigen binding protein or portion thereof, an enzyme, and a growth factor receptor or agonist or antagonist thereof. In some embodiments, the first and second proteins of interest are subunits of a multi-subunit protein. In some embodiments, the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain. In some embodiments, the first and / or second proteins of interest are subunits of a viral particle or the first and / or second nucleic acids of interest are part of a viral genome. In some embodiments, the first nucleic acid constructs encode at least a second protein of interest in addition to the first protein of interest. In some embodiments, the first protein of interest and second protein of interest are encoded in a bicistronic construct. In some embodiments, the first and second proteins of interest are subunits of a multi-subunit protein. In some embodiments, one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.In some embodiments, the at least first nucleic acid constructs further comprise at least the following elements in operable association in 5’ to 3’ order: an internal promoter sequence; a nucleic acid sequence encoding the first protein of interest operably linked to the internal promoter; and a poly A signal sequence.In some embodiments, the at least second nucleic acid constructs further comprise at least the following elements in operable association in 5’ to 3’ order: an internal promoter sequence; a nucleic acid sequence encoding the selectable marker operably linked to the internal promoter; and a poly A signal sequence.In some embodiments, the nucleic acid construct comprises an extending packaging region (EPR) between the 5’ promoter and the selectable marker. In some embodiments, the EPR comprises multiple potential Kozak sequences and / or ATG translation start sites. In some embodiments, the promoter sequence is selected from the group consisting of SIN- LTR, SV40, EFla, E. coli lac, E. col trp, phage lambda PL, phage lambda PR. T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences. In some embodiments,Atorney Docket No. CATA-43085.601 the first promoter sequence is not a retroviral LTR promoter. In some embodiments, the nucleic acid expression constructs are provided in a vector. In some embodiments, the vector is a plasmid vector.In some embodiments, the host cell comprises a nucleic acid construct encoding an enzy me that facilitates insertion of the nucleic acid expression construct at the dock site. In some embodiments, the nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the dock site is provided in a vector. In some embodiments, the vector is a plasmid vector. In some embodiments, the enzyme is selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.In some embodiments, the host cell genome comprises from 5 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 5 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 5 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 25 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 25 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 25 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the integrated docking sites are independently positioned throughout the host cell genome. In some embodiments, the host cell genome comprises from 50 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 50 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In some embodiments, the host cell genome comprises from 50 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element.In some embodiments, the integrated docking sites are independently positioned throughout the host cell genome.In some embodiments, the dock site insertion element is targeted by enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase. In some embodiments, the dock site insertion element is selected from the group consisting of a recombinase dock site insertion element and a HDR dock site insertion element. In some embodiments, the dock site insertion element is a recombinase dock site insertion element. InAtorney Docket No. CATA-43085.601 some embodiments, the recombinase dock site insertion element comprises an atachment site (at). In some embodiments, the atachment site (at) is selected from the group consisting of attB and atP and atR and atL. In some embodiments, the recombinase dock site insertion element comprises a LoxP sequence. In some embodiments, the recombinase dock site insertion element is a Flp Recombination Target (FRT) site. In some embodiments, the dock site insertion element is a HDR dock site insertion element. In some embodiments, the HDR dock site insertion element comprises one or two dock site homology arms. In some embodiments, the HDR dock site insertion element further comprises one or more sequences homologous to a guide RNA sequence. In some embodiments, the dock site homology7arms are from about 30 to 1000 bases in length. In some embodiments, the integrase dock site insertion element comprises an AAVS1 safe harbor locus sequence.In some embodiments, each docking site is flanked by exogenous integrating vector sequences. In some embodiments, the exogenous integrating vector sequences are selected from the group consisting of viral vector sequences and transposon vector sequences.In some embodiments, the host cell further comprises an expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase. In some embodiments, the expression construct encoding an exogenous enzy me selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is provided in an episomal expression vector. In some embodiments, the expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is integrated into the host cell genome.In some embodiments, the dock site insertion element is positioned to facilitate cassete exchange. In some embodiments, each docking site comprises two dock site insertion elements. In some embodiments, the two dock site insertion elements are positioned to facilitate cassete exchange. In some embodiments, the two dock site insertion elements flank sequences encoding a selectable marker, an enzy me, or a combination thereof.In some embodiments, the host cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells. HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 line transformed by SV40, baby hamster kidney cells, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts. MDBK cells, VERO cells, NS0 cells, and human hepatoma line cells. In some embodiments, the host cell isAtorney Docket No. CATA-43085.601 selected from the group consisting of a Chinese Hamster Ovary (CHO) cells, a HEK 293 cells and a CAP cells. In some embodiments, the host cell is not a CHO cell. In some embodiments, the host cell is a GS knockout cell line. In some embodiments, the host cell is a DHFR knockout cell line. In some embodiments, the host cells are grown in a medium comprising an inhibitor of the selectable marker. In some embodiments, the selectable marker is GS and the inhibitor is phosphinothricin or methionine sulphoximine (Msx). In some embodiments, the selectable marker is DHFR and the inhibitor is methotrexate.In some embodiments, the methods further comprise the step of culturing the host cells under conditions such that first protein of interest (and / or second, third, fourth, fifth, sixth, seventh, eighth, etc. protein of interest) is expressed and purifying the protein(s) of interest from the host cell culture.In a fifth aspect, the present invention provides host cells made by any of the foregoing methods.In a sixth aspect, the present invention provides a host cell comprising: integrated first nucleic acid constructs encoding a first protein or nucleic acid of interest: and integrated second nucleic acid constructs encoding a first selectable marker; wherein the at least integrated first nucleic acid constructs and the at least second integrated nucleic acid constructs are integrated into the genome of the host cell at a ratio selected from the group consisting of 5000:1 to 5:1, 5000: 1 to 10:1, 5000:1 to 20: 1, 5000:1 to 30:1, 5000:1 to 40:1. 5000: 1 to 50: 1.4000: 1 to 5: 1, 4000: 1 to 10: 1, 4000: 1 to 20: 1.4000: 1 to 30: 1, 4000: 1 to 40: 1,4000:1 to 50:1,3000:1 to 5:1, 3000:1 to 10:1, 3000:1 to 20:1,3000:1 to 30:1,3000:1 to 40:1,3000:1 to 50:1, 2000:1 to 5:1, 2000:1 to 10:1, 2000:1 to 20:1, 2000:1 to 30:1, 2000:1 to 40:1,2000:1 to 50:1.1000:1 to 5:1, 1000:1 to 10:1, 1000:1 to 20:1, 1000:1 to 30:1. 1000:1 to 40:1,1000:1 to 50:1.500:1 to 5:1.500:1 to 10:1, 500:1 to 20:1.500:1 to 30:1, 500:1 to 40:1.500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1, 200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1, 200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100: 1 to 50:1, 20:1 to 5:1, 15:1 to 5:1, and 12:1 to 7:1.In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200:1 to 5:1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200:1 to 10:1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 20: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 30: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200:1 to 40:1. In some embodiments, the ratio of firstAtorney Docket No. CATA-43085.601 nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 50: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 10: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 20: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 30: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 40: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 50: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 20: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 15: 1 to 5: 1 . In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 12: 1 to 7: 1.In some preferred embodiments, the first nucleic acid constructs do not comprise a selectable marker. In some preferred embodiments, the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs. In some preferred embodiments, the second nucleic acid constructs further comprise a nucleic acid sequence encoding the first protein or nucleic acid of interest.The present invention is not limited to the use of any particular selectable marker(s). In some embodiments, the selectable marker is GS. In some embodiments, the selectable marker is DHFR.In some preferred embodiments, the host cells further comprise at least integrated third nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a second protein or nucleic acid of interest. In some preferred embodiments, the ratio of third nucleic acid constructs to second nucleic acid constructs is selected from the group consisting of 200: 1 to 5:1, 200: 1 to 10: 1, 200: 1 to 20:1, 200: 1 to 30: 1, 200:1 to 40:1, 200: 1 to 50: 1, 100:1 to 5: 1, 100: 1 to 10:1, 100: 1 to 20: 1, 100:1 to 30: 1, 100: l to 40: l, 100: l to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1, and 12: 1 to 7: 1.In some embodiments, the host cell comprises from 20 to 10,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 1,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 500 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20Atorney Docket No. CATA-43085.601 to 250 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 20 to 200 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 10,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 1,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 500 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 250 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 50 to 200 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 10,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 1,000 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 500 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 250 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios. In some embodiments, the host cell comprises from 100 to 200 copies of the at least first and second (or third, etc.) nucleic acid constructs at the indicated ratios.In some preferred embodiments, the first protein of interest is selected from the group consisting of an antigen binding protein or portion thereof, an enzyme, and a growth factor receptor or agonist or antagonist thereof. In some preferred embodiments, the first and second proteins of interest are subunits of a multi-subunit protein. In some preferred embodiments, one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain. In some preferred embodiments, the first and / or second proteins of interest are subunits of a viral particle or the first and / or second nucleic acids of interest are part of a viral genome.In some preferred embodiments, the first nucleic acid constructs encode at least a second protein of interest in addition to the first protein of interest. In some preferred embodiments, the first protein of interest and second protein of interest are encoded in a bicistronic construct. In some preferred embodiments, the first and second proteins of interest are subunits of a multi-subunit protein. In some preferred embodiments, one of the first andAtorney Docket No. CATA-43085.601 second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.In some preferred embodiments, the at least first nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: a promoter sequence; a nucleic acid sequence encoding the first protein of interest operably linked to the promoter; and a poly A signal sequence.In some preferred embodiments, the promoter sequence is selected from the group consisting of SV40, EFla, E. coll lac, E. coll trp, phage lambda PL, phage lambda PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.In some preferred embodiments, the at least second nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: a promoter sequence; the nucleic acid sequence encoding the first selectable marker operably linked to the promoter; and a poly A signal sequence.In some preferred embodiments, wherein the promoter sequence is selected from the group consisting of SIN-LTR, SV40, EFla, E. coli lac, E. coli trp, phage lambda PL, phage lambda PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.In some preferred embodiments, the at least second nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: the nucleic acid sequence encoding the selectable marker; and a poly A signal sequence, wherein the nucleic acid sequence encoding the selectable marker is not operably linked to a promoter.In some preferred embodiments, the at least second nucleic acid constructs do not include a polyA signal sequence operably linked to the nucleic acid sequence encoding the selectable marker and / or do not include a promoter sequence operably linked to the nucleic acid sequence encoding the selectable marker.In some preferred embodiments, the promoter sequence is not a retroviral LTR promoter.In some preferred embodiments, the host cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 line transformed by SV40, baby hamster kidney cells, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liverAtorney Docket No. CATA-43085.601 cells, mouse mammary tumor, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, VERO cells, NSO cells, and human hepatoma line cells.In some preferred embodiments, the host cell is selected from the group consisting of a Chinese Hamster Ovary (CHO) cell, aHEK 293 cell and a CAP cell. In some preferred embodiments, the host cell is not a CHO cell. In some preferred embodiments, the host cell is a GS knockout cell line. In some preferred embodiments, the host cell is a DHFR knockout cell line.In some preferred embodiments, the first and second nucleic acid constructs are integrated retroviral vectors.In some preferred embodiments, the first and second nucleic acid constructs are integrated transposon vectors.In some preferred embodiments, the first and second nucleic acid constructs are boat constructs that are integrated at docking sites that are integrated into the genome of the host cell.In some preferred embodiments, the present invention provides a cell culture comprising host cells of any of claims comprising the vectors in the rations described for this aspect.In some preferred embodiments, the present invention provides a process for producing a protein of interest comprising culturing host cells as described for this aspect under conditions such that the protein(s) of interest are expressed and purifying the protein(s) of interest from the host cell culture. In some preferred embodiments, the host cells are grow n in a medium comprising an inhibitor of the selectable marker. In some preferred embodiments, the selectable marker is GS and the inhibitor is phosphinothricin or methionine sulphoximine (Msx). In some preferred embodiments, the selectable marker is DHFR and the inhibitor is methotrexate.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1. Sequence of Recombinase Expression Plasmid. (SEQ ID NO:!).FIG. 2. Sequence of neatGS-Anyway Boat Plasmid. (SEQ ID NO:2).FIG. 3. Sequence of GSpA-Anyway Boat Plasmid. (SEQ ID NO:3).FIG. 4. Sequence of pGS-Anyway Boat Plasmid. (SEQ ID NO:4).FIG. 5. Sequence of neatNeo-Anyway Boat Plasmid. (SEQ ID NO:5).FIG. 6. Sequence of pNeo-Anyway Boat Plasmid. (SEQ ID NO:6).FIG. 7. Sequence of neatNeo-Highway-HC Boat Plasmid. (SEQ ID NO:7).Atorney Docket No. CATA-43085.601FIG. 8. Sequence of neatNeo-Highway LC Boat Plasmid. (SEQ ID NO: 8).FIG. 9. Sequence of GSpA-Null Boat Plasmid. (SEQ ID NO:9).DEFINITIONSTo facilitate understanding of the invention, a number of terms are defined below.As used herein, the term "host cell" refers to any eukaryotic cell (e.g.. mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.As used herein, the term “genome” refers to the genetic material (e.g., chromosomes) of an organism.The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, expression of a protein of interest in a host cell, expression of a ribozyme, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g.. reporter genes, selection marker genes, oncogenes, drug resistance genes, grow th factors, etc.), and noncoding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).As used herein, the term “product of interest” refers to a protein or nucleic acid product, such as a viral backbone or genome, encoded by a nucleic acid of interest. In some preferred embodiments, a plurality' of products of interest are expressed in a host cell.As used herein, the term “protein of interest” refers to a protein encoded by a nucleic acid of interest.Atorney Docket No. CATA-43085.601As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA or RNA sequence thus codes for the amino acid sequence.The term "promoter," "promoter element," or "promoter sequence" as used herein, refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is ty pically, though not necessarily, located 5' (z.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236: 1237

[1987] ). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell ty pes (for review see, Voss et al., Trends Biochem. Sci., 11 :287

[1986] ; and Maniatis et al., supra). For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema et al. , EMBO J. 4:761

[1985] ). Two other examples of promoter / enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor la gene (Uetsuki et al. , J. Biol. Chem., 264:5791

[1989] ; Kim etal.. Gene 91 :217

[1990] ; and Mizushima and Nagata, Nuc. Acids. Res., 18:5322

[1990] ) and the long terminal repeats of the Rous sarcoma virus (Gorman et al.. Proc. Natl. Acad. Sci. USA 79:6777

[1982] ) and the human cytomegalovirus (Boshart et al., Cell 41:521

[1985] ).As used herein, the term "promoter / enhancer" denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element, see above for aAtorney Docket No. CATA-43085.601 discussion of these functions). For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer / promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of that gene is directed by the linked enhancer / promoter.As used herein, the term '‘long terminal repeat” of "LTR" refers to transcriptional control elements located in or isolated from the U3 region 5' and 3' of a retroviral genome. As is known in the art, long terminal repeats may be used as control elements in retroviral vectors, or isolated from the retroviral genome and used to control expression from other types of vectors.As used herein, the terms "complementary'" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "5'-A-G-T-3'," is complementary’ to the sequence "3'-T-C-A-5'." Complementarity’ may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity’ between the nucleic acids. The degree of complementarity’ between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.The terms "homology’" and "percent identity " when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology’ (i.e., partial identity) or complete homology (i.e., complete identity). A partially complementary sequence is one that at least partially inhibits a completely complementary’ sequence from hybridizing to a target nucleic acid sequence and is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary’ sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe (i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest) will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permited; low stringency conditions require that the binding of two sequences toAtorney Docket No. CATA-43085.601 one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.The terms "in operable combination," "in operable order," and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.As used herein, the term “selectable marker ’ refers to a gene that encodes an enzymatic activity or other protein that confers the ability to grow in medium lacking what would otherwise be an essential nutrient; in addition, a selectable marker may confer resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.As used herein, the term “retrovirus" refers to a retroviral particle which is capable of entering a cell (i.e., the particle contains a membrane-associated protein such as an envelope protein or a viral G glycoprotein which can bind to the host cell surface and facilitate entry of the viral particle into the cytoplasm of the host cell) and integrating the retroviral genome (as a double-stranded provirus) into the genome of the host cell. The term "retrovirus" encompasses Oncovirinae (e.g., Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus (MoMSV), and Mouse mammary tumor virus (MMTV). Spumavirinae, and Lentiviridae (e.g.. Human immunodeficiency virus, Simian immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis-encephalitis virus; See. e.g., U.S. Pat. Nos. 5,994,136 and 6,013.516, both of which are incorporated herein by reference).As used herein, the term "retroviral vector" refers to a retrovirus that has been modified to express a gene of interest. Retroviral vectors can be used to transfer genes efficiently into host cells by exploiting the viral infectious process. Foreign or heterologous genes cloned (i.e., inserted using molecular biological techniques) into the retroviral genome can be delivered efficiently to host cells that are susceptible to infection by the retrovirus. Through well-known genetic manipulations, the replicative capacity of the retroviral genome can be destroyed. The resulting replication-defective vectors can be used to introduce new genetic material to a cell but they are unable to replicate. A helper virus or packaging cell line can be used to permit vector particle assembly and egress from the cell. Such retroviral vectors comprise a replication-deficient retroviral genome containing a nucleic acid sequence encoding at least one gene of interest (i.e., a polycistronic nucleic acid sequence can encodeAtorney Docket No. CATA-43085.601 more than one gene of interest), a 5' retroviral long terminal repeat (5' LTR); and a 3' retroviral long terminal repeat (3' LTR).As used herein, the term "lenti virus vector” refers to retroviral vectors derived from the Lentiviridae family (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus) that are capable of integrating into non-dividing cells (See, e.g., U.S. Pat. Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).As used herein, the term ’‘transposon” refers to transposable elements (e.g., Tn5, Tn7, and TnlO) that can move or transpose from one position to another in a genome. In general, the transposition is controlled by a transposase. The term "transposon vector," as used herein, refers to a vector encoding a nucleic acid of interest flanked by the terminal ends of transposon. Examples of transposon vectors include, but are not limited to, those described in U.S. Pat. Nos. 6,027,722; 5,958,775; 5,968,785; 5,965,443; and 5,719,055, all of which are incorporated herein by reference.As used herein, the term “adeno-associated virus (AAV) vector” refers to a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV- 2, AAV-3, AAV -4, AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-ty pe genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences.AAV vectors can be constructed using recombinant techniques that are known in the art to include one or more heterologous nucleotide sequences flanked on both ends (5' and 3') with functional AAV ITRs. In the practice of the invention, an AAV vector can include at least one AAV ITR and a suitable promoter sequence positioned upstream of the heterologous nucleotide sequence and at least one AAV ITR positioned downstream of the heterologous sequence. A "recombinant AAV vector plasmid" refers to one type of recombinant AAV vector wherein the vector comprises a plasmid. As with AAV vectors in general, 5' and 3' ITRs flank the selected heterologous nucleotide sequence.As used herein, the term “adenoviral vector” refers to a non-enveloped doublestranded DNA vector comprising an adenovirus backbone.As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their normal environment, isolated or separated. An "isolated nucleic acid sequence" is therefore a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are normally associated.Atorney Docket No. CATA-43085.601DETAILED DESCRIPTION OF THE INVENTIONThe present invention provides host cells and vector systems that allow precise tuning of expression of selectable marker expression so that host cell growth and expression of desired proteins or nucleic acid products from the host cell can be optimized. In some particularly preferred embodiments, an expression construct encoding a selectable marker, and most preferably where the selectable marker in the construct is not associated with a nucleic acid of interest or nucleic acid encoding a protein of interest, and an expression construct(s) encoding one or more gene of interest are introduced into host cells at defined ratios. In some particularly preferred embodiments, the host cell lines contain multiple dock sites for insertion of the nucleic acid constructs. Cell lines containing multiple dock sites and expression constructs for use with the cells are described in PCT / US21 / 35403 and PCT / US21 / 35404, both of which are incorporated by reference herein in their entirety7.The present invention addresses potential issues associated with optimization of host cell growth and expression of gene products (such as proteins of interest or nucleic acid products) from the host cell. For example, the present system optimizes selectable marker expression to increase growth rates, VCD, and pool stability7. The present system also optimizes selectable marker expression by minimizing transcript read through. Further, the system of the present invention can be infinitely tuned to provide the desired level of selectable marker expression and thus can be easily transferred to cell lines other than CHO cells. Finally, the system is useful for driving increased product of interest copy number. For example, without the digital selection allowed by the instant systems, for a given selectable marker cassete any cells with a threshold level of the constructs containing the selectable marker will survive. As a result, the probability of finding clones comprising a high number of the constructs can be relatively low. With the present system, the ratio of constructs encoding selectable markers and constructs without the selectable marker (i.e., constructs encoding a product of interest) can be tuned to greatly increase the probability of clones in the pools w ith a high copy number of the constructs encoding the product of interest. For example, if the given selectable marker cassete yields 20 construct copies on average in pools w hen present on all plasmids, the probability of isolating clones w ith 200 constructs is low7. However, with the digital selection allowed by the instant systems, the selectable marker cassete construct to product of interest construct is provided to the cells at a 1 : 10 ratio, most clones in the selected pools should have at least 200 product of interest constructs.Atorney Docket No. CATA-43085.601Accordingly, in some embodiments, the present invention provides host cells comprising: a plurality of docking sites integrated into the genome of the host cell, each docking site comprising at least one dock site insertion element; at least integrated first nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a first protein or nucleic acid of interest, and at least integrated second nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion and encoding a first selectable marker, wherein the at least integrated first nucleic acid constructs and the at least second integrated nucleic acid constructs are integrated at the plurality' of docking sites at a ratio of first nucleic acid constructs to second nucleic acid constructs of from 500: 1 to 1 :500. In some preferred embodiments, the first nucleic acid constructs do not comprise a selectable marker. In other preferred embodiments, the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs. In further embodiments, additional selectable markers (e.g., third, fourth, fifth or more) may be incorporated into constructs as needed.In some preferred embodiments, the host cells comprise multiple exogenous nucleic acids encoding multiple products of interest (e.g., a protein of interest of nucleic acid of interest), for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 products of interest.The present invention is not limited to the expression of any particular product of interest. In some preferred embodiments, the product or products of interest are proteins. In some preferred embodiments, one protein of interest may be expressed. In some preferred embodiments, two or more proteins of interest may be expressed. In some preferred embodiments, two proteins of interest may be expressed. In some preferred embodiments, three proteins of interest may be expressed. In some preferred embodiments, four proteins of interest may be expressed. In some preferred embodiments, five proteins of interest may be expressed. In some preferred embodiments, the protein of interest is selected from the group consisting of an Fc-fusion protein, an enzy me, an albumin fusion, a growth factor, a protein receptor, a single chain antibody (scFv), a single chain-Fc (scFv-Fc), a diabody, and minibody (scFv-CH3). Fab, single chain Fab (scFab). an immunoglobulin heavy chain, and an immunoglobulin light chain and other antigen binding proteins. In general, the protein or proteins of interest may be any pharmaceutical or industrial protein for which expression and production via a host culture is desired. In some preferred embodiments, the protein of interest is a biopharmaceutical protein. In some preferred embodiments, the protein of interest is an immunoglobulin heavy7and / or light chain. In some embodiments, the protein ofAtorney Docket No. CATA-43085.601 interest is an immunoglobulin fragment or single chain antibody. In some preferred embodiments, where expression of an immunoglobulin is desired, the host cell further comprises a third exogenous nucleic acid sequence encoding a second protein of interest operably linked to a promoter sequence and a secretion signal sequence, wherein the first protein of interest is an immunoglobulin light chain sequence and the second protein of interest is an immunoglobulin heavy chain sequence. Where more than one protein of interest is expressed in the host cell, the different proteins of interest may be expressed from different nucleic acid constructs (which are described in more detail below), or they may be provided in a one or more polycistronic nucleic acid constructs.In some preferred embodiments, the product or products of interest are nucleic acid products, for example nucleic acids that are or form part of a virus or exosome. In some preferred embodiments, the viral nucleic acids are selected from the group consisting of retroviral nucleic acids, lentiviral nucleic acids, adenoviral nucleic acids, and adeno- associated virus (AAV) nucleic acids. The viral nucleic acids may preferably encode a viral backbone, genome or other nucleic acid needed for viral replication or packaging. In some preferred embodiments, product of interest is a retroviral backbone or lentiviral backbone. In some preferred embodiments, the nucleic acid of interest may be co-expressed with a protein of interest, for example a viral capsid or envelope protein, such as a retrovirus, lentivirus or AAV capsid or envelope protein. In some preferred embodiments, where the viral backbone is a lentiviral or retroviral backbone, the capsid or envelope protein may be a protein that allows pseudotyping such as the VSV-G protein.In some preferred embodiments, the product of interest is one or more AAV nucleic acids. In some preferred embodiments, the AAV nucleic acids may preferably include a pHelper sequence and / or an adenoviral backbone sequence with or without a gene of interest. In some preferred embodiments, the AAV nucleic acid sequence(s) may be co-expressed with sequences of interest encoding AAV Rep and Cap proteins, which can, for example, be the first and second or second and third proteins or products of interest in the embodiments described above or below. Suitable AAV sequences are described, for example, in U.S. Pat. Nos. 5.622,856; 5.945.335; 6,001.650; 6,004.797; 6,027,931; 6.376,237; 6.365,403; 6,482,633; 6,897,063; 7,037,713; 7,638,120; 6,759,237; 8,906,675; 7,282,199; 7,906,111; and 9,790,472, each of which incorporated herein by reference in its entirety.In some preferred embodiments, one nucleic acid product of interest may be expressed. In some preferred embodiments, two or more nucleic acid products of interest may be expressed. In some preferred embodiments, two nucleic acid products of interest may beAtorney Docket No. CATA-43085.601 expressed. In some preferred embodiments, three nucleic acid products of interest may be expressed. In some preferred embodiments, four nucleic acid products of interest may be expressed. In some preferred embodiments, five nucleic acid products of interest may be expressed. Where more than one nucleic acid product of interest is expressed in the host cell, the different nucleic acids of interest may be expressed from different nucleic acid constructs (which are described in more detail below), or they may be provided in a one or more polycistronic nucleic acid constructs.In some preferred embodiments, the nucleic acid constructs are stably integrated into the genome of the host cell. In some preferred embodiments, the nucleic acid constructs are stably integrated into the genome of the host cell at a docking site as described in further detail below.The host cells may preferably comprise a plurality of integrated nucleic acid constructs. In some preferred embodiments, the present invention provides methods for integrating two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) nucleic acid constructs into a host cell line at a desired ratios, and host cells produced by such processes.The ratio of the integrated sequences in the host cell may be varied. In some embodiments, the ratio of first nucleic acid constructs (e.g., constructs that encode a protein of interest) to second nucleic acid constructs (e.g., constructs that encide the first selectable marker) is from 200: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 10: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 20: 1 . In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 30: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 40: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 50: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 10: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 20: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 30: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 40: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 50: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs isAtorney Docket No. CATA-43085.601 from 20: 1 to 5:1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 15: 1 to 5: 1. In some embodiments, the ratio of first nucleic acid constructs to second nucleic acid constructs is from 12: 1 to 7: 1.In some embodiments, the host cells comprise from about 50 to about 300 integrated first nucleic acid constructs (e.g., constructs that encode the product of interest) and from about 2 to about 30 integrated copies of the second nucleic acid constructs (e g., constructs that encode the selectable marker). In some embodiments, the host cells comprise from about 50 to about 250 integrated first nucleic acid constructs and from about 2 to about 25 integrated second nucleic acid constructs. In some embodiments, the host cells comprise from about 50 to about 200 integrated first nucleic acid constructs and from about 2 to about 25 integrated second nucleic acid constructs. In some embodiments, the host cells comprise from about 50 to about 200 integrated first nucleic acid constructs and from about 2 to about 20 integrated second nucleic acid constructs.Where third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, etc., nucleic acid constructs encoding additional products of interest are integrated into the host cell, their ratio to the second nucleic acid construct encoding a selectable marker may also range from 200: 1 to 5: 1, 200:1 to 10:1, 200: 1 to 20: 1, 200: 1 to 30: 1, 200: 1 to 40: 1, 200: 1 to 50: 1, 100: 1 to 5:1, 100: 1 to 10: 1, 100:1 to 20: 1, 100: 1 to 30: 1, 100:1 to 40:1, 100: 1 to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1 and 12: 1 to 7: 1.In some preferred embodiments, the present invention provides host cells comprising a plurality of docking sites integrated into the genome of the host cell, each docking site comprising at least one dock site insertion element; at least first nucleic acid constructs encoding a first protein or nucleic acid of interest and at least second nucleic acid constructs encoding a selectable marker at a ratio of first nucleic acid constructs to second nucleic acid constructs selected from the group consisting of 200: 1 to 5: 1, 200: 1 to 10: 1, 200: 1 to 20: 1, 200: 1 to 30: 1, 200:1 to 40:1, 200: 1 to 50: 1, 100:1 to 5: 1, 100: 1 to 10: 1, 100: 1 to 20: 1, 100:1 to 30: 1, 100: 1 to 40: 1, 100: 1 to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1 and 12: 1 to 7: 1. In some embodiments, the host cells comprise from about 50 to about 300 integrated first nucleic acid constructs (e.g., constructs that encode the product of interest) and from about 2 to about 30 integrated copies of the second nucleic acid constructs (e.g., constructs that encode the selectable marker). In some embodiments, the host cells comprise from about 50 to about 250 integrated first nucleic acid constructs and from about 2 to about 25 integrated second nucleic acid constructs. In some embodiments, the host cells comprise from about 50 to about 200 integrated first nucleic acid constructs and from about 2 to about 25 integratedAtorney Docket No. CATA-43085.601 second nucleic acid constructs. In some embodiments, the host cells comprise from about 50 to about 200 integrated first nucleic acid constructs and from about 2 to about 20 integrated second nucleic acid constructs.In further aspects, the present invention provides methods to express any two or more proteins or nucleic acids from corresponding nucleic acid constructs that can be incorporated into the docking sites in the host cell. In some embodiments, at least one of the proteins is an enzyme. In some preferred embodiments, the first and second proteins of interest are subunits of a multi -subunit protein, such as the heavy and light chains of an immunoglobulin. In some preferred embodiments, the nucleic acids of interest are viral nucleic acids, such as components of an adenoviral genome, AAV genome, or retroviral genome. In some embodiments, the present invention allows for co-integration of an expression construct encoding a first subunit of a protein of interest with an expression construct encoding a second subunit of a protein of interest where the expression construct encoding one of the subunits is provided at an increased ratio as compared to the expression construct encoding the other subunits(s). Exemplary ratios are provided below. In some preferred embodiments, the first and second proteins of interest are subunits or components of a viral particle, e.g., the REP and CAP proteins of an AAV vector. In some preferred embodiments, the nucleic acids of interest are viral nucleic acids, such as components of an adenoviral genome, AAV genome, or retroviral genome. In some embodiments, the present invention allows for co-integration of an expression construct encoding a first subunit of a viral particle with an expression construct encoding a second subunit of a viral particle where the expression construct encoding one of the subunits is provided at an increased ratio as compared to the expression construct encoding the other subunits(s). Exemplary ratios are provided below .In some preferred embodiments, the methods comprise introducing at least first nucleic acid constructs encoding a first protein or nucleic acid of interest and second nucleic acid constructs encoding a selectable marker at a ratio of first nucleic acid constructs to second nucleic acid constructs selected from the group consisting of 5000: 1 to 5: 1, 5000: 1 to 10:1.5000:1 to 20:1.5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to 50:1, 4000:1 to 5:1.4000:1 to10:1, 4000:1 to 20:1, 4000:1 to 30:1, 4000:1 to 40:1, 4000:1 to 50:1, 3000:1 to 5:1, 3000:1 to10:1, 3000:1 to 20:1, 3000:1 to 30:1, 3000:1 to 40:1, 3000:1 to 50:1, 2000:1 to 5:1, 2000:1 to10:1,2000:1 to 20:1,2000:1 to 30:1, 2000:1 to 40:1,2000:1 to 50:1, 1000:1 to 5:1. 1000:1 to10:1, 1000:1 to 20:1. 1000:1 to 30:1, 1000:1 to 40:1, 1000:1 to 50:1, 500:1 to 5:1, 500:1 to 10:1, 500:1 to 20:1, 500:1 to 30:1, 500:1 to 40:1, 500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1,Atorney Docket No. CATA-43085.601200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1, 200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1,20:1 to 5:1.15:1 to 5:1 and 12:1 to 7:1 into a host cell having genome comprising from 1, 10, 20, 50 or 100 to 200, 25, 500 or 1000 integrated docking sites, each docking site comprising at least one dock site insertion element and the nucleic acid constructs each comprising at least one insertion element compatible with the at least one dock site insertion element in the integrated docking sites. In some embodiments, the methods further comprise introducing third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, etc., nucleic acid constructs encoding additional products of interest into the host cell, wherein their ratio to the second nucleic acid construct encoding a selectable marker may also range from 5000:1 to 5:1, 5000:1 to 10:1, 5000:1 to 20: 1, 5000: 1 to 30:1, 5000:1 to 40:1.5000:1 to 50:1, 4000:1 to 5:1, 4000:1 to 10:1.4000:1 to 20:1, 4000:1 to 30:1,4000:1 to 40:1, 4000:1 to 50:1, 3000:1 to 5:1, 3000:1 to 10:1, 3000:1 to 20:1, 3000:1 to 30:1,3000:1 to 40:1, 3000:1 to 50:1, 2000:1 to 5:1, 2000:1 to 10:1, 2000:1 to 20:1, 2000:1 to 30:1,2000:1 to 40:1,2000:1 to 50:1, 1000:1 to 5:1, 1000:1 to 10:1, 1000:1 to 20:1. 1000:1 to 30:1,1000:1 to 40:1.1000:1 to 50:1, 500:1 to 5:1, 500:1 to 10:1, 500:1 to 20:1, 500:1 to 30:1, 500:1 to 40:1, 500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1, 200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1,200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1, and 20:1 to 5:1, 15:1 to 5:1 and 12:1 to 7:1.In some embodiments, the methods comprise introducing at least first nucleic acid constructs encoding a product of interest and second nucleic acid constructs encoding a selectable marker into host cells comprising docking sites compatible with the first and second constructs under conditions such that from about 50 to about 300 integrated first nucleic acid constructs (e.g., constructs that encode the product of interest) and from about 2 to about 30 integrated copies of the second nucleic acid constructs (e.g., constructs that encode the selectable marker) are integrated at the dock sites in the host cell. In some embodiments, the methods comprise introducing at least first nucleic acid constructs encoding a product of interest and second nucleic acid constructs encoding a selectable marker into host cells comprising docking sites compatible with the first and second constructs under conditions such that from about 50 to about 250 integrated first nucleic acid constructs and from about 2 to about 25 integrated copies of the second nucleic acid constructs are integrated at the dock sites in the host cell. In some embodiments, the methods comprise introducing at least first nucleic acid constructs encoding a product of interest and second nucleic acid constructs encoding a selectable marker into host cells comprising docking sites compatible with the first and second constructs under conditions such that fromAtorney Docket No. CATA-43085.601 about 50 to about 200 integrated first nucleic acid constructs and from about 2 to about 25 integrated copies of the second nucleic acid constructs are integrated at the dock sites in the host cell. In some embodiments, the methods comprise introducing at least first nucleic acid constructs encoding a product of interest and second nucleic acid constructs encoding a selectable marker into host cells comprising docking sites compatible with the first and second constructs under conditions such that from about 50 to about 200 integrated first nucleic acid constructs and from about 2 to about 20 integrated copies of the second nucleic acid constructs are integrated at the dock sites in the host cell.In some preferred embodiments, the present invention provides methods for production of a product(s) of interest. In these embodiments, the host cells as described above are cultured in a culture medium under conditions such that the first product of interest and / or two or more products of interest and / or an assembled complex thereof is produced. The product of interest is then isolated or purified from the culture medium. In some embodiments, the product (or products) of interest is operably associated with a signal sequence so the product (or products) are secreted into the culture medium and are then isolated therefrom.In some preferred embodiments, the host cells (and cultures of host cells) are engineered to comprise a plurality7of integrated docking sites. For example, in some preferred embodiments, the genomes of the host cells of the present invention preferably comprise from 1 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cells comprises from 1 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cells comprises from 5 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cells comprises from 5 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 5 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 10 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 20 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 50 to 250 integrated docking sites, each docking site comprising at least oneAtorney Docket No. CATA-43085.601 dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 10 to 200 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 20 to 200 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 50 to 200 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 5 to 100 integrated docking sites, each docking site comprising at least one dock site insertion element. In other preferred embodiments, the genome of the host cell comprises from 5 to 50 integrated docking sites, each docking site comprising at least one dock site insertion element. In some preferred embodiments, the integrated docking sites are independent integrated docking sites that are separated from one another and positioned at independent sites within the genome. For example, the integrated docking sites may preferably be spread across a number of chromosome sin the genome. In other embodiments, the integrated docking sites may be present as concatemers which comprise multiple copies of the same DNA sequence linked in series. Cell lines containing multiple dock sites and expression constructs for use with the cells are described in PCT / US21 / 35403 and PCT / US21 / 35404, both of which are incorporated by reference herein in their entirety.The integrated docking sites preferably comprise one or more insertion elements (which may be termed a “dock site insertion element.”) The dock site insertion elements are preferably nucleic acid sequences that facilitate insertion of a nucleic acid sequence encoding a protein of interest at the dock site. Nucleic acid constructs that can be inserted into the dock sites in the host cells of the present invention are described in detail below.The present invention is not limited to the use of any particular insertion elements. Indeed the use of a variety of insertion elements is contemplated. In some preferred embodiments, the insertion element is a recombinase dock site insertion element. Recombinase dock site insertion elements are nucleic acid sequences that are recognized and utilized by recombinase enzymes.For example, in some preferred embodiments, the recombinase dock site insertion element comprises an atachment site (at). In some particularly preferred embodiments, the attachment site is atP. These atachment sites are utilized by the PhiC31 integrase, which is a recombinase enzy me and which can be provided in the host cell via a vector in preferred embodiments. These dock sites serve as acceptors for integration of nucleic acid constructsAtorney Docket No. CATA-43085.601 comprising an atB atachment site. In other preferred embodiments, atR and atL atachment sites are utilizedIn other preferred embodiments, the recombinase dock site insertion element comprises an Flp Recombination Target (FRT) site. These sites are utilized by the enzyme flippase, which is a recombinase enzyme and which can be provided in the host cell via a vector in preferred embodiments. These dock sites serve as acceptors for integration of nucleic acid constructs comprising at the FRT site.In other preferred embodiments, the recombinase dock site insertion element comprises a LoxP site. These sites are utilized by the Cre recombinase which can be provided in the host cell via a vector in preferred embodiments. These dock sites serve as acceptors for integration of nucleic acid constructs comprising the LoxP site.In other preferred embodiments, the insertion element is an HDR (homology directed repair) dock site insertion element. HDR dock site insertion elements are nucleic acid sequences that provide an area of homology (a “homology arm"’) that base pair with corresponding homology arms on the nucleic acid construct that is inserted at the site. These systems are preferably used with endonucleases that introduce double stranded breaks at a targeted site or sites, preferably flanked by the homology arms. In some embodiments, the HDR dock site insertion element is an AAVS1 safe harbor locus. In these embodiments, the dock site is used utilized by the Rep 78 endonuclease (nickase) which may be introduced into the host cell via a vector. The Rep 78 protein nickase promotes site-specific integration of nucleic acid sequences bearing homology arms corresponding to the AAVS1 safe harbor locus.In other preferred embodiments, the HDR dock site insertion element comprises one or more homology arms that are exogenous sequences of from 30 to 1000 base pairs in length. These dock sites are preferably used in conjunction with CRISPR gene editing systems. In some embodiments, the dock site further comprises one or more sequences that are homologous to guide RNA sequences. In these embodiments, the nucleic acid construct that is inserted at the dock site preferably comprises homology arms that are homologous to and base pair with the homology arms in the dock site. For utilization with CRISPR gene editing systems, a CRISPR gene editing system-compatible nuclease is introduced into the host cell. The CRISPR gene editing system-compatible nuclease may be a wild-type endonuclease that creates a double-stranded break at a position determined by the guide RNA (and within the docking site) or a mutated nuclease (i.e., a nickase) that creates a single stranded break at a staggered positions within the dock site defined by two guide RNAs.Atorney Docket No. CATA-43085.601Suitable nucleases are described in detail below in the discussion of nucleic acid expression constructs.In some preferred embodiments, the docking site may preferably comprise a suitable promoter so that a promoter trap scheme is utilized when suitable nucleic acid constructs are introduced at the docking site. In some preferred embodiments, the trap is an ATG trap where the start codon for the selectable marker is provided in the dock in an exon that gets spliced onto the rest of the selectable marker gene once it is properly inserted into the dock. It is contemplated that random integration events are much less likely to express the marker. Suitable promoters include, but are not limited to, SIN-LTR, SV40, EFla, E. coli lac, E. coli trp, phage lambda PL, phage lambda PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences. In some preferred embodiments the promoter sequence is oriented at the dock site so that the promoter will drive expression from an inserted nucleic acid construct. In some preferred embodiments, the promoter is oriented 5’ to the docking site. In some particularly preferred embodiments, the promoter is a SIN LTR. In these embodiments, the SIN-LTR and EPR are positioned 5’ to the dock site and a SIN LTR is positioned 3’ to the dock site.The docking sites may be introduced into any suitable host cell line. Suitable host cell lines include, but are not limited to, Chinese hamster ovary cells (CHO-K1, ATCC CC1-61); bovine mammary epithelial cells (ATCC CRL 10274; bovine mammary epithelial cells); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for grow th in suspension culture; see, e.g., Graham et al., J. Gen Virol., 36:59

[1977] ); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251

[1980] ); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Set., 383:44-68

[1982] ); MRC 5 cells; FS4 cells; rat fibroblasts (208F cells); MDBK cells (bovine kidney cells); CAP (CEVEC's Amniocyte Production) cells; VERO cells, NS0 cells, and a human hepatoma line (Hep G2).In some particularly preferred embodiments, the host cells are modified so that they are deficient, or are naturally deficient, in an enzyme activity that is required for growth orAtorney Docket No. CATA-43085.601 survival of the cells in the presence of a selection agent and which is provided by the selectable marker. For example. Chinese Hamster Ovary (CHO) cells have been modified to be deficient for GS. In some preferred embodiments where vector includes a GS selectable marker, the host cell line is deficient in GS. In some particularly preferred embodiments, the GS deficient host cell line is the CHOZN® GS" ' cell line available from Merck KGaA. In other embodiments, where the selectable marker is, for example, DHFR, the cell line may preferably be deficient for DHFR activity (i.e., DHFR"). Suitable DHFR- cell lines include but are not limited to CHO-DG44 and derivatives thereof.The docking site sequences may be introduced into the host cells by any suitable genome modification system. In some preferred embodiments, the docking sites are incorporated into the host cells via the use of integrating vectors. The use of integrating vectors to introduce high copy numbers of a sequence of interest, such as a docking site, is described in detail in US Pat. Nos. 6,852,510 and 7,332,333 as well as US Publ. Nos. 20030092882, 20030224415, 20040235173 and 20050100952, all which are incorporated herein by reference in their entirety.According to the present invention, host cells such as those described above are transduced or transfected with integrating vectors comprising a dock site under conditions such that multiple copies of the dock site are integrated into the genome of the host cell. Examples of integrating vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adeno-associated viral vectors, and transposon vectors.In some preferred embodiments, nucleic acid constructs for expression of a product of interest are introduced into the host cell lines containing multiple docking sites. As discussed above, in preferred embodiments, the nucleic acid constructs preferably comprise nucleic acid sequences (which may be termed “expression construct insertion elements”) that are compatible with the dock site insertion elements as described above.Accordingly, in some preferred embodiments, the present invention provides nucleic acid expression constructs for use in expressing a product of interest in a host cell, and in particular to expression of two or more products of interest where the nucleic acid expression constructs encoding the two or more products of interest are integrated into the genome of the host cell at desired ratios as described in detail above.In some preferred embodiments, where the dock site does not comprise a promoter, the nucleic acid expression constructs, for example, comprise the following elements in operable association, most preferably in 5?to 3’ order:Atorney Docket No. CATA-43085.601 first promoter sequence - nucleic acid sequence encoding a first product of interest - poly A signal sequence. first promoter sequence - nucleic acid sequence encoding a second product of interest- poly A signal sequence. first promoter sequence - nucleic acid sequence encoding a third product of interest - poly A signal sequence. first promoter sequence - nucleic acid sequence encoding a fourth product of interest - poly A signal sequence.In some preferred embodiments, these nucleic acid constructs are used in defined ratios (as described above) to a nucleic acid construct that expresses a selectable marker sequence (5' to 3’ order): first promoter sequence - nucleic acid sequence encoding a selectable marker - poly A signal sequence.In some preferred embodiments, where the dock site comprises an exogenous promoter, the nucleic acid expression constructs, for example, comprise the following elements in operable association, most preferably in 5’ to 3’ order: nucleic acid sequence encoding a first product of interest - poly A signal sequence. nucleic acid sequence encoding a second product of interest - poly A signal sequence. nucleic acid sequence encoding a third product of interest - poly A signal sequence. nucleic acid sequence encoding a fourth product of interest - poly A signal sequence.In some preferred embodiments, the nucleic acid constructs encoding a product of interest are used in defined ratios (as described above) to a nucleic acid construct that expresses a selectable marker sequence (5’ to 3’ order):Atorney Docket No. CATA-43085.601 nucleic acid sequence encoding a selectable marker - poly A signal sequence, wherein the construct does not include a promoter operably linked to the nucleic acid sequence encoding a selectable marker.In some preferred embodiments, the nucleic acid constructs encoding a product of interest are used in defined ratios (as described above) to a nucleic acid construct that expresses a selectable marker sequence (5’ to 3’ order): nucleic acid sequence encoding a selectable marker, wherein the construct does not include a promoter operably linked to the nucleic acid sequence encoding a selectable marker or a polyA sequence in operable association with the nucleic acid sequence encoding a selectable marker.In some preferred embodiments, the nucleic acid constructs encoding a product of interest are used in defined ratios (as described above) to a nucleic acid construct that expresses a selectable marker sequence (5’ to 3’ order): promoter - nucleic acid sequence encoding a selectable marker polyA.In embodiments, where the selectable marker constructs inserted at the dock sites do not comprise a promoter, it is contemplated that expression of the genes encoded within the constructs may be driven by a promoter associated with the dock site. For example, when the dock site has been integrated into the host cell with a retroviral vector, the dock site into which the construct lacking a promoter is inserted can have retroviral LTR that serves as a promoter for the promoterless construct.In some particularly preferred embodiments, the nucleic acid expression constructs further comprises at least one expression construct insertion element at a position or positions selected from the group consisting of 5’ to the first promoter. 3’ to the poly A signal sequence, between the first promoter and the poly A signal sequence, and both 5' to the first promoter and 3’ to the poly A signal sequence. Suitable constructs are shown in the following non-limiting examples:Atorney Docket No. CATA-43085.601 expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - poly A signal sequence first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - poly A signal sequence - expression construct insertion element expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - poly A signal sequence - expression construct insertion element. first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence)- nucleic acid sequence encoding a first product of interest - poly A signal sequence. expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a second product of interest - poly A signal sequence first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a second product of interest - poly A signal sequence - expression construct insertion element expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a second product of interest - poly A signal sequence - expression construct insertion element. first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence)- expression construct insertion element -Atorney Docket No. CATA-43085.601- nucleic acid sequence encoding a second product of interest - poly A signal sequence.In some preferred embodiments, these nucleic acid constructs are used in defined ratios (as described above) to a nucleic acid construct wherein the nucleic acid sequence encoding a product of interest is replaced with a selectable marker sequence.In some preferred embodiments, the constructs may include nucleic acid sequences encoding multiple products of interest, for example 2, 3 ,4 or 5 (or more) products of interest. Suitable constructs for expressing two products of interest are show n in the following nonlimiting examples. These expression constructs may be used at different ratios in conjunction with expression constructs encoding an additional third product of interest, or as exemplified below, third and fourth products of interest. expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence or IRES - nucleic acid sequence encoding a second product of interest - WPRE (optional) - poly A signal sequence first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - intron (optional) - nucleic acid sequence encoding a second product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - intron (optional) - nucleic acid sequence encoding a second product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element.Atorney Docket No. CATA-43085.601 first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - expression construct insertion element - nucleic acid sequence encoding a first product of interest - WPRE - poly A signal sequence - third promoter sequence or IRES - nucleic acid sequence encoding a second product of interest - WPRE - poly A signal sequence expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - nucleic acid sequence encoding a second product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element. expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a first product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - intron- nucleic acid sequence encoding a second product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element. expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a third product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence or IRES - nucleic acid sequence encoding a fourth product of interest - WPRE (optional) - poly A signal sequence first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a third product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - intron (optional) - nucleic acid sequence encoding a fourth product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion elementAtorney Docket No. CATA-43085.601 expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a third product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - intron (optional) - nucleic acid sequence encoding a fourth product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element. first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) expression construct insertion element - nucleic acid sequence encoding a third product of interest - WPRE - poly A signal sequence - third promoter sequence or IRES - nucleic acid sequence encoding a fourth product of interest - WPRE - poly A signal sequence expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a third product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - nucleic acid sequence encoding a fourth product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element. expression construct insertion element - first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a third product of interest - WPRE (optional) - poly A signal sequence - third promoter sequence - intron- nucleic acid sequence encoding a fourth product of interest - WPRE (optional) - poly A signal sequence - expression construct insertion element.In some preferred embodiments, these nucleic acid constructs are used in defined ratios (as described above) to a nucleic acid construct that expresses a selectable marker sequence (5’ to 3’ order): first promoter sequence (optional depending on whether the dock site already comprises an exogenous promoter sequence) - nucleic acid sequence encoding a selectable marker - poly A signal sequence,Atorney Docket No. CATA-43085.601 wherein the construct comprises one or more expression construct insertion elements positioned at a position or positions selected from the group consisting of 5’ to the first promoter, 3’ to the poly A signal sequence, between the first promoter and the poly A signal sequence, and both 5’ to the first promoter and 3’ to the poly A signal sequence.In some embodiments, the mixtures of different constructs are utilized. In some preferred embodiments, the mixture of different constructs may comprise constructs as described above and constructs starting with the internal or second promoter (i.e., starting after and not including the selectable marker). It is contemplated that by using mixtures of constructs, some which do not include selectable markers, higher insertion rates may be achieved.As discussed above, any suitable products of interest may be expressed via the methos of the present invention. Exemplary products of interest include immunoglobulins, single chain antibodies, anticoagulant proteins, blood factor proteins, bone morphogenetic proteins, engineered protein scaffolds, enzymes, Fc fusion proteins, growth factors, hormones, interferons, interleukins, antigens, and thrombolytic proteins.In other preferred embodiments, the constructs of the present invention may be utilized to express viral vector sequences as the product of interest. Viral vector expression sequences that may be included in the constructs of the present invention include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors and AAV vectors as described elsewhere herein In some preferred embodiments, the retroviral vectors themselves include a nucleic acid sequence encoding a protein of interest as described above that is expressed by the vector. In some particularly preferred embodiments, the protein of interest that is expressed by the vector is an antigen sequence for use in a vaccine.In some preferred embodiments, the expression construct insertion elements are elements that find use in conjunction with or are recognized by transposons, integrases, recombinases or CRISPR systems. Suitable insertion elements include, but are not limited to, inverted terminal repeats, integrase atachment sites (at), and homologous recombination arms which in the context of the constructs described herein can be described as homologous recombination insertion elements.The nucleic acid constructs may be utilized with many different vectors and vectors systems. These vectors and vectors system may preferably be used to introduce the nucleic acid expression constructs into the host cells described above. Suitable vectors and vectors systems include, but are not limited to, viral gene insertion technologies such as retroviral,Atorney Docket No. CATA-43085.601 lentiviral and AAV systems as well as non-viral gene insertion technologies such as transposase, recombinase, integrase or CRISPR gene insertion. Specific examples of technologies / enzymes that can be used with nucleic acid constructs of the present invention include piggyback transposase systems, sleeping beauty transposase systems, Most transposase systems, Tol2 transposase systems, Leapin transposase systems, Lambda recombinase systems, FLP / FRT systems, Cre / Lox systems, MMLV integrase systems, Rep 78 integrase systems and CRISPR systems which can include nucleases or nickases as well as guide sequences. In some preferred embodiments, the system is a nucleic acid integration system with the proviso that the system is not a retroviral or lentiviral systems utilizing a retroviral or lentiviral LTR.As discussed above, in some preferred embodiments, the expression construct insertion element comprises an atachment site (at). In some particular preferred embodiments, the atachment site is atB. These atachment sites are utilized by the PhiC31 integrase, which is a recombinase enzy me and which can be provided in the host cell via a vector in preferred embodiments. These sites facilitate integration of the nucleic acid constructs into a dock site comprising atP atachment site. In other preferred embodiments, attR and attL atachment sites may be utilized.In other preferred embodiments, the expression construct insertion element comprises an Flp Recombination Target (FRT) site. These sites are utilized by the enzyme flippase, which is a recombinase enzyme and which can be provided in the host cell via a vector in preferred embodiments. These sites serve facilitate integration of nucleic acid constructs into dock sites comprising corresponding FRT sites.In other preferred embodiments, the expression construct insertion element comprises a LoxP site. These sites are utilized by the Cre recombinase which can be provided in the host cell via a vector in preferred embodiments. These sites facilitate integration of nucleic acid constructs into dock sites comprising corresponding LoxP sites.In other preferred embodiments, the expression construct insertion element is an HDR (homology directed repair) expression construct insertion element. HDR expression construct insertion elements are nucleic acid sequences that provide an area of homology (a ‘"homology arm”) that base pair with corresponding homology arms in the dock site. These systems are preferably used with endonucleases that introduce double stranded breaks at a targeted site or sites, preferably flanked by the homology arms. In some embodiments, the HDR expression construct insertion element comprises AAVS1 safe harbor locus homology arms. In these embodiments, the expression construct is specifically integrated in a dock site comprising theAtorney Docket No. CATA-43085.601AAVS1 safe harbor locus. The integration is facilitated by the Rep 78 endonuclease (nickase) which may be introduced into the host cell via a vector. The Rep 78 protein nickase promotes site-specific integration of nucleic acid sequences bearing homology arms corresponding to the AAVS1 safe harbor locus.In other preferred embodiments, the HDR expression construct insertion element comprises one or more homology arms that are exogenous sequences of from 30 to 1000 base pairs in length. These expression constructs are preferably used in conjunction with CRISPR gene editing systems. In these embodiments, the nucleic acid construct is inserted at dock sites that comprise homology7arms that are homologous to and base pair with the homology arms in the nucleic acid construct. For utilization with CRISPR gene editing systems, a CRISPR gene editing system-compatible nuclease is introduced into the host cell. The CRISPR gene editing system-compatible nuclease may be a wild-ty pe endonuclease that creates a double-stranded break at a position determined by the guide RNA (and within the docking site) or a mutated nuclease (i.e., a nickase) that creates a single stranded break at a staggered positions within the dock site defined by two guide RNAs. Suitable nucleases are described in detail below in the discussion of nucleic acid expression constructs.As discussed above, integration at the dock sites generally requires expression of an exogenous enzyme in the host cell. Suitable enzy mes include, but are not limited to, recombinases (including integrases), endonucleases, and nickases. Accordingly, in some embodiments, host cells of the present invention comprise an exogenous nucleic acid sequence (or expression construct) for expression of a recombinase (including integrases), an endonuclease, and a nickase In some embodiments, constructs for expressing the exogenous enzymes may be stably integrated into the genome of the host cell. In other embodiments, vectors for expressing the exogenous enzymes are transiently introduced into the host cell, for example with an extrachromosomal vector such as a plasmid.In some embodiments, both the vectors comprising exogenous enzy me and the vectors comprising the nucleic acid constructs for expression of the protein of interest are transiently introduced into the host cell, for example by transfection. In these embodiments, the preferred ratio of the vectors encoding the exogenous enzyme to the gene of interest vectors is from 1 : 1000 to 1 : 10. In some more preferred embodiments, the ratio is from 1 : 100 to 1:750. In some still more preferred embodiments, the ratio is from 1:400 to 1 :600. This is surprising as the literature for other integrase systems generally indicates that a higher level of vector encoding the exogenous enzyme to the gene of interest construct is required.Atorney Docket No. CATA-43085.601In some preferred embodiments, the integrase is the phiC31 integrase (BioCat GmbH, Heidelberg, DE or System Biosciences, Palo Alto, CA)). The phiC31 integrase is a sequence-specific recombinase encoded within the genome of the bacteriophage phiC31. The phiC31 integrase mediates recombination between two 34 base pair sequences termed atachment sites (at), one found in the phage and the other in the host. This serine integrase has been shown to function efficiently in many different cell types including mammalian cells. In the presence of phiC31 integrase, an atB- containing donor plasmid can be unidirectional integrated into a target genome through recombination at sites with sequence similarity to the native atP site (termed pseudo-atP sites). phiC31 integrase can integrate a plasmid of any size, as a single copy, and requires no cofactors. The integrated transgenes are stably expressed and heritable.Other suitable recombinase-based systems include CRISPR gene editing systems, CRE-Lox, FLP-FRT, and lambda recombinase systems.Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. The system consists of a single enzyme, Cre recombinase, which recombines a pair of short target sequences called the Lox sequences. This system can be implemented without inserting any extra supporting proteins or sequences. The Cre enzy me and the original Lox site called the LoxP sequence are derived from bacteriophage P 1. See, e.g.. Targeted integration of DNA using mutant lox sites in embryonic stem cells. Araki, et al. Nucleic Acids Res, Feb 1997, Vol. 25, Issue 4, pp. 868-872; High-Resolution Labeling and Functional Manipulation of Specific Neuron Types in Mouse Brain by Cre-Activated Viral Gene Expression. Kuhlman, et al. PLos One, Apr 2008, Vol. 3, e2005; When reverse genetics meets physiology’: the use of site-specific recombinases in mice. Tranche, et al. FEBS Leters. Aug 2002. Vol. 529. Issue 1, PP- 116-121.The FLP-FRT recombination system is another site-directed recombination technology7very’ conceptually similar to Cre-lox, with flippase (Flp) and the short flippase recognition target (FRT) site being analogous to Cre and loxP, respectively. See, e.g., Candice et al., Cre / loxP, Flp / FRT Systems and Pluripotent Stem Cell Lines (2012) Topics in Current Genetics, vol 23. The FLP-FRT technology can be an effective alternative to Cre-lox,Atorney Docket No. CATA-43085.601 and has also been used in conjunction with it, allowing for two separate recombination events to be controlled in parallel.The nucleic acid constructs of the present invention may be used in conjunction with CRISPR homologous recombination (HDR) systems. HDR is initiated by the presence of double strand breaks (DSBs) in DNA. The CRISPR / Cas9 system is preferably used to create targeted double stranded breaks via a guide RNA sequence so that the nucleic acid construct of the invention can be inserted. See, e.g.. Zhang et al., Efficient precise knockin with a double cut HDR donor after CRISPR / Cas9-mediated double-stranded DNA cleavage (2017) Genome Biol. 18:35; Mali et al., Cas9 as a versatile tool for engineering biology. Nature Methods 10, 957-963 (2013); Mali et al., RNA-Guided Human Genome Engineering via Cas9. Science339(6121), 823-826 (2013); Ran et al., Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 155(2), 479-480(2013). Suitable guide RNA sequences (gRNAs) may be designed as is known in the art. In some preferred embodiments, CRISPR systems for HDR utilize either one or two guide sequences. When one guide RNA sequence is utilized, it preferred to use a nuclease such as a Cas9 nuclease which makes a single double stranded break guided by the guide RNA sequence. When two guide sequences are utilized, it is preferred to use a nickase, which can be a mutated Cas9 nuclease which only makes single stranded breaks in the target DNA sequence guided by each of the guide RNA sequences. The single stranded breaks are preferably positioned at staggered points on different strands (i.e., the sense and antisense strands) of the target DNA sequence. This arrangement generally improves HDR efficiency.In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the contextAtorney Docket No. CATA-43085.601 of formation of a CRISPR complex, “target sequence'’ refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence”. In aspects of the invention, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination.Typically , in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20. 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity’ is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectorsAtorney Docket No. CATA-43085.601 providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to (‘’upstream” of) or 3' with respect to ("dow nstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.Non-limiting examples of Cas proteins useful in the present invention include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl. Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4. Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known: for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzy me directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D 10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. In aspects of the invention, nickases may be used for genome editing via homologous recombination.Atorney Docket No. CATA-43085.601In some preferred embodiments, the HDR insertion element comprises AAVS1 safe harbor locus homology arms and are used in conjunction with Rep 78 endonuclease (nickase). The adeno-associated virus serotype 2 (AAV 2) Rep 78 protein is a strand-specific endonuclease (nickase) that promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. See, e.g., Ramachandra et al., Efficient recombinase-mediated cassete exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors (2011) Nucleic Acids Research, 39(16):eI07; WO1998027207).As indicated above, in some preferred embodiments, the nucleic acid constructs of the present invention preferably comprise promoter sequences. Suitable promoter sequences include, but are not limited to the MMLV LTR promoter, the MoMuSV LTR promoter, the RSV LTR promoter, the SIN LTR promoter, the SV40 promoter, cytomegalovirus (CMV) immediate early promoter, herpes simplex virus (HSV) thymidine kinase promoter, alphalactalbumin promoter, mouse metallothionein-I promoter, dihydrofolate reductase promoter, the P-actin promoter, phosphoglycerol kinase (PGK) promoter, and the EFla promoter sequences, and combinations thereof. In some preferred embodiments, the promoter sequence is not a retroviral LTR promoter, i.e., the first promoter is a promoter sequence other than a retroviral LTR promoter sequence.In some preferred embodiments, one of the nucleic acid constructs encodes a selectable marker. Suitable selectable markers include but are not limited to glutamine synthetase (GS), dihydrofolate reductase (DHFR) and the like. These genes are described in U.S. Pat. Nos. 5,770,359; 5,827,739; 4,399,216; 4,634,665; 5,149,636; and 6,455,275; all of which are incorporated herein by reference. In some preferred embodiments, the selectable marker that is utilized is compatible with a host cell line that is deficient in the production of the enzyme encoded by the selectable marker nucleic acid sequence. Suitable host cell lines are described in more detail below. In other embodiments, the selectable marker is an antibiotic resistance marker, i.e., a gene that produces a protein that provides cells expressing this protein with resistance to an antibiotic. Suitable antibiotic resistance markers include genes that provide resistance to neomycin (neomycin resistance gene (neo)), hygromycin (hygromycin B phosphotransferase gene), puromycin (puromycin N-acetyl-transferase), and the like.In other embodiments of the present invention, where secretion of the protein of interest is desired, the nucleic acid constructs include a signal peptide sequence in operable association with the protein of interest. The sequences of several suitable signal peptides areAtorney Docket No. CATA-43085.601 known to those in the art, including, but not limited to, those derived from tissue plasminogen activator, human growth hormone, lactoferrin, alpha-casein, and alpha-lactalbumin.In other embodiments of the present invention, the nucleic acid constructs include an RNA export element (See, e.g., U.S. Pat. Nos. 5,914,267; 6,136,597; and 5,686,120; and WO99 / 14310, all of which are incorporated herein by reference) either 3' or 5' to the nucleic acid sequence encoding the protein of interest. It is contemplated that the use of RNA export elements allows high levels of expression of the protein of interest without incorporating splice signals or introns in the nucleic acid sequence encoding the protein of interest.In still other embodiments, the nucleic acid constructs include at least one internal ribosome entry site (IRES) sequence. The sequences of several suitable IRES's are available, including, but not limited to. those derived from foot and mouth disease virus (FDV), encephalomyocarditis virus, and poliovirus. The IRES sequence can be interposed between two transcriptional units (e.g., nucleic acids encoding different proteins of interest or subunits of a multi-subunit protein such as an antibody) to form a polycistronic sequence so that the two transcriptional units are transcribed from the same promoter.In some preferred embodiments, the nucleic acid constructs are incorporated into a nucleic acid expression vector. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively -linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility inAtorney Docket No. CATA-43085.601 recombinant DNA techniques are often in the form of plasmids. Other suitable vectors include, but are not limited to, cosmids and Yeast Artificial Chromosomes.Accordingly, suitable nucleic acid expression vectors include, but are not limited to, transposon vectors as described above, as well as plasmid vectors, retroviral vectors, lentiviral vectors, AAV vectors, phage vectors, etc.). It is contemplated that any vector maybe used as long as it is replicable and viable in the host. In preferred embodiments, the vectors are mammalian expression vectors that comprise among other elements described herein an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.Suitable plasmid vectors that may be adapted to incorporate the nucleic acid constructs of the present invention include specific plasmids systems for transposon vectors, FLP-FLT systems, Cre-lox systems, CRISPR-Cas9 systems, recombinase systems and integrase systems as well as plasmid vectors derived from pCIneo, pVAXl, pACT, Gatewayplasmids, pAdvantage. pBIND, pG51uc, pTNT, pTarget, pCat3. pSI, pCMV, pSV and the like.In some embodiments, the present invention provides host cells and host cell culture wherein the host cells express the protein of interest from the nucleic acid constructs described above. In preferred embodiment, the host cells a mammalian host cells. A number of mammalian host cell lines are know n in the art. In general, these host cells are capable of growth and survival when placed in either monolayer culture or in suspension culture in a medium containing the appropriate nutrients and grow th factors, as is described in more detail below. Typically, the cells are capable of expressing and secreting large quantities of a particular protein of interest into the culture medium. Examples of suitable mammalian host cells include, but are not limited to Chinese hamster ovary cells (CHO-K1, ATCC CC1-61); bovine mammary epithelial cells (ATCC CRL 10274; bovine mammary epithelial cells); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; see. e.g., Graham et al., J. Gen Virol.. 36:59

[1977] ); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251

[1980] ); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumorAtorney Docket No. CATA-43085.601(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68

[1982] ); MRC 5 cells; FS4 cells: rat fibroblasts (208F cells); MDBK cells (bovine kidney cells); CAP (CEVEC's Amniocyte Production) cells; and a human hepatoma line (Hep G2).In some particularly preferred embodiments, the host cells are modified so that they are deficient, or are naturally deficient, in an enzyme activity that is required for growth or survival of the cells in the presence of a selection agent and which is provided by the selectable marker. For example. Chinese Hamster Ovary (CHO) cells have been modified to be deficient for GS. In some preferred embodiments where vector includes a GS selectable marker, the host cell line is deficient in GS. In some particularly preferred embodiments, the GS deficient host cell line is the CHOZN® GS" ' cell line available from Merck KGaA. In other embodiments, where the selectable marker is, for example, DHFR, the cell line may preferably be deficient for DHFR activity (i.e., DHFR"). Suitable DHFR- cell lines include but are not limited to CHO-DG44 and derivatives thereof.The nucleic acid constructs and vectors of the present invention may be introduced into host cells by any suitable means such as by transfection, transformation or transduction. In some embodiments, after transfection or transduction, the cells are allowed to multiply, and are then trypsinized and re-plated. Individual colonies are then selected to provide clonally selected cell lines. In still further embodiments, the clonally selected cell lines are screened by Southern blotting or PCR assays to verify that the desired number of integration events has occurred. It is also contemplated that clonal selection allows the identification of superior protein producing cell lines. In other embodiments, the cells are not clonally selected following transfection.In some embodiments, the nucleic acid constructs encoding different proteins of interest are introduced into the host cells, for example by transfection or electroporation. The nucleic acid constructs encoding different proteins of interest can be introduced into the host cells at the same time or in a serial manner (e.g., a nucleic acid construct encoding a first protein of interest is introduced, a period of time is allowed to pass, and then a nucleic acid construct encoding a second protein of interest is introduced).It will be understood that the present invention is not limited to the use of host cells comprising docking sites and boat constructs as described above. Accordingly, in some embodiments, the present invention provides a host cell comprising: at least integrated first nucleic acid constructs encoding a first protein or nucleic acid of interest: and at least integrated second nucleic acid constructs encoding a first selectable marker; wherein the at least integrated first nucleic acid constructs and the at least second integrated nucleic acidAtorney Docket No. CATA-43085.601 constructs are integrated into the genome of the host cell selected from the group consisting of 5000:1 to 5:1, 5000:1 to 10:1.5000:1 to 20:1, 5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to 50:1,4000:1 to 5:1, 4000:1 to 10:1,4000:1 to 20:1,4000:1 to 30:1, 4000:1 to 40:1,4000:1 to50:1, 3000:1 to 5:1, 3000:1 to 10:1, 3000:1 to 20:1, 3000:1 to 30:1, 3000:1 to 40:1, 3000:1 to50:1, 2000:1 to 5:1, 2000:1 to 10:1, 2000:1 to 20:1, 2000:1 to 30:1, 2000:1 to 40:1, 2000:1 to50:1, 1000:1 to 5:1, 1000:1 to 10:1, 1000:1 to 20:1, 1000:1 to 30:1, 1000:1 to 40:1. 1000:1 to50:1.500:1 to 5:1.500:1 to 10:1, 500:1 to 20:1.500:1 to 30:1, 500:1 to 40:1.500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1, 200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1, 200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1, 20:1 to 5:1, 15:1 to 5:1, and 12:1 to 7:1. As above, in some embodiments, the first nucleic acid constructs do not comprise a selectable marker. In some embodiments, the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs. In some embodiments, the second nucleic acid constructs further comprise a nucleic acid sequence encoding the first protein or nucleic acid of interest. In some embodiments, the at least first and second nucleic acid constructs are integrated retroviral vectors. In some embodiments, the at least first and second nucleic acid constructs are integrated transposon vectors. In some embodiments, the at least first and second nucleic acid constructs are integrated at docking sites that are integrated into the genome of the host cell. It will be understood that the expression constructs described above can be adapted for use in, for example, retroviral vectors and transposon vectors by using elements appropriate to such vectors as is known in the art.In some embodiments of the present invention, following transformation of a suitable host strain and grow th of the host strain to an appropriate cell density in media, the protein of interest is secreted during culture of the host cells. In some preferred embodiments where amplifiable markers are utilized, it is contemplated that culture of transduced host cells in a medium comprising an inhibitor of the gene. Suitable inhibitors include, but are not limited to methotrexate for inhibition of DHFR and methionine sulphoximine (Msx) or phosphinothricin for inhibition of GS. It is contemplated that as concentrations of these inhibitors are increased in a cell culture system, cells with higher copy numbers of the amplifiable marker (and thus the genes or genes of interest) or which contain higher- producing insertions are selected.Accordingly, the host cells containing vectors as described above are preferably cultured according to methods known in the art. Suitable culture conditions for mammalian cells are well known in the art (See e.g., J. Immunol. Methods (1983) 56:221-234

[1983] ,Atorney Docket No. CATA-43085.601Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. Oxford University Press. New York

[1992] ).The host cell cultures of the present invention are prepared in a media suitable for the particular cell being cultured. Commercially available media such as ActiPro media (HyClone), ExCell Advanced Fed Batch Medium (SAFC), Ham's F10 (Sigma, St. Louis, MO). Minimal Essential Medium (MEM, Sigma). RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM. Sigma) are exemplary nutrient solutions. Suitable media are also described in U.S. Pat. Nos. 4,767,704: 4,657,866; 4,927,762; 5,122,469; 4,560,655; and WO 90 / 03430 and WO 87 / 00195; the disclosures of which are herein incorporated by reference. Any of these media may be supplemented as necessary' with serum, hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamycin (gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source. In some preferred embodiments where selectable markers such as GS are utilized, for example, the media will lack glutamine. Any other necessary' supplements may also be included at appropriate concentrations that would be known to those skilled in the art.The present invention also contemplates the use of a variety of culture systems (e.g., petri dishes, 96 well plates, roller botles, and bioreactors) for the transfected host cells. For example, the transfected host cells can be cultured in a perfusion system. Perfusion culture refers to providing a continuous flow of culture medium through a culture maintained at high cell density. The cells are suspended and do not require a solid support to grow on. Generally, fresh nutrients must be supplied continuously with concomitant removal of toxic metabolites and, ideally, selective removal of dead cells. Filtering, entrapment and microcapsulation methods are all suitable for refreshing the culture environment at sufficient rates.As another example, in some embodiments a fed batch culture procedure can be employed. In the preferred fed batch culture the mammalian host, cells and culture medium are supplied to a culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and / or product harvest before termination of culture. The fed batch culture can include, for example, a semi-continuous fed batch culture, wherein periodically whole culture (including cells and medium) is removed and replaced by fresh medium. Fed batch culture isAtorney Docket No. CATA-43085.601 distinguished from simple batch culture in which all components for cell culturing (including the cells and all culture nutrients) are supplied to the culturing vessel at the start of the culturing process. Fed batch culture can be further distinguished from perfusion culturing insofar as the supernatant is not removed from the culturing vessel during the process (in perfusion culturing, the cells are restrained in the culture by, e.g., filtration, encapsulation, anchoring to microcarriers etc. and the culture medium is continuously or intermitently introduced and removed from the culturing vessel). In some particularly preferred embodiments, the batch cultures are performed in roller botles.Further, the cells of the culture may be propagated according to any scheme or routine that may be suitable for the particular host cell and the particular production plan contemplated. Therefore, the present invention contemplates a single step or multiple step culture procedure. In a single step culture, the host cells are inoculated into a culture environment and the processes of the instant invention are employed during a single production phase of the cell culture. Alternatively, a multi-stage culture is envisioned. In the multi-stage culture cells may be cultivated in a number of steps or phases. For instance, cells may be grown in a first step or growth phase culture wherein cells, possibly removed from storage, are inoculated into a medium suitable for promoting growth and high viability. The cells may be maintained in the growth phase for a suitable period of time by the addition of fresh medium to the host cell culture.Fed batch or continuous cell culture conditions are devised to enhance growth of the mammalian cells in the growth phase of the cell culture. In the growth phase cells are grown under conditions and for a period of time that is maximized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dCh) and the like, are those used with the particular host and will be apparent to the ordinarily skilled artisan. Generally, the pH is adjusted to a level between about 6.5 and 7.5 using either an acid (e.g., CO2) or a base (e.g., Na2CCh or NaOH). A suitable temperature range for culturing mammalian cells such as CHO cells is between about 30° to 38° C and a suitable dCh is between 5-90% of air saturation.Following the polypeptide production phase, the polypeptide of interest is recovered from the culture medium using techniques that are well established in the art. The protein of interest preferably is recovered from the culture medium as a secreted polypeptide (e.g., the secretion of the protein of interest is directed by a signal peptide sequence), although it also may be recovered from host cell lysates. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The polypeptide thereafter is purified fromAtorney Docket No. CATA-43085.601 contaminant soluble proteins and polypeptides, with the following procedures being exemplary of suitable purification procedures: by fractionation on immunoaffinity or ionexchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and protein A Sepharose columns to remove contaminants such as IgG. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification. Additionally, the protein of interest can be fused in frame to a marker sequence that allows for purification of the protein of interest. Non-limiting examples of marker sequences include a hexa-histidine tag, which may be supplied by a vector, preferably a pQE- 9 vector, and a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (See e.g., Wilson et al., Cell, 37:767

[1984] ). One skilled in the art will appreciate that purification methods suitable for the polypeptide of interest may require modification to account for changes in the character of the polypeptide upon expression in recombinant cell culture.In some preferred embodiments, the nucleic acid constructs are incorporated into systems. In some embodiments, the systems comprise multiple nucleic acid constructs or vectors as described above which are intended for introduction into a host cell. In other preferred embodiments, the systems comprise one or more multiple nucleic acid constructs or vectors as described above which are intended for introduction into a host cell in addition to a nucleic acid or vector that encodes an enzyme that is necessary for incorporation of the nucleic acid constructs into a host cell genome. Exemplary7enzymes include, but are not limited to, transposes for use with transposon vector systems, integrases for use in systems which utilize integration sequences such as the PhiC31 system, MMLV systems, and the like, recombinases for use in vector systems such as Cre-loc, FLP-FRT and the like, and Cas9 nucleases for use in CRISPR based systems.EXPERIMENTALThe GPEx Lightning cell line development technology7, as well as other cell line development technologies, rely on the relatively weak expression of a selectable marker to achieve high integrated copy numbers of plasmids expressing a therapeutic GOT resulting in high volumetric titer in fed-batch production- see Table 1 for descriptions of all terms and used throughout this section . The GPEx Lightning system utilizes a parental Dock cell line containing 100-200 Dock sites throughout theAtorney Docket No. CATA-43085.601CHO genome. To create a cell line that produces high titer of a therapeutic protein, this parental Dock line is transfected with a plasmid expressing the PhiC31 Recombinase along with a separate Boat plasmid(s) containing a PhiC31attB sequence, the product GOI (s), and a relatively weak selectable marker expression cassette that lacks a promoter and polyadenylation sequence. The Recombinase protein mediates integration of Boat plasmids into the Dock sites through recombination between the attP and attB sequences creating a pool of cells with a range of integrated Boat copy number. Upon removal of glutamine from the media, cells with low Boat copy number do not express sufficient GS to grow in the absence of glutamine resulting in an enrichment for cells with relatively high Boat copy number- typically 30-100 copies.Like most cell line development technologies, the GPEx Lightning technology utilizes what we will refer to as “Analog selection” in which all Boat plasmids contain the same selectable marker expression cassette. The Analog selection method is limited in three ways. First, altering the expression from the selectable marker expression cassette to further increase integrated Boat copy number requires cloning of additional expression cassettes, empirical testing of each additional expression cassette, and iterative optimization which can be laborious, time consuming, unpredictable, and prone to failure. Second, if integration efficiency is improved, for example through increased Dock number in the GPEx Lightning Parental Cell Line or the use of higher fidelity recombinase or transposase enzymes, the integrated copy number achieved by a given selectable marker expression cassette is unlikely to produce higher integrated Boat copy numbers. Third, selectable marker expression cassettes may not perform the same in other medias or cell lines, ie HEK. CAP. or NSO, requiring additional time consuming and laborious testing and optimization.Here we provide evidence for an alternative method, termed “Digital selection”, in which only a small portion of Boat plasmids contain the selectable marker expression cassette while the remaining Boat plasmid lack a selectable marker expression cassette but still express the GOI. We demonstrate that the portion of Boat plasmids containing the selectable marker that is transfected into the GPEx Lightning Parental Cells is faithfully recapitulated in the integrated Boat copies after selection. By simply altering the portion of plasmids containing the selectable marker expression cassette prior to transfection we are able to independently modulate tire copy number and thus expression of the selectable marker and GOI. This results in a 2-3 fold increase integrated GOI copy number in Digital selection pools relative to Analog selection pools in which all Boat plasmids contain the same selectable marker expression cassette. Surprisingly, when comparing productivity of Digital and Analog selection pools with similar integrated Boat copy number, we find that Digital selection pools display different growth characteristics and are frequently more productive in terms of both volumetric titer and cell specific productivity than Analog selection pools.Table 1. Descriptions of Terms Used. Terms are listed in alphabetical order.Atorney Docket No. CATA-43085.601Attorney Docket No. CATA-43085.601EXAMPLE 1This example provides data examining the use of Digital selection process to reduce the copy number and thus expression of the selectable marker relative to the GOI copy number and expression. Boat plasmids encoding either GS or Neo selectable marker expression cassettes as well as a GOI, Anyway (encoding an Fc Fusion protein) are describedAtorney Docket No. CATA-43085.601 in Tables 1 and 2 and the sequences for all plasmids used in these Examples is provided in Figures 1-9 ate the end of this section. The expression strength of the selectable markers in these boats was altered to the inclusion / exclusion of a strong promoter, which increases transcription, and a poly adenylation sequence, which stabilizes the mRNA and increases translation.The Boats were combined as indicated in Table 3 (left 4 columns) and introduced into host GPEx Lightning Dock cells (Clone 1F7) along with a recombinase plasmid via transfection. 3 million cells were transfected in 300 ul CHOGro® Expression Medium (MirusBio) with 4 ng of recombinase plasmid and a total of 2 ug of the indicated GS and Neo containing Boats and 8 ul ExpiFectamine™ CHO Transfection Reagent (ThermoFisher Scientific) precomplexed in 200 ul of OptiPRO™ SFM media (ThermoFisher Scientific). After 2-4 hours of transfection in 50 ml TPP® Tubespin bioreactors (Techno Plastics Products) at 37 degrees C and 250 RPM, transfection media was removed by centrifugation and replaced with 10 ml FB media supplemented with 6mM glutamine and 1% ClonaCell™- CHO ACF Supplement (STEMCELL™ Technologies). After 3-5 days of recovery’, media was removed by centrifugation and all 21 pools were passaged to 2 million cells per ml in 10 ml of 3 different medias for selection:1. FB media lacking glutamine, indicated by “(-)Q ”, for GS selection2. FB media supplemented with 6 mM Q and 1 mg / ml G418 for neomycin-resistance selection3. FB media lacking glutamine and supplemented with 1 mg / ml G418 for dual GS and neomycin-resistance selectionPools were selected for 16 days with media changes every 4-6 days, cryopreserved, and analyzed by QPCR for copy number quantification. Primers sets used for QPCR were specific to atR, the GS CDS in the Boat plasmid (not endogenous GS), Neo, and an internal refence gene Bl,4-GalT. Delta-Ct values were converted to copy number using the following formula: copy number = 2AdeltaCt. The atR amplicon uses a forward primer that anneals in the Dock, upstream of the recombination site, and a reverse primer that anneals in the Boat downstream of the recombination site. Thus the attR amplicon is only formed when the Boat is properly inserted into the Dock. The atR amplicon is the same in all Boats so it is used to measure the total number of GS and Neo Boats that have been integrated into Dock sites. Since all Boat plasmids in this example also contain the GOI, atR number also represents the total GOI copy number. Due to variability in QPCR, the Neo primers used apparently underrepresented the number of Neo copies inserted- atR and Neo copy numbers in Pools 7,Attorney Docket No. CATA-43085.60114, and 21 selected with G418 should logically be equal but Neo values were consistently lower. We therefore performed regression analysis using the attR (59, 25, and 29 respectively) and Neo copy numbers (42, 13, and 16 respectively) from these pools and corrected the Neo copy numbers for all pools using the following equation: Corrected Neo copy number = -0.015 x (observed Neo copy number)2+ 2.0488 x(observed Neo copy number)Integrated copy number for GS, Neo, and attR as well as the portion of GS and Neo copies in the selected pools are shown in Table 3. Table 2. Naming Conventions and Genetic Architecture of Plasmids. Marker indicates the selectable marker used (GS or Neo). GOI indicates the product gene used (Anyway (Examples 1 and 2) or Highway HC and Highway LC (Example 3))Table 3. Total (attR), GS Boat, and Neo Boat inserted copy numbers for each Boat after (-)Q or G418 selection. Blank cells indicate pools did not survive selection. Note the pNeo Boat was used in pools 8-14 and 15-21. * indicates values are corrected (see above).Atorney Docket No. CATA-43085.601For (-)Q selection, the pools with GSpA (pools 8-14) and pGS (pools 15-21) showed that as the portion of these plasmids is reduced, the total integrated copy number, indicated by atR, increases. GS and Neo copy numbers indicate this was the combined result of decreasing GS copy numbers, as could be expected, but increasing Neo copy number. This result is surprising since Neo Boat integration does not contribute to resistance. Interestingly, the portion of integrated GS and Neo Boats in the selected pools aligned well with the portion of these Boats in the initial transfection mixture. Surprisingly, dilution of the GSpA and pGS Boat plasmids resulted in 2-4 fold higher total integrated copy number compared to the Analog selection pools (Pools 8 and 15).Similarly, with G418 selection, for the pools with neatNeo (pools 1-7) and pNeo (pools 8-21) as the portion of these plasmids is reduced, the total integrated copy number, indicated by atR, increases. GS and Neo copy numbers indicate this was the combined result of decreasing Neo copy numbers, as could be expected, but increasing GS copy number. This result is again surprising since GS Boat integration does not contribute to G418 resistance. Again, the portion of integrated GS and Neo Boats in the selected pools aligned well with the portion of these Boats in the initial transfection mixture. Again surprisingly, dilution of the neatNeo and pNeo Boat plasmids resulted in 0.3-0.7 fold higher total integrated copy number compared to the Analog selection pools (Pools 7, 14, and 21). This indicates that the increased total integrated copy number with dilution of the relevant Boat plasmid is not specific to the GS selection.To determine if dual selection for both Boat plasmids could achieve even higher integrated copy number, we also performed selection with media that lacked glutamine andAttorney Docket No. CATA-43085.601 contained 1 mg / ml G418. QPCR was performed and analyzed as above. Integrated copy number for GS, Neo. and attR as well as the portion of GS and Neo copies in the selected pools are shown in Table 4. Table 4. Total (attR), GS Boat, and Neo Boat inserted copy numbers for each Boat after dual (-)Q and G418 selection. Blank cells indicate pools did not survive selection. * indicates values are corrected (see above).Surprisingly, after selection several pools (Pools 3, 4, 19, 20) had higher total integrated copy number, indicated by attR, than with either selection method alone. Again, the portion of integrated GS and Neo Boats in the selected pools aligned well with the portion of these Boats in the initial transfection mixture. Together, these data indicate that:Atorney Docket No. CATA-43085.6011. Relative integration of GS and Neo Boats occurs in a predictable and consistent fashion based on their portions in the transfection mixture and these portions remain consistent in selected pools after selection with either or both selectable markers.2. By adjusting the portions of two plasmids containing two different selectable marker cassetes, but both expressing the same GOI, and performing selection for either selectable marker alone it is possible to achieve up to 4-fold higher total integrated copy number than pools produced using Analog selection with the same Boat plasmids.3. By adjusting the portions of two plasmids containing two different selectable marker cassetes, but both expressing the same GOI, and performing selection for both marker at once, it is possible to achieve even higher total integrated copy number than either individual Digital selection method or the best performing Analog selection method (Pool 1).Thus by simply decreasing the portion of the Boat plasmids that contain the relevant selection marker, pools with up to 4-fold higher total integrated copy number relative the traditional Analog selection approach. If it is even possible to achieve such a benefit using the Analog selection approach, it would require significant time and effort to create and test Boat constructs with further reduced expression of the selectable marker.EXAMPLE 2Results in Example 1 suggest that ratios between 50% and 1% of the GSpA plasmid might result in even higher total integrated copy number. In this experiment we also sought to determine if the Digital selection effect was influenced by the media used in selection. This experiment was performed as in Example 1 with the following exceptions:1. The portion of GSpA Boat plasmid ranged from 100% to .2% of the total plasmid transfected and the balance comprised of the neatNeo-Anyway Boat plasmid.2. Only (-)Q selection was performed.3. Selection was performed in both FB media and Cellist media, each lacking glutamine4. Neo copy number values were not corrected as in Example 1 due to the lack of a reference pool that contains only Neo Boat insertion events.Atorney Docket No. CATA-43085.601After selection, pools were analyzed by qPCR to determine the integrated copy number (atR), GS plasmid copy number, and Neo plasmid copy number as in Example 1 with the following exceptions:1. GS copy numbers were corrected using Pool 1 in which all inserted atR copies must also be GS copies. The atR copy number in this pool was 67 and the observed GS copy number was 128. We therefore adjusted all of the observed GS copy numbers by multiplying them by 67 / 128.2. Neo copy numbers were not adjusted as in Example 1 as no appropriate pools were created (where AtR copy number must equal Neo copy number) to make this such a correction. Neo copy numbers reported appear to overrepresent the actual Neo copy number. However we still observe the expected trend of increasing Neo copy number as the portion of Neo plasmid transfected increases in these pools.QPCR results in Table 5 again indicated that dilution of the GSpA Boat plasmid with a Boat plasmid that also contains the GOI but lacks the GS gene can lead to 2-3x more inserted copies after selection than Analog selection pools made using only the GSpA plasmid (Pool 1). Further, this effect was also demonstrated in Cellist media indicating that is not specific to FB media.Table 5. Total (atR), GS Boat (corrected), and Neo Boat inserted copy numbers for each Boat after (-)Q selection. FB and Cellist indicate the media used in selection. * indicates values are corrected (see above).Attorney Docket No. CATA-43085.601To determine the productivity of pools made using the Digital selection method compared to pools made using Analog selection, we performed generic fed-batch production on pools 1 -9 and another Analog selection pool with similar copy number to pools 3 and 5. This latter pool was made using the neat-GS expression cassette which has very weak expression resulting in high integrated copy number after selection relative to GS cassettes with stronger expression- compare attR copy# in Pools 1 to Pools 8 and 15 selected with (-)Q in Example 1. All vessels were inoculated at 1E6 cells per ml in 15 mis of fresh Acti media and cultured for 10 days, glucose was fed daily to maintain cultures above 4 g / L, and cultures were fed 2.5% CB7a and 0.25% CB7b on days 3 and 4, 3.5% / 0.35% on day 5, and 4% / 0.4% on days 6 through 9, respectively.Table 6. Results of 10-day fed-batch production in Acti media with variable CellBoost7a / 7b feeding strategy.Surprisingly, the top Digital selection pool produced approximately 40% higher titer than the neatGS Analog selection pool despite having very similar integrated copy numberAtorney Docket No. CATA-43085.601 and growth. This indicates that pools made using Digital selection have higher production capacity per integrated copy than pools made using Analog selection.To determine if the relatively higher titer from Digital selection pools compared to the equivalent copy number neat-GS Analog selection pool is specific to this media and feeding strategy we performed fed-batch production using 4 different medias and feeding strategies. Since pool 4 has slightly higher integrated copies than the neatGS Analog selection pool, we decided to use pool 5 for this study since it has slightly fewer integrated copies but still outperformed the neatGS Analog selection pool. All vessels were inoculated at 1E6 cells per ml in 15 mis of fresh media and glucose w as fed daily to maintain cultures above 4 g / L. The following generic fed-batch strategies were used:1. Acti media was supplemented with 0.25% CB4 and cultures were fed 2.5% CB7a and .25% CB7b daily starting on day 2.2. Cellist media cultures were fed 3% F7 Feed daily starting on day 3.3. FB cultures were fed 5% Modifeed daily starting on day 2.4. G12 was supplemented with 0.25% CB4 and cultures were fed 4% F12.7 on days1 and 2 and 5% daily starting on day 5.Table 7. Results of 7-day fed-batch production across 4 different feeding strategies. DS = representative Digital selection pool (Pool 5); AS = Analog selection pool which is the same 100% neatGS pool used in Table 6. Acti is strategy 1 above, Cellist is strategy 2, FB is strategy 3 and G12 is strategy 4,Atorney Docket No. CATA-43085.601Several unexpected differences were noted between the Digital selection and Analog selection pools. The Digital selection pool grew to a higher viable cell density and in turn had higher cumulative cell density in some media / feed strategies (Acti and FB) but equivalent (G12) or lower (Cellist) in others. However, titer was markedly higher for the Digital selection pool in all medias (albeit marginally for G12) despite its slightly lower number of integrated Boat copies (atR#) and Qp per copy was significantly higher in all medias.In summary7, this example shows that fine-tuning the portion of GSpA plasmid relative to the plasmid lacking GS can result in further increased integrated copy number. Suprisingly, productivity of pools produced with Digital selection can produce higher titer, Qp, and Qp per integrated copy compared to Analog selection pools with similar integrated copy number.EXAMPLE 3The above two examples expressed an Fc-fusion protein, Anyway. To determine if the Digital selection effect (increased total integrated copy number achieved by adjusting the portion of transfected plasmid that contains the selectable marker and GOI relative to a plasmid that only contains the GOI) could also be demonstrated with another product (GOI) we performed Digital selection with Boat plasmids expressing a IgGl -kappa monoclonal antibody (mAb). In this example we also tested another variation of Digital selection whereby the GOI(s) and selectable marker (GSpA) are not present on the same plasmid. We used a total of 3 Boat plasmids - one that expresses the mAh Heavy7Chain (HC) but not the relevant selectable marker (neatNeo-Highway-HC), one that expressed the mAb Light Chain (LC) but not contain the relevant selectable marker (neatNeo-Highway-LC), and one that expresses only the relevant selectable marker but does not express a GOI (GSpA-null). Transfection and selection were performed as in Example 1 with the following exceptions:1. Boat plasmid compositions described aboveAtorney Docket No. CATA-43085.6012. In addition to selection in FB media, selection was also performed in Acti media supplemented with 0.25% CB4.3. Instead of both plasmids containing the GOI, we created a new plasmid that lacks the GOI expression cassete but still contains GSpA. This plasmid was mixed with 2 other plasmids that both lack GS but express either mAh Heavy Chain or Light Chain.4. Pools were selected in both FB and Acti5. GS copy numbers were corrected using Pool 1 for FB media (Pools 1-7). In this pool all atR copies must also be GS copies. The atR copy number in this pool was 39 and the observed GS copy number was 106. We therefore adjusted all of the observed GS copy numbers by multiplying them by 39 / 126. Similarly for Pools 1 1-17, we used the Pool 11 atR (29) and observed GS copy number (44) to correct the GS copy by multiplying the observed GS copy numbers by 29 / 44.After selection, QPCR results in Table 8 indicated that dilution of the GSpA plasmid (which lacks a product gene) with 2 plasmids that express the mAh Heavy Chain or Light Chain (but lack GS) resulted in pools with 0.7-2.7x more inserted Boat copies (atR#) than pools made using only the GSpA plasmid (Pool 1 and 11). This result is consistent with Examples 1 and2. In both medias, as the portion of GSpA was reduced, we observed a significant increase in the total number of HC and LC copies inserted that was maximal in the 6.3% GSpA pool for both medias.Table 8. Total (atR), HC Boat, and LC Boat inserted copy numbers for each Boat after (-)Q selection. FB and Acti indicate the media used in selection. * indicates values are corrected (see above).Attorney Docket No. CATA-43085.601To determine if this in the increased copy number across this also results in increased productivity, as in Example 2, we performed fed-batch production on pools 11-17 using the same method as Example 2, Table 6.Similar to Example 2, we found that pools with higher total HC and LC copy number also had higher titer see table 9. Interestingly, cell growth, as indicated by maximum viable cell density and cumulative cell density, was also different across these pools. Table 9. Results of 10-day fed-batch production in Acti media with variable CB7a / CB7b feeding strategy.Taken together, this example demonstrates Digital selection can also be used to increase inserted Boat copy number and productivity7for mAb products by placing the gene(s) of interest and selectable marker on separate Boat plasmids and altering the portion of selectable marker Boat plasmid at transfection.

Claims

1. Atorney Docket No. CATA-43085.601CLAIMSWhat is claimed is:

1. A host cell comprising: a plurality’ of docking sites integrated into the genome of the host cell, each docking site comprising at least one dock site insertion element; at least integrated first nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a first protein or nucleic acid of interest; and at least integrated second nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion and encoding a first selectable marker; wherein the at least integrated first nucleic acid constructs and the at least second integrated nucleic acid constructs are integrated at the plurality of docking sites at a ratio of first nucleic acid constructs to second nucleic acid constructs of from 500: 1 to 1:500.

2. The host cell of claim 1, wherein the first nucleic acid constructs do not comprise a selectable marker.

3. The host cell of claim 1, wherein the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs.

4. The host cell of any of claims 1 to 3. wherein the second nucleic acid constructs further comprise a nucleic acid sequence encoding the first protein or nucleic acid of interest.

5. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 5: 1.

6. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 10: 1.

7. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 20: 1.Atorney Docket No. CATA-43085.6018. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 30: 1.

9. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 40: 1.

10. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 200: 1 to 50: 1.

11. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 5: 1.

12. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 10: 1.

13. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 20: 1.

14. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 30: 1 .

15. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 40: 1.

16. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 100: 1 to 50: 1.

17. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 20: 1 to 5:1.

18. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 15: 1 to 5: 1.Atorney Docket No. CATA-43085.60119. The host cell of any one of claims 1 to 4, wherein the ratio of first nucleic acid constructs to second nucleic acid constructs is from 12: 1 to 7: 1.

20. A host cell of any one of claims 1 to 19, further comprising at least integrated third nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a second protein or nucleic acid of interest.

21. The host cell of any one of claims 1 to 20, wherein the ratio of third nucleic acid constructs to second nucleic acid constructs is selected from the group consisting of 200:1 to 5: 1, 200: 1 to 10: 1, 200: 1 to 20: 1, 200: 1 to 30: 1, 200: 1 to 40: 1, 200: 1 to 50: 1, 100: 1 to 5: 1, 100: 1 to 10: 1, 100: 1 to 20:

1. 100: 1 to 30: 1, 100: 1 to 40:

1. 100: 1 to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1, and 12: 1 to 7: 1.

22. The host cell of any one of claims 1 to 21, wherein the first protein of interest is selected from the group consisting of an antigen binding protein or portion thereof, an enzyme, and a growth factor receptor or agonist or antagonist thereof.

23. The host cell of claim 22, wherein the first and second proteins of interest are subunits of a multi-subunit protein.

24. The host cell of claim 23, wherein one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.

25. The host cell of any one of claims 23 to 24, wherein the first and / or second proteins of interest are subunits of a viral particle or the first and / or second nucleic acids of interest are part of a viral genome.

26. The host cell of any one of claims 1 to 19. wherein the first nucleic acid constructs encode at least a second protein of interest in addition to the first protein of interest.

27. The host cell of claim 26, wherein the first protein of interest and second protein of interest are encoded in a bicistronic construct.Atorney Docket No. CATA-43085.60128. The host cell of any one of claims 26 to 27, wherein the first and second proteins of interest are subunits of a multi-subunit protein.

29. The host cell of claim 27, wherein one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.

30. The host cell of any one of claims 1 to 29, wherein the at least first nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: a promoter sequence; a nucleic acid sequence encoding the first protein of interest operably linked to the promoter; and a poly A signal sequence.

31. The host cell of any one of claims 1 to 29, wherein the at least second nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3:order: a promoter sequence; the nucleic acid sequence encoding the first selectable marker operably linked to the promoter; and a poly A signal sequence.

32. The host cell of claim 30, wherein the promoter sequence is selected from the group consisting of SV40, EFla, E. coli lac, E. coll trp, phage lambda PL, phage lambda PR, T3, T7. cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.

33. The host cell of claim 32, wherein the promoter sequence is selected from the group consisting of SIN-LTR, SV40. EFla, E. coli lac, E. coli trp, phage lambda PL, phage lambda PR. T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.

34. The host cell of any one of claims 1 to 29, wherein the at least second nucleic acid constructs comprise at least the following elements in operable association in 5?to 3?order: the nucleic acid sequence encoding the selectable marker; andAtorney Docket No. CATA-43085.601 a poly A signal sequence, wherein the nucleic acid sequence encoding the selectable marker is not operably linked to a promoter.

35. The host cell of any one of claims 1 to 29, wherein the at least second nucleic acid constructs do not include a polyA signal sequence operably linked to the nucleic acid sequence encoding the selectable marker and / or do not include a promoter sequence operably linked to the nucleic acid sequence encoding the selectable marker.

36. The host cell of any one of claims 1 to 35, wherein the promoter sequence is not a retroviral LTR promoter.

37. The host cell of any one of claims 1 to 36, wherein the nucleic acid expression constructs are provided in a vector.

38. The host cell of claim 37, wherein the vector is a plasmid vector.

39. The host cell of any one of claims 1 to 38, wherein the host cell comprises a nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the dock site.

40. The host cell of claim 38, wherein the nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the dock site is provided in a vector, as a protein, or as an mRNA.

41. The host cell of claim 40, wherein the vector is a plasmid vector.

42. The host cell of any one of claims 39 to 41, wherein the enzyme is selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.

43. The host cell of any one of claims 1 to 42, wherein the host cell genome comprises from 5 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element.Atorney Docket No. CATA-43085.60144. The host cell of any one of claims 1 to 43, wherein the host cell genome comprises from 5 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element.

45. The host cell of any one of claims 1 to 44, wherein the host cell genome comprises from 5 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element.

46. The host cell of any one of claims 1 to 45, wherein the integrated docking sites are independently positioned throughout the host cell genome.

47. The host cell of any one of claims 1 to 46, wherein the dock site insertion element is targeted by enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.

48. The host cell of any one of claims 1 to 47, wherein the dock site insertion element is selected from the group consisting of a recombinase dock site insertion element and a HDR dock site insertion element.

49. The host cell of claim 48, wherein the dock site insertion element is a recombinase dock site insertion element.

50. The host cell of claim 49, wherein the recombinase dock site insertion element comprises an atachment site (at).

51. The host cell of claim 45, wherein the atachment site (at) is selected from the group consisting of atB and atP and atR and atL.

52. The host cell of claim 49, wherein the recombinase dock site insertion element comprises a LoxP sequence.

53. The host cell of claim 50, wherein the recombinase dock site insertion element is a Flp Recombination Target (FRT) site.Atorney Docket No. CATA-43085.60154. The host cell of claim 48, wherein the dock site insertion element is a HDR dock site insertion element.

55. The host cell of claim 54, wherein the HDR dock site insertion element comprises one or two dock site homology arms.

56. The host cell of claim 55, wherein the HDR dock site insertion element further comprises one or more sequences homologous to a guide RNA sequence.

57. The host cell of any of claims 55 to 56, wherein the dock site homology' arms are from about 30 to 1000 bases in length.

58. The host cell of claim 57, wherein the integrase dock site insertion element comprises an AAV S 1 safe harbor locus sequence.

59. The host cell of any one of claims 1 to 58, wherein each docking site is flanked by exogenous integrating vector sequences.

60. The host cell of claim 59, wherein the exogenous integrating vector sequences are selected from the group consisting of viral vector sequences and transposon vector sequences.

61. The host cell of any one of claims 1 to 60, wherein the host cell further comprises an expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.

62. The host cell of claim 61, wherein the expression construct encoding an exogenous enzy me selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is provided in an episomal expression vector.

63. The host cell of claim 61, wherein the expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is integrated into the host cell genome.Atorney Docket No. CATA-43085.60164. The host cell of any one of claims 1 to 63, wherein the dock site insertion element is positioned to facilitate cassete exchange.

65. The host cell of any one of claims 1 to 64, wherein each docking site comprises two dock site insertion elements.

66. The host cell of claim 65, wherein the two dock site insertion elements are positioned to facilitate cassete exchange.

67. The host cell of any one of claims 65 to 66, wherein the two dock site insertion elements flank sequences encoding a selectable marker, an enzyme, or a combination thereof.

68. The host cell of any one of claims 1 to 67, wherein the host cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 line transformed by SV40, baby hamster kidney cells, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, VERO cells, NSO cells, and human hepatoma line cells.

69. The host cell of claim 68, wherein the host cell is selected from the group consisting of a Chinese Hamster Ovary (CHO) cell, a HEK 293 cell and a CAP cell.

70. The host cell of any one of claims 1 to 69, wherein the host cell is not a CHO cell.

71. The host cell of any one of claims 68 to 70, wherein the host cell is a GS knockout cell.

72. The host cell of any one of claims 68 to 70, wherein the host cell is a DHFR knockout cell.

73. A cell culture comprising host cells of any of claims 1 to 72.Atorney Docket No. CATA-43085.60174. A process for producing a protein of interest comprising culturing host cells according to any of claims 1 to 72 under conditions such that the protein(s) of interest are expressed and purifying the protein(s) of interest from the host cell culture.

75. The process of claim 74, wherein the host cells are grown in a medium comprising an inhibitor of the selectable marker.

76. The process of claim 74, wherein the selectable marker is GS and the inhibitor is phosphinothricin or methionine sulphoximine (Msx).

77. The process of claim 74, wherein the selectable marker is DHFR and the inhibitor is methotrexate.

78. A method comprising: introducing at least first nucleic acid constructs encoding a first protein or nucleic acid of interest and second nucleic acid constructs encoding a selectable marker at a ratio of first nucleic acid constructs to second nucleic acid constructs selected from the group consisting of 5000:1 to 5:1, 5000:1 to 10:1, 5000:1 to 20:1, 5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to 50:1,4000:1 to 5:1, 4000:1 to 10:1,4000:1 to 20:1,4000:1 to 30:1, 4000:1 to 40:1.4000:1 to50: 1.3000: 1 to 5: 1, 3000: 1 to 10:1, 3000: 1 to 20: 1, 3000: 1 to 30: 1.3000: 1 to 40: 1.3000: 1 to50:1,2000:1 to 5:1, 2000:1 to 10:1,2000:1 to 20:1,2000:1 to 30: 1, 2000:1 to 40:1,2000:1 to50:1, 1000:1 to 5:1, 1000:1 to 10:1, 1000:1 to 20:1, 1000:1 to 30:1, 1000:1 to 40:1, 1000:1 to50:1, 500:1 to 5:1, 500:1 to 10:1, 500:1 to 20:1, 500:1 to 30:1, 500:1 to 40:1, 500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1, 200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1, 200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1, 20:1 to 5:1, 15:1 to 5:1, and 12:1 to 7:1 into a host cell having genome comprising from 10 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element and the nucleic acid constructs each comprising at least one insertion element compatible with the at least one dock site insertion element in the integrated docking sites.

79. The method of claim 78, wherein the first nucleic acid constructs do not comprise a selectable marker.Atorney Docket No. CATA-43085.60180. The method of claim 78, wherein the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs.

81. The method of any one of claims 78 to 80, wherein the second nucleic acid constructs further comprise a nucleic acid sequence encoding the first protein or nucleic acid of interest.

82. The method of any one of claims 78 to 81, further comprising introducing at least a third nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a second protein or nucleic acid of interest into the host cell, wherein the ratio of third nucleic acid constructs to second nucleic acid constructs is selected from the group consisting of 5000:1 to 5:1, 5000:1 to 10:1, 5000:1 to 20:1, 5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to 50:1, 4000:1 to 5:1, 4000:1 to 10:1, 4000:1 to 20:1, 4000:1 to30:1, 4000:1 to 40:1, 4000:1 to 50:1, 3000:1 to 5:1, 3000:1 to 10:1, 3000:1 to 20:1, 3000:1 to30:1, 3000:1 to 40:1.3000:1 to 50:1, 2000:1 to 5:1, 2000:1 to 10:1, 2000:1 to 20:1.2000:1 to30:1, 2000:1 to 40:1,2000:1 to 50:1, 1000:1 to 5:1, 1000:1 to 10:1, 1000:1 to 20:1, 1000:1 to30:1, 1000:1 to 40:1, 1000:1 to 50:1, 500:1 to 5:1, 500:1 to 10:1, 500:1 to 20:1, 500:1 to 30:1, 500:1 to 40:1, 500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1, 200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1,200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1, 20:1 to 5:1, 15:1 to 5:

1. and 12:1 to 7:1.

83. The method of any one of claims 78 to 81, wherein the first protein of interest is selected from the group consisting of an antigen binding protein or portion thereof, an enzyme, and a growth factor receptor or agonist or antagonist thereof.

84. The method of claim 82, wherein the first and second proteins of interest are subunits of a multi-subunit protein.

85. The method of claim 84. wherein one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.Atorney Docket No. CATA-43085.60186. The method of any one of claims 84 to 85, wherein the first and / or second proteins of interest are subunits of a viral particle or the first and / or second nucleic acids of interest are part of a viral genome.

87. The method of any one of claims 78 to 81, wherein the first nucleic acid constructs encode at least a second protein of interest in addition to the first protein of interest.

88. The method of claim 87, wherein the first protein of interest and second protein of interest are encoded in a bicistronic construct.

89. The method of any one of claims 86 to 88. wherein the first and second proteins of interest are subunits of a multi -subunit protein.

90. The method of claim 85, wherein one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.

91. The method of any one of claims 78 to 90, wherein the at least first nucleic acid constructs further comprise at least the following elements in operable association in 5’ to 3‘ order: a promoter sequence; a nucleic acid sequence encoding the first protein of interest operably linked to the promoter sequence; and a poly A signal sequence.

92. The method of any one of claims 78 to 90, wherein the at least second nucleic acid constructs further comprise at least the following elements in operable association in 5’ to 3 ' order: a promoter sequence; a nucleic acid sequence encoding the selectable marker operably linked to the promoter sequence; and a poly A signal sequence.Atorney Docket No. CATA-43085.60193. The method of claim 91, wherein the promoter sequence is selected from the group consisting of SV40, EFla, E. coll lac, E. coli trp, phage lambda PL, phage lambda PR. T3. T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.

94. The method of claim 92, wherein the promoter sequence is selected from the group consisting of SIN-LTR, SV40. EFla, E. coli lac, E. coli trp. phage lambda PL. phage lambda PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.

95. The method of any one of claims 78 to 90. wherein the at least second nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3:order: the nucleic acid sequence encoding the selectable marker; and a poly A signal sequence, wherein the nucleic acid sequence encoding the selectable marker is not operably linked to a promoter.

96. The method of any one of claims 78 to 90, wherein the at least second nucleic acid constructs do not include a polyA tail operably linked to the nucleic acid sequence encoding the selectable marker and / or do not include a promoter sequence operably linked to the nucleic acid sequence encoding the selectable marker.

97. The method of any one of claims 78 to 96, wherein the first promoter sequence is not a retroviral LTR promoter.

98. The method of any one of claims 78 to 97, wherein the nucleic acid expression constructs are provided in a vector.

99. The method of claim 98. wherein the vector is a plasmid vector.

100. The method of any one of claims 78 to 99, wherein the host cell comprises a nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the dock site.Atorney Docket No. CATA-43085.601101. The method of claim 100, wherein the nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the dock site is provided in a vector.

102. The method of claim 101, wherein the vector is a plasmid vector.

103. The method of any one of claims 100 to 101, wherein the enzyme is selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.

104. The method of any one of claims 78 to 103, wherein the host cell genome comprises from 5 to 1000 integrated docking sites, each docking site comprising at least one dock site insertion element.

105. The method of any one of claims 78 to 104, wherein the host cell genome comprises from 5 to 500 integrated docking sites, each docking site comprising at least one dock site insertion element.

106. The method of any one of claims 78 to 105, wherein the host cell genome comprises from 5 to 250 integrated docking sites, each docking site comprising at least one dock site insertion element.

107. The method of any one of claims 78 to 106, wherein the integrated docking sites are independently positioned throughout the host cell genome.

108. The method of any one of claims 78 to 107, wherein the dock site insertion element is targeted by enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.

109. The method of any one of claims 78 to 108, wherein the dock site insertion element is selected from the group consisting of a recombinase dock site insertion element and a HDR dock site insertion element.

110. The method of claim 109, wherein the dock site insertion element is a recombinase dock site insertion element.Atorney Docket No. CATA-43085.601111. The method of claim 110, wherein the recombinase dock site insertion element comprises an atachment site (at).

112. The method of claim 111, wherein the attachment site (at) is selected from the group consisting of atB and atP and atR and atL.

113. The method of claim 110, wherein the recombinase dock site insertion element comprises a LoxP sequence.

114. The method of claim 110, wherein the recombinase dock site insertion element is a Flp Recombination Target (FRT) site.

115. The method of claim 109, wherein the dock site insertion element is a Homology Directed Repair (HDR) dock site insertion element.

116. The method of claim 115, wherein the HDR dock site insertion element comprises one or two dock site homology7arms.

117. The method of claim 116, wherein the HDR dock site insertion element further comprises one or more sequences homologous to a guide RNA sequence.

118. The method of any of claims 116 to 117, wherein the dock site homology arms are from about 30 to 1000 bases in length.

119. The method of claim 118, wherein the integrase dock site insertion element comprises an AAV S 1 safe harbor locus sequence.

120. The method of any one of claims 78 to 119, wherein each docking site is flanked by exogenous integrating vector sequences.

121. The method of claim 120, wherein the exogenous integrating vector sequences are selected from the group consisting of viral vector sequences and transposon vector sequences.Atorney Docket No. CATA-43085.601122. The method of any one of claims 78 to 121, wherein the host cell further comprises an expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase.

123. The method of claim 122, wherein the expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is provided in an episomal expression vector.

124. The method of claim 123, wherein the expression construct encoding an exogenous enzyme selected from the group consisting of an integrase, a recombinase, a nuclease and a nickase is integrated into the host cell genome.

125. The method of any one of claims 78 to 124, wherein the dock site insertion element is positioned to facilitate cassete exchange.

126. The method of any one of claims 78 to 125, wherein each docking site comprises two dock site insertion elements.

127. The method of claim 126, wherein the two dock site insertion elements are positioned to facilitate cassete exchange.

128. The method of any one of claims 126 to 127, wherein the two dock site insertion elements flank sequences encoding a selectable marker, an enzyme, or a combination thereof.

129. The method of any one of claims 78 to 126, w herein the host cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, monkey kidney CV1 line transformed by SV40, baby hamster kidney cells, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor, TRI cells, MRC 5 cells. FS4 cells, rat fibroblasts, MDBK cells. VERO cells. NSO cells and human hepatoma line cells.

130. The method of claim 129, wherein the host cell is selected from the group consisting of a Chinese Hamster Ovar ' (CHO) cell, a HEK 293 cell and a CAP cell.Atorney Docket No. CATA-43085.601131. The method of any one of claims 78 to 130, wherein the host cell is not a CHO cell.

132. The method of any one of claims 127 to 131, wherein the host cell is a GS knockout cell.

133. The method of any one of claims 129 to 131, wherein the host cell is a DHFR knockout cell.

134. The method of any one of claims 77 to 133, wherein the host cells are grown in a medium comprising an inhibitor of the selectable marker.

135. The method of claim 134, wherein the selectable marker is GS and the inhibitor is phosphinothricin or methionine sulphoximine (Msx).

136. The method of claim 134, wherein the selectable marker is DHFR and the inhibitor is methotrexate.

137. A cell culture comprising host cells made the method on any of claims 78 to 136.

138. A host cell comprising: integrated first nucleic acid constructs encoding a first protein or nucleic acid of interest; and integrated second nucleic acid constructs encoding a first selectable marker; wherein the at least integrated first nucleic acid constructs and the at least second integrated nucleic acid constructs are integrated into the genome of the host cell selected from the group consisting of 5000:1 to 5:1, 5000:1 to 10:1, 5000:1 to 20:1, 5000:1 to 30:1, 5000:1 to 40:1, 5000:1 to 50:1, 4000:1 to 5:1, 4000:1 to 10:1, 4000:1 to 20:1, 4000:1 to 30:1, 4000:1 to 40:1, 4000:1 to 50:1, 3000:1 to 5:1, 3000:1 to 10:1, 3000:1 to 20:1, 3000:1 to 30:1, 3000:1 to 40:1, 3000:1 to 50:1, 2000:1 to 5:1, 2000:1 to 10:1.2000:1 to 20:1, 2000:1 to 30:1, 2000:1 to 40:1,2000:1 to 50:1, 1000:1 to 5:1, 1000:1 to 10:1, 1000:1 to 20:1, 1000:1 to 30:1, 1000:1 to 40:1, 1000:1 to 50:1, 500:1 to 5:1, 500:1 to 10:1, 500:1 to 20:1, 500:1 to 30:1, 500:1 to 40:1, 500:1 to 50:1, 200:1 to 5:1, 200:1 to 10:1, 200:1 to 20:1, 200:1 to 30:1, 200:1 to 40:1, 200:1 to 50:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 20:1, 100:1 to 30:1, 100:1 to 40:1, 100:1 to 50:1,20:1 to 5:1, 15:1 to 5:1, and 12:1 to 7:1.Atorney Docket No. CATA-43085.601139. The host cell of claim 138. wherein the first nucleic acid constructs do not comprise a selectable marker.

140. The host cell of claim 138, wherein the first nucleic acid constructs comprise a second selectable marker that is different from the first selectable marker in the second nucleic acid constructs.

141. The host cell of any of claims 138 to 140, wherein the second nucleic acid constructs further comprise a nucleic acid sequence encoding the first protein or nucleic acid of interest.

142. A host cell of any one of claims 138 to 141, further comprising at least integrated third nucleic acid constructs comprising at least one insertion element compatible with the dock site insertion element and encoding a second protein or nucleic acid of interest.

143. The host cell of any one of claims 138 to 142, wherein the ratio of third nucleic acid constructs to second nucleic acid constructs is selected from the group consisting of 200:1 to 5: 1, 200: 1 to 10: 1, 200: 1 to 20: 1, 200: 1 to 30: 1, 200: 1 to 40: 1, 200: 1 to 50: 1, 100: 1 to 5: 1, 100: 1 to 10: 1, 100:1 to 20: 1, 100: 1 to 30: 1, 100:1 to 40:

1. 100: 1 to 50: 1, 20: 1 to 5: 1, 15: 1 to 5: 1, and 12: 1 to 7: 1.

144. The host cell of any one of claims 138 to 143, wherein the first protein of interest is selected from the group consisting of an antigen binding protein or portion thereof, an enzyme, and a growth factor receptor or agonist or antagonist thereof.

145. The host cell of claim 144, wherein the first and second proteins of interest are subunits of a multi-subunit protein.

146. The host cell of claim 145. wherein one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.Atorney Docket No. CATA-43085.601147. The host cell of any one of claims 144 to 145, wherein the first and / or second proteins of interest are subunits of a viral particle or the first and / or second nucleic acids of interest are part of a viral genome.

148. The host cell of any one of claims 138 to 143, wherein the first nucleic acid constructs encode at least a second protein of interest in addition to the first protein of interest.

149. The host cell of claim 148, wherein the first protein of interest and second protein of interest are encoded in a bicistronic construct.

150. The host cell of any one of claims 148 to 149, wherein the first and second proteins of interest are subunits of a multi -subunit protein.

151. The host cell of claim 149, wherein one of the first and second proteins of interest is an antibody heavy chain and the other of the first and second proteins of interest is an antibody light chain.

152. The host cell of any one of claims 138 to 151, wherein the at least first nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: a promoter sequence; a nucleic acid sequence encoding the first protein of interest operably linked to the promoter; and a poly A signal sequence.

153. The host cell of any one of claims 138 to 151, wherein the at least second nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: a promoter sequence; the nucleic acid sequence encoding the first selectable marker operably linked to the promoter; and a poly A signal sequence.

154. The host cell of claim 152, wherein the promoter sequence is selected from the group consisting of SV40, EFla. E. coli lac, E. coh trp, phage lambda PL, phage lambda PR. T3.Atorney Docket No. CATA-43085.601T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.

155. The host cell of claim 153, wherein the promoter sequence is selected from the group consisting of SIN-LTR, SV40, EFla, E. coli lac, E. coli trp, phage lambda PL, phage lambda PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, alpha-lactalbumin, and mouse metallothionein-I promoter sequences.

156. The host cell of any one of claims 138 to 155, wherein the at least second nucleic acid constructs comprise at least the following elements in operable association in 5’ to 3’ order: the nucleic acid sequence encoding the selectable marker; and a poly A signal sequence, wherein the nucleic acid sequence encoding the selectable marker is not operably linked to a promoter.

157. The host cell of any one of claims 138 to 155, wherein the at least second nucleic acid constructs do not include a polyA signal sequence operably linked to the nucleic acid sequence encoding the selectable marker and / or do not include a promoter sequence operably linked to the nucleic acid sequence encoding the selectable marker.

158. The host cell of any one of claims 138 to 157, wherein the promoter sequence is not a retroviral LTR promoter.

159. The host cell of any one of claims 138 to 158. wherein the host cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells, HEK 293 cells. CAP cells, bovine mammary epithelial cells, monkey kidney CV1 line transformed by SV40, baby hamster kidney cells, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, VERO cells, NSO cells, and human hepatoma line cells.

160. The host cell of claim 159, wherein the host cell is selected from the group consisting of a Chinese Hamster Ovary (CHO) cell, a HEK 293 cell and a CAP cell.

161. The host cell of any one of claims 138 to 160. wherein the host cell is not a CHO cell.Atorney Docket No. CATA-43085.601162. The host cell of any one of claims 159 to 161, wherein the host cell is a GS knockout cell.

163. The host cell of any one of claims 159 to 161, wherein the host cell is a DHFR knockout cell.

164. The host cell line of any one of claims 138 to 163, wherein the first and second nucleic acid constructs are integrated retroviral vectors.

165. The host cell line of any one of claims 138 to 163, wherein the first and second nucleic acid constructs are integrated transposon vectors.

166. The host cell line of any one of claims 138 to 1 3, wherein the first and second nucleic acid constructs are integrated at docking sites that are integrated into the genome of the host cell.

167. A cell culture comprising host cells of any of claims 138 to 166.

168. A process for producing a protein of interest comprising culturing host cells according to any of claims 138 to 167 under conditions such that the protein(s) of interest are expressed and purifying the protein(s) of interest from the host cell culture.

169. The process of claim 167, wherein the host cells are grown in a medium comprising an inhibitor of the selectable marker.

170. The process of claim 167, wherein the selectable marker is GS and the inhibitor is phosphinothricin or methionine sulphoximine (Msx).

171. The process of claim 167, wherein the selectable marker is DHFR and the inhibitor is methotrexate.

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