Patent Publication Number: US-2023135673-A1

Title: Systems, methods, and compositions for site-specific genetic engineering using programmable addition via site-specific targeting elements (paste)

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 17/451,734, filed Oct. 21, 2021, which claims the benefit of U.S. Provisional Patent Application Ser. No. 63/222,550, filed Jul. 16, 2021 and U.S. Provisional Patent Application Ser. No. 63/094,803, filed Oct. 21, 2020. The entire contents of the above-referenced patent applications are incorporated by reference in their entirety herein. 
    
    
     FIELD OF DISCLOSURE 
     The subject matter disclosed herein is generally directed to systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE) for the treatment of diseases and diagnostics. 
     BACKGROUND 
     Editing genomes using the RNA-guided DNA targeting principle of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) immunity has been widely exploited and has become a powerful genome editing means for a wide variety of applications. The main advantage of CRISPR-Cas system lies in the minimal requirement for programmable DNA interference: an endonuclease, such as a Cas9, Cas12, or any programmable nucleases, guided by a customizable dual-RNA structure. Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand. The CRISPR/Cas9 protein-RNA complex is localized on the target by a guide RNA (guide RNA), then cleaved to generate a DNA double strand break (dsDNA break, DSB). After cleavage, DNA repair mechanisms are activated to repair the cleaved strand. Repair mechanisms are generally from one of two types: non-homologous end joining (NHEJ) or homologous recombination (HR). In general, NHEJ dominates the repair, and, being error prone, generates random indels (insertions or deletions) causing frame shift mutations, among others. In contrast, HR has a more precise repairing capability and is potentially capable of incorporating the exact substitution or insertion. To enhance HR, several techniques have been tried, for example: combination of fusion proteins of Cas9 nuclease with homology-directed repair (HDR) effectors to enforce their localization at DSBs, introducing an overlapping homology arm, or suppression of NHEJ. Most of these techniques rely on the host DNA repair systems. 
     Recently, new guided editors have been developed, such as guided prime editors (PE) PE1, PE2, and PE3, e.g., Liu, D. et al., Nature 2019, 576, 149-157. These PEs are reverse transcriptase (RT) fused with Cas 9 H 840A nickase (Cas9n (H840A)), and the genome editing is achieved using a prime-editing guide RNA (pegRNA). Despite these developments, programmable gene integration is still generally dependent on cellular pathways or repair processes. 
     Therefore, there is a need for more effective tools for gene editing and delivery. 
     SUMMARY 
     The present disclosure provides a method of site-specific integration of a nucleic acid into a cell genome. The method comprises incorporating an integration site at a desired location in the cell genome by introducing into the cell a DNA binding nuclease linked to a reverse transcriptase domain, wherein the DNA binding nuclease comprises a nickase activity; and a guide RNA (gRNA) comprising a primer binding sequence linked to an integration sequence, wherein the gRNA interacts with the DNA binding nuclease and targets the desired location in the cell genome, wherein the DNA binding nuclease nicks a strand of the cell genome and the reverse transcriptase domain incorporates the integration sequence of the gRNA into the nicked site, thereby providing the integration site at the desired location of the cell genome. The method further comprises integrating the nucleic acid into the cell genome by introducing into the cell a DNA or RNA strand comprising the nucleic acid linked to a sequence that is complementary or associated to the integration site, and an integration enzyme, wherein the integration enzyme incorporates the nucleic acid into the cell genome at the integration site by integration, recombination, or reverse transcription of the sequence that is complementary or associated to the integration site, thereby introducing the nucleic acid into the desired location of the cell genome of the cell. 
     In some embodiments, the gRNA can be hybridized to a complementary strand of the cell genome to the genomic strand that is nicked by the DNA binding nuclease. 
     In some embodiments, the integration enzyme can be introduced as a peptide or a nucleic acid encoding the same. 
     In some embodiments, the DNA binding nuclease can be introduced as a peptide or a nucleic acid encoding the same. 
     In some embodiments, the DNA or RNA strand comprising the nucleic acid can be introduced into the cell as a minicircle, a plasmid, mRNA or a linear DNA. 
     In some embodiments, the DNA or RNA strand comprising the nucleic acid can be between 1000 bp and 10,000 bp. 
     In some embodiments, the DNA or RNA strand comprising the nucleic acid can be more than 10,000 bp. 
     In some embodiments, the DNA or RNA strand comprising the nucleic acid can be less than 1000 bp. 
     In some embodiments, the DNA comprising the nucleic acid can be introduced into the cell as a minicircle. 
     In some embodiment, the minicircle cannot comprise sequences of a bacterial origin. 
     In some embodiments, the DNA binding nuclease can be linked to a reverse transcriptase domain and the integration enzyme can be linked via a linker. The linker can be cleavable. The linker can be non-cleavable. The linker can be replaced by two associating binding domains of the DNA binding nuclease linked to a reverse transcriptase. 
     In some embodiments, the integration enzyme can be selected from the group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, To12 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof. 
     In some embodiments, the integration enzyme can be Bxb1 or a mutant thereof. 
     In some embodiments, the integration site can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site. 
     In some embodiments, the DNA binding nuclease comprising a nickase activity can be selected from Cas9-D10A, Cas9-H840A, and Cas12a/b nickase. 
     In some embodiments, the reverse transcriptase domain can be selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), and  Eubacterium  rectale maturase RT (MarathonRT). 
     In some embodiments, the reverse transcriptase domain can comprise a mutation relative to the wild-type sequence. 
     In some embodiments, the M-MLV reverse transcriptase domain can comprise one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P and L603W. 
     In some embodiments, the method can further comprise introducing a second nicking guide RNA (ngRNA). The ngRNA can direct nicking at 90 bases downstream of the gRNA nick on a complementary strand. 
     In some embodiments, the gRNA, the nucleic acid encoding the DNA binding nuclease, the reverse transcriptase, the DNA comprising nucleic acid linked to a complementary integration site, the integration enzyme, and optionally the ngRNA can be introduced into a cell in a single reaction. 
     In some embodiments, the gRNA, the nucleic acid encoding the DNA binding nuclease, the reverse transcriptase, the DNA comprising nucleic acid linked to a complementary integration site, the integration enzyme, and optionally the ngRNA can be introduced using a virus, a RNP, an mRNA, a lipid, or a polymeric nanoparticle. 
     In some embodiments, the nucleic acid can be a reporter gene. The reporter gene can be a fluorescent protein. 
     In some embodiments, the cell can be a dividing cell. 
     In some embodiments, the cell can be a non-dividing cell. 
     In some embodiments, the desired location in the cell genome can be the locus of a mutated gene. 
     In some embodiments, the nucleic acid can be a degradation tag for programmable knockdown of proteins in the presence of small molecules. 
     In some embodiments, the cell can be a mammalian cell, a bacterial cell or a plant cell. 
     In some embodiments, nucleic acid can be a T-cell receptor (TCR), a chimeric antigen receptor (CAR), an interleukin, a cytokine, or an immune checkpoint gene for integration into a T-cell or natural killer (NK) cell. The TCR, the CAR, the interleukin, the cytokine, or the immune checkpoint gene can be incorporated into the target site of the T-cell or NK cell genome using a minicircle DNA. 
     In some embodiments, the nucleic acid can be a beta hemoglobin (HBB) gene and the cell can be a hematopoietic stem cell (HSC). The HBB gene can be incorporated into the target site in the HSC genome using a minicircle DNA. The nucleic acid can be a gene responsible for beta thalassemia or sickle cell anemia. 
     In some embodiments, the nucleic acid can be a metabolic gene. The metabolic gene can be involved in alpha-1 antitrypsin deficiency or ornithine transcarbamylase (OTC) deficiency. The metabolic gene can be a gene involved in inherited diseases. 
     In some embodiments, the nucleic acid can be a gene involved in an inherited disease or an inherited syndrome. The inherited disease can be cystic fibrosis, familial hypercholesterolemia, adenosine deaminase (ADA) deficiency, X-linked SCID (X-SCID), Wiskott-Aldrich syndrome (WAS), hemochromatosis, Tay-Sachs, fragile X syndrome, Huntington&#39;s disease, Marfan syndrome, phenylketonuria, or muscular dystrophy. 
     The present disclosure provides a vector comprising a nucleic acid encoding a DNA binding nuclease comprising a nickase activity C-terminally linked to a reverse transcriptase linked to an integration enzyme via a linker. 
     In some embodiments, the linker can be cleavable. 
     In some embodiments, the linker can be non-cleavable. 
     In some embodiments, the linker can comprise two associating binding domains of the DNA binding nuclease linked to a reverse transcriptase. 
     In some embodiments, the integration enzyme can comprise a conditional activation domain or conditional expression domain. 
     In some embodiments, the integration enzyme can be fused to an estrogen receptor. 
     In some embodiments, the DNA binding nuclease comprising a nickase activity can be selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a/b. 
     In some embodiments, the reverse transcriptase can be a M-MLV reverse transcriptase, a AMV-RT, MarathonRT, or a RTX. The reverse transcriptase can be a modified M-MLV reverse transcriptase relative to the wildtype M-MLV reverse transcriptase. The M-MLV reverse transcriptase domain can comprise one or more of the mutations selected from the group consisting of D200N, T306K, W313F, T330P and L603W. 
     In some embodiments, the integration enzyme can be selected from the group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, To12 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof. 
     In some embodiments, the recombinase or integrase can be Bxb1 or a mutant thereof. 
     The present disclosure provides a cell comprising a vector comprising a nucleic acid encoding a DNA binding nuclease comprising a nickase activity C-terminally linked to a reverse transcriptase linked to an integration enzyme via a linker. The cell further comprises a gRNA comprising a primer binding sequence, an integration sequence, and a guide sequence, wherein the gRNA can interact with the encoded nuclease comprising a nickase activity. The cell further comprising a DNA minicircle comprising a nucleic acid and a sequence recognized by the encoded integrase, recombinase, or reverse transcriptase. The cell further comprising a nicking guide RNA (ngRNA) capable of binding the encoded nuclease comprising a nickase activity, and wherein the ngRNA targets a sequence away from the gRNA. 
     In some embodiments, the minicircle cannot comprise a sequence of bacterial origin. 
     In some embodiments, the integration enzyme can be selected from the group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, WO, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof. 
     In some embodiments, the integration enzyme can be Bxb1 or a mutant thereof. 
     In some embodiments, the DNA binding nuclease comprising a nickase activity can be selected from the group consisting of Cas9-D10A, Cas9-H840A and Cas12a. 
     In some embodiments, the reverse transcriptase can be a M-MLV reverse transcriptase. The reverse transcriptase can be a modified M-MLV reverse transcriptase. The amino acid sequence of the M-MLV reverse transcriptase can comprise one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W. 
     In some embodiments, the cell can further comprise introducing ngRNA to the cell. The ngRNA can be a +90 ngRNA. The +90 ngRNA can direct nicking at 90 bases downstream of the gRNA nick on a complementary strand. 
     The present disclosure provides a polypeptide comprising a DNA binding nuclease comprising a nickase activity C-terminally linked to a reverse transcriptase linked to an integration enzyme via a linker. 
     In some embodiments, the linker can be cleavable. 
     In some embodiments, the linker can be non-cleavable. 
     In some embodiments, the integration enzyme can be fused to an estrogen receptor. 
     In some embodiments, the DNA binding nuclease comprising a nickase activity can be selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a/b. 
     In some embodiments, the reverse transcriptase can be a M-MLV reverse transcriptase, a AMV-RT, a MarathonRT, or a XRT. The reverse transcriptase can be a modified M-MLV relative to a wild-type M-MLV reverse transcriptase. The M-MLV reverse transcriptase domain can comprise one or more of mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W. 
     In some embodiments, the integration enzyme can be selected from group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof. 
     In some embodiments, the integration enzyme can be Bxb1 or a mutant thereof. 
     The present disclosure provides a gRNA that specifically binds to a DNA binding nuclease comprising nickase activity, the gRNA comprising a primer binding site, which hybridizes to a nicked DNA strand, a recognition site for an integration enzyme, and a target recognition sequence recognizing a target site in a cell genome and hybridizing to a genomic strand complementary to the strand that is nicked by the DNA binding nuclease. 
     In some embodiments, the DNA binding nuclease comprising a nickase activity can be selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a/b. 
     In some embodiments, the primer binding site can hybridize to the 3′ end of the nicked DNA strand. 
     In some embodiments, the recognition site for the integration enzyme can be selected from an attB site, an attP site, an attL site, an attR site, a lox71 site, and a FRT site. 
     In some embodiments, the recognition site for the integration enzyme can be a Bxb1 site. 
     The present disclosure provides a method of site-specific integration of two or more nucleic acids into a cell genome. The method comprises incorporating two integration sites at desired locations in the cell genome by introducing into the cell a DNA binding nuclease linked to a reverse transcriptase domain, wherein the DNA binding nuclease comprises a nickase activity, and two guide RNAs (gRNAs), each comprising, a primer binding sequence, linked to a unique integration sequence, wherein the gRNA interacts with the DNA binding nuclease and targets the desired locations in the cell genome, wherein the DNA binding nuclease nicks a strand of the cell genome and the reverse transcriptase domain incorporates each of the integration sequence of the gRNA into the nicked site, thereby providing the integration site at the desired locations of the cell genome. The method further comprises integrating the nucleic acid by introducing into the cell two or more DNA or RNA comprising the nucleic acids, wherein each DNA is flanked by orthogonal integration sites, and an integration enzyme, wherein the integration enzyme incorporates the nucleic acids into the cell genome at the integration sites by integrase, recombinase, or reverse transcriptase of the sequence that is complementary or associated to the integration site, thereby introducing the nucleic acids into the desired locations of the cell genome of the cell. 
     In some embodiments, each of the two different integration sites inserted into the cell genome can be attB sequences comprising different palindromic or non-palindromic central dinucleotide. 
     In some embodiments, each of the two different integration sites inserted into the cell genome can be attP sequences comprising different palindromic or non-palindromic central dinucleotide. 
     In some embodiments, the integration enzyme can enable each of the two or more DNA or RNA comprising the nucleic acids to directionally enable integration of the nucleic acids into a genome via recombination of a pair of orthogonal attB site sequence and an attP site sequence. 
     In some embodiments, the integration enzyme can be selected from the group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R1, R2, R3, R4, R5, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof. 
     In some embodiments, the integration enzyme can be Bxb1 or a mutant thereof. 
     In some embodiments, the DNA comprising genes can be genes involved in a cell maintenance pathway, cell-division, or a signal transduction pathway. 
     In some embodiments, the reverse transcriptase domain can comprise Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), or  Eubacterium  rectale maturase RT (MarathonRT). 
     In some embodiments, the DNA binding nuclease comprising a nickase activity can be selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a/b. 
     In some embodiments, the pair of an attB site sequence and an attP site sequence can be selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 and SEQ ID NO: 35 and SEQ ID NO: 36. 
     The present disclosure provides a cell comprising a vector comprising a nucleic acid encoding a DNA binding nuclease comprising a nickase activity, wherein the DNA binding nuclease is C-terminally linked to a reverse transcriptase, wherein the reverse transcriptase is linked to a recombinase or integrase via a linker. The cell further comprises two guide RNAs (gRNAs) comprising a primer binding sequence, an integration sequence and a guide sequence, wherein the gRNA can interact with the encoded DNA binding nuclease comprising a nickase activity. The cell further comprises two or more DNA or RNA strands comprising a nucleic acid and a pair of flanking attB site sequence and an attP site sequence recognized by the encoded integrase or recombinase. The cell optionally further comprises a nicking guide RNA (ngRNA) capable of binding the encoded nuclease comprising a nickase activity, and wherein the ngRNA targets a sequence away from the gRNA. 
     The present disclosure provides a cell comprising a modified genome, wherein the modification comprises incorporation of two orthogonal integration sites within the cell genome by introducing into the cell a: vector comprising a nucleic acid encoding a DNA binding nuclease comprising a nickase activity, wherein the DNA binding nuclease is C-terminally linked to a reverse transcriptase; two guide RNAs (gRNAs), each comprising a primer binding sequence, a genomic integration sequence, and a guide sequence, wherein the gRNA can interact with the encoded nuclease comprising a nickase activity; and optionally a nicking guide RNA (ngRNA) capable of binding the encoded nuclease comprising a nickase activity, and wherein the ngRNA targets a sequence away from the gRNA. 
     The present disclosure provides a method of integrating two or more nucleic acids into the cell genome of cell of claim  90 , the method comprising introducing into the cell: two or more DNA, each comprising a nucleic acid and a pair of flanking orthogonal integration site sequences; an integration enzyme that can recognize the integration site sequence enabling directional linking of the two or more DNA comprising nucleic acid; and enabling incorporation of the nucleic acids into the cell genome by integrating the 5′ orthogonal integration sequence of the first DNA with the first genomic integration sequence and 3′ orthogonal integration sequence of the last DNA with the last genomic integration sequence, thereby incorporating the two or more nucleic acids into the cell genome. 
     The present disclosure provides a cell comprising a modified genome, wherein the modification comprises incorporation of two orthogonal integration sites within the cell genome by introducing into the cell: a vector comprising a nucleic acid encoding a DNA binding nuclease comprising a nickase activity, wherein the DNA binding nuclease is C-terminally linked to a reverse transcriptase; two guide RNAs (gRNAs), each comprising a primer binding sequence, a genomic integration sequence, and a guide sequence, wherein the gRNA can interact with the encoded nuclease comprising a nickase activity; and optionally a nicking guide RNA (ngRNA) capable of binding the encoded nuclease comprising a nickase activity, and wherein the ngRNA targets a sequence away from the gRNA; two or more DNA or RNA comprising the nucleic acids, wherein each DNA is flanked by orthogonal integration sites; and an integration enzyme, wherein the integration enzyme incorporates the nucleic acids into the cell genome at the integration sites. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Aspects, features, benefits and advantages of the embodiments described herein will be apparent with regard to the following description, appended claims, and accompanying drawings where: 
         FIG.  1    shows a schematic diagram of a concept of Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  2    shows a schematic diagram of a prime editing process according to embodiments of the present teachings; 
         FIG.  3    shows the percent integration of green fluorescent protein (GFP) in the lentiviral integrated lox71 site in HEK293FT cell line in the presence of various plasmids according to embodiments of the present teachings; 
         FIG.  4    shows the percent editing of the HEK293FT genome for incorporation of various lengths of lox71 or lox66 according to embodiments of the present teachings; 
         FIG.  5 A  shows the percent editing of lox71 site with different PE/Cre vectors according to embodiments of the present teachings; 
         FIG.  5 B  shows the percent integration of GFP at the lox71 site in HEK293FT cell genome according to embodiments of the present teachings; 
         FIG.  6    shows a schematic representation of using Bxb1 to integrate a nucleic acid into the genome according to embodiments of the present teachings; 
         FIG.  7    shows the percent integration of GFP or Gluc into the attB locus using Bxb1 Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  8    shows the percent editing of various HEK3 targeting pegRNA Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  9 A  shows a fluorescent image of cells wherein the SUPT16H marker is tagged with EGFP using PASTE according to embodiments of the present teachings; 
         FIG.  9 B  shows a fluorescent image of cells wherein the SRRM2 marker is tagged with EGFP using Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  9 C  shows a fluorescent image of cells wherein the LAMNB1 marker is tagged with EGFP using Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  9 D  shows a fluorescent image of cells wherein the NOLC1 marker is tagged with EGFP using Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  9 E  shows a fluorescent image of cells wherein the NOLC1 marker is tagged with EGFP using Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  9 F  shows a fluorescent image of cells wherein the NOLC1 marker is tagged with EGFP using Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  9 G  shows a fluorescent image of cells wherein the DEPDC4 marker is tagged with EGFP using Programmable Addition via Site-Specific Targeting Elements (PASTE) according to embodiments of the present teachings; 
         FIG.  10 A  shows comparisons of lipofectamine aided transfection in blue with electroporation aided transfection in red for the addition of the Bxb1 attB site at the ACTB N-terminal site in the genome using PASTE according to embodiments of the present teachings; 
         FIG.  10 B  shows comparisons of lipofectamine aided transfection in blue with electroporation aided transfection in red for EGFP integration at the ACTB N-terminal site in the genome using PASTE according to embodiments of the present teachings; 
         FIG.  11    shows a diagram of the integration of EGFP and Gluc with various HEK3 targeting pegRNAs according to embodiments of the present teachings; 
         FIG.  12    shows a schematic diagram of the using φC31 as the integration enzyme, according to embodiments of the present teachings; 
         FIG.  13    shows a schematic diagram of multiplexing involving inserting multiple genes of interest in multiple loci using unique guide RNAs that incorporated exterior flanking attB sites according to embodiments of the present teachings; 
         FIG.  14 A  shows a diagram of the orthogonal editing with the right GT-EGFP according to embodiments of the present teachings; 
         FIG.  14 B  shows a diagram of the orthogonal editing with the right GA-mCherry according to embodiments of the present teachings; 
         FIG.  15 A  shows a fluorescent image of a multiplexing of ACTB-EGFP and NOLC1-mCherry according to embodiments of the present teachings 
         FIG.  15 B  shows a fluorescent image of a multiplexing of ACTB-EGFP and LAMNB1-mCherry according to embodiments of the present teachings; 
         FIG.  16 A  shows next generation sequencing results of 9×9 attP and attB central dinucleotide variants and their edit percentage wherein the orthogonality of attB/attP combinations for potential multiplexing applications is shown according to embodiments of the present teachings; 
         FIG.  16 B  shows an heatmap of 9×9 attP and attB central dinucleotide variants and their edit percentage according to embodiments of the present teachings; 
         FIG.  17    shows integration of SERPINA and CPS1 into Albumin loci using Albumin guide-pegRNA in HEK293FT cells according to embodiments of the present teachings; 
         FIG.  18    shows schematics for different nucleic acids for engineering T-cells according to embodiments of the present teachings; 
         FIG.  19    shows the editing efficiency for EGFP integration at the ACTB locus in primary T-cells according to embodiments of the present teachings; 
         FIG.  20    shows editing in TRAC locus in HEK293FT with different pegRNA according to embodiments of the present teachings; 
         FIG.  21 A  shows the attB integration at the ACTB locus using nicking guides 1 and 2 according to embodiments of the present teachings; 
         FIG.  21 B  shows the EGFP integration at the ACTB locus using nicking guides 1 and 2 according to embodiments of the present teachings; 
         FIG.  21 C  shows the EGFP integration at an ACTB site according to embodiments of the present teachings; 
         FIG.  22 A  shows PASTE editing in liver hepatocellular carcinoma cell line HEPG2 according to embodiments of the present teachings; 
         FIG.  22 B  shows PASTE editing of chronic myelogenous leukemia cell line K562 according to embodiments of the present teachings; 
         FIG.  23 A  shows the attB addition with targeting and non-targeting guides according to embodiments of the present teachings; 
         FIG.  23 B  shows the EGFP integration with targeting and non-targeting guides according to embodiments of the present teachings; 
         FIG.  23 C  shows the EGFP integration for mutagenized Bxb1 according to embodiments of the present teachings; 
         FIG.  24 A  shows a schematic of the design parameters for the pegRNA according to embodiments of the present teachings; 
         FIG.  24 B  shows a schematic of the design parameters for nicking guide RNA according to embodiments of the present teachings; 
         FIG.  25 A  shows the integration of EGFP at the ACTD locus with different PBS and RT lengths according to embodiments of the present teachings; 
         FIG.  25 B  shows the integration of EGFP at the LMNB1 loci with different PBS and RT lengths according to embodiments of the present teachings; 
         FIG.  25 C  shows the integration of EGFP at the NOLC1 loci with different PBS and RT lengths according to embodiments of the present teachings; 
         FIG.  25 D  shows the integration of EGFP at the GRSF1 locus with different PBS and RT lengths and different nicking guides according to embodiments of the present teachings; 
         FIG.  25 E  shows EGFP integration with mutant attP sites according to embodiments of the present teachings; 
         FIG.  25 F  shows the PASTE editing of an expanded panel of genes according to embodiments of the present teachings; 
         FIG.  26 A  shows the PASTE EGPF editing at the ACTB locus according to embodiments of the present teachings; 
         FIG.  26 B  shows the HITI EGPF editing at the ACTB locus according to embodiments of the present teachings; 
         FIG.  26 C  shows the comparison between the PASTE and HITI editing a panel of 14 genes according to embodiments of the present teachings; 
         FIG.  26 D  shows PASTE Bxb1 off-target integrations according to embodiments of the present teachings; 
         FIG.  26 E  shows PASTE Cas9 off-target integrations according to embodiments of the present teachings; 
         FIG.  26 F  shows the EGFP integration for gene inserts of different sizes according to embodiments of the present teachings; 
         FIG.  27 A  shows the orthogonality between selected sets of attB and attP sites according to embodiments of the present teachings; 
         FIG.  27 B  shows the orthogonality between selected sets of attB and attP sites according to embodiments of the present teachings; 
         FIG.  27 C  shows a schematic for the orthogonal PASTE editing using engineered di-nucleotide combinations according to embodiments of the present teachings; 
         FIG.  28 A  shows fluorescent images of the GFP tagging of ACTB and SUPT16H genes with PASTE according to embodiments of the present teachings; 
         FIG.  28 B  shows fluorescent images of the GFP tagging of NOLC1 and SRRM2 genes with PASTE according to embodiments of the present teachings; 
         FIG.  28 C  shows fluorescent images of the GFP tagging of LMNB1 and DEPDC4 genes with PASTE according to embodiments of the present teachings; 
         FIG.  28 D  shows the orthogonal gene integration at three endogenous sites with PASTE according to embodiments of the present teachings; 
         FIG.  28 E  shows the multiplexed insertion via one-plex, two-plex, and three-plex gene insertion at three endogenous sites via PASTE according to embodiments of the present teachings; 
         FIG.  28 F  shows fluorescent images of two single cells with multiplexed gene tagging of ACTB (EGFP) and NOLC1 (mCherry) using PASTE according to embodiments of the present teachings; 
         FIG.  28 G  shows fluorescent images two single cells with multiplexed gene tagging of ACTB (EGFP) and LMNB1 (mCherry) using PASTE according to embodiments of the present teachings; 
         FIG.  29 A  shows the prime editing efficiency of Bxb1 attB site insertion at the ACTB locus according to embodiments of the present teachings; 
         FIG.  29 B  shows the prime editing efficiency at inserting Bxb1 attB sites of different lengths at the ACTB locus according to embodiments of the present teachings; 
         FIG.  29 C  shows the prime editing efficiency of inserting attB sequences from different integrases, wherein both orientations of landing sites are profiled (F, forward; and R, reverse) according to embodiments of the present teachings; 
         FIG.  29 D  shows the prime editing efficiency of inserting attB sequences from Bxb1 integrase and Cre recombinase, wherein both orientations of landing sites are profiled (F, forward; and R, reverse) according to embodiments of the present teachings; 
         FIG.  29 E  shows a schematic of PASTE insertion at the ACTB locus showing guide and target sequences according to embodiments of the present teachings.  FIG.  29 E  discloses SEQ ID NOS 428-431, respectively, in order of appearance; 
         FIG.  29 F  shows a comparison of PASTE integration efficiency of GFP with a panel of integrases targeting the 5′ end of the ACTB locus, wherein both orientations of landing sites are profiled (F, forward; and R, reverse) according to embodiments of the present teachings; 
         FIG.  29 G  shows a comparison of GFP cargo integration efficiency between Bxb1 integrases and Cre recombinase according to embodiments of the present teachings; 
         FIG.  29 H  shows the dependence of PASTE editing activity on different prime and integrase components according to embodiments of the present teachings; 
         FIG.  29 I  shows a titration of a single vector PASTE system (SpCas9-RT-P2A-Bxb1) on integrase efficiency according to embodiments of the present teachings; 
         FIG.  29 J  shows the effect of cargo size on PASTE insertion efficiency at the endogenous ACTB target according to embodiments of the present teachings; 
         FIG.  29 K  shows a gel electrophoresis showing complete insertion by PASTE for multiple cargo sizes according to embodiments of the present teachings; 
         FIG.  30 A  shows a schematic of PASTE integration, including resulting attR and attL sites that are generated and PCR primers for assaying the integration junctions according to embodiments of the present teachings; 
         FIG.  30 B  shows a PCR and gel electrophoresis readout of left integration junction from PASTE insertion of GFP at the ACTB locus, wherein the insertion is analyzed for in-frame and out-of-frame GFP integration experiments as well as for a no prime control and expected sizes of the PCR fragments are shown using the primers shown in the schematic in subpanel  FIG.  30 A  according to embodiments of the present teachings; 
         FIG.  30 C  shows a PCR and gel electrophoresis readout of right integration junction from PASTE insertion of GFP at the ACTB locus, wherein the insertion is analyzed for in-frame and out-of-frame GFP integration experiments as well as for a no prime control and the expected sizes of the PCR fragments are shown using the primers shown in the schematic in subpanel  FIG.  30 A  according to embodiments of the present teachings; 
         FIG.  30 D  shows a Sanger sequencing shown for the right integration junction for an in-frame fusion of GFP via PASTE to the N-terminus of ACTB according to embodiments of the present teachings; 
         FIG.  30 E  shows a Sanger sequencing shown for the left integration junction for an in-frame fusion of GFP via PASTE to the N-terminus of ACTB according to embodiments of the present teachings; 
         FIG.  31 A  shows a schematic of various parameters that affect PASTE integration of ˜1 kb GFP insert, wherein on the pegRNA, the PBS, RT, and attB lengths can alter the efficiency of attB insertion, and nicking guide selection also affects overall gene integration efficiency according to embodiments of the present teachings; 
         FIG.  31 B  shows the impact of PBS and RT length on PASTE integration of GFP at the ACTB locus according to embodiments of the present teachings; 
         FIG.  31 C  shows the impact of PBS and RT length on PASTE integration of GFP at the LMNB1 locus according to embodiments of the present teachings; 
         FIG.  31 D  shows the impact of attB length on PASTE integration of GFP at the ACTB locus according to embodiments of the present teachings; 
         FIG.  31 E  shows the impact of attB length on PASTE integration of GFP at the LMNB1 locus according to embodiments of the present teachings; 
         FIG.  31 F  shows the impact of attB length on PASTE integration of GFP at the NOLC1 locus according to embodiments of the present teachings; 
         FIG.  31 G  shows the impact of minimal PBS, RT, and attB lengths on PASTE integration efficiency of GFP at the ACTB locus according to embodiments of the present teachings; 
         FIG.  31 H  shows the impact of minimal PBS, RT, and attB lengths on PASTE integration efficiency of GFP at the LMNB1 locus according to embodiments of the present teachings; 
         FIG.  31 I  shows the PASTE integration of GFP at the LMNB1 locus in the presence and absence of nicking guide, prime, and Bxb1 with a minimally compact pegRNA containing a 38 bp attB compared to a longer pegRNA design according to embodiments of the present teachings; 
         FIG.  32 A  shows the PASTE insertion efficiency at ACTB and LMNB1 loci with two different nicking guide designs according to embodiments of the present teachings; 
         FIG.  32 B  shows the PASTE editing efficiency at ACTB and LMNB1 with target and non-targeting spacers and matched pegRNAs with and without Bxb1 expression according to embodiments of the present teachings; 
         FIG.  33 A  shows the PASTE integration of GFP at the ACTB locus with different Bxb1 catalytic mutants according to embodiments of the present teachings; 
         FIG.  33 B  shows the PASTE integration of GFP at the ACTB locus with different RT catalytic mutants according to embodiments of the present teachings; 
         FIG.  34 A  shows the GFP integration by PASTE at a panel of endogenous genomic loci according to embodiments of the present teachings; 
         FIG.  34 B  shows the integration of a panel of different gene cargo at ACTB locus via PASTE according to embodiments of the present teachings; 
         FIG.  34 C  shows the integration efficiency of therapeutically relevant genes at the ACTB locus according to embodiments of the present teachings; 
         FIG.  34 D  shows the endogenous protein tagging with GFP via PASTE by in-frame endogenous gene tagging at the ACTB loci and SRRM2 loci according to embodiments of the present teachings; 
         FIG.  34 E  shows the endogenous protein tagging with GFP via PASTE by in-frame endogenous gene tagging at the NOLC1 loci and LMNB1 loci according to embodiments of the present teachings; 
         FIG.  35    shows the integration of a panel of different gene cargo at LMNB1 locus via PASTE according to embodiments of the present teachings; 
         FIG.  36 A  shows the PASTE integration efficiency for all 16 central dinucleotide attB/attP sequence pairs with a 5 kb GFP template at the ACTB locus according to embodiments of the present teachings; 
         FIG.  36 B  shows a schematic of the pooled attB/attP dinucleotide orthogonality assay, wherein each attB dinucleotide sequence is co-transfected with a barcoded pool of all 16 attP dinucleotide sequences and Bxb1 integrase, relative integration efficiencies are determined by next generation sequencing of barcodes, and all 16 attB dinucleotides are profiled in an arrayed format with attP pools according to embodiments of the present teachings; 
         FIG.  36 C  shows the relative insertion preferences for all possible attB/attP dinucleotide pairs determined by the pooled orthogonality assay according to embodiments of the present teachings; 
         FIG.  36 D  shows the orthogonality of top 4 attB/attP dinucleotide pairs evaluated for GFP integration with PASTE at the ACTB locus according to embodiments of the present teachings; 
         FIG.  37    shows the orthogonality of Bxb1 dinucleotides as measured by a pooled reporter assay, wherein each web logo motif shows the relative integration of different attP sequences in a pool at a denoted attB sequence with the listed dinucleotide according to embodiments of the present teachings; 
         FIG.  38 A  shows a schematic of multiplexed integration of different cargo sets at specific genomic loci, wherein three fluorescent cargos (GFP, mCherry, and YFP) are inserted orthogonally at three different loci (ACTB, LMNB1, NOLC1) for in-frame gene tagging according to embodiments of the present teachings; 
         FIG.  38 B  shows the efficiency of multiplexed PASTE insertion of combinations of fluorophores at ACTB, LMNB1, and NOLC1 loci according to embodiments of the present teachings; 
         FIG.  39 A  shows the GFP integration efficiency at a panel of genomic loci by PASTE compared to insertion rates by homology-independent targeted integration (HITI) according to embodiments of the present teachings; 
         FIG.  39 B  shows a comparison of unintended indel generation by PASTE and HITI at the ACTB and LMNB1 target sites, wherein the on-target EGFP integration rate observed compared to unintended indels is shown according to embodiments of the present teachings; 
         FIG.  39 C  shows the integration of a GFP template by PASTE at the ACTB locus compared to homology-directed repair (HDR) at the same target, wherein the quantification is by single-cell clone counting, wherein targeting and non-targeting guides were used for HDR insertion, and wherein for PASTE targeting and non-targeting refers to the presence or absence of the SpCas9-RT protein respectively according to embodiments of the present teachings; 
         FIG.  39 D  shows the comparison of unintended indel generation by PASTE and HDR based EGFP insertion at the ACTB target site, wherein the average indel rate measured across all single-cell clones generated is showed according to embodiments of the present teachings; 
         FIG.  39 E  shows a schematic for Bxb1 and Cas9 off-target identification and a detection assay according to embodiments of the present teachings; 
         FIG.  39 F  shows the GFP integration activity at predicted Bxb1 off-target sites in the human genome according to embodiments of the present teachings; 
         FIG.  39 G  shows the GFP integrations activity at predicted PASTE ACTB Cas9 guide off target sites according to embodiments of the present teachings; 
         FIG.  39 H  shows the GFP integration activity at predicted HITI ACTB Cas9 guide off-target sites according to embodiments of the present teachings; 
         FIG.  39 I  shows a schematic of next-generation sequencing method to assay genome-wide off-target integration sites by PASTE according to embodiments of the present teachings; 
         FIG.  39 J  shows the alignment of reads at the on-target ACTB site using a genome-wide integration assay, wherein expected on-target integration outcomes are shown according to embodiments of the present teachings; 
         FIG.  39 K  shows the analysis of on-target and off-target integration events across 3 single-cell clones for PASTE and 3 single-cell clones for no prime condition according to embodiments of the present teachings; 
         FIG.  39 L  shows a Manhattan plot of integration events for a representative single-cell clone with PASTE editing, wherein the on-target site is at the ACTB gene on chromosome 7 according to embodiments of the present teachings; 
         FIG.  40 A  shows a comparison of indel rates generated by PASTE and HITI mediated insertion of EGFP at the ACTB and LMNB1 loci in HepG2 cells according to embodiments of the present teachings; 
         FIG.  40 B  shows the validation of ddPCR assays for detecting editing at predicted Bxb1 offtarget sites using synthetic amplicons according to embodiments of the present teachings; 
         FIG.  40 C  shows the validation of ddPCR assays for detecting editing at predicted PASTE ACTB Cas9 guide off-target sites using synthetic amplicons according to embodiments of the present teachings; 
         FIG.  40 D  shows the validation of ddPCR assays for detecting editing at predicted HITI ACTB Cas9 guide off-target sites using synthetic amplicons according to embodiments of the present teachings; 
         FIG.  41 A  shows a number of significant differentially regulated genes in HEK293FT cells expressing Bxb1 integrase, PASTE targeting ACTB integration of EGFP, or Prime editing targeting ACTB for EGFP insertion without Bxb1 expression according to embodiments of the present teachings; 
         FIG.  41 B  shows Volcano plots depicting the fold expression change of sequenced mRNAs versus significance (p-value), wherein each dot represents a unique mRNA transcript and significant transcripts are shaded according to either upregulation (red) or downregulation (blue), and wherein fold expression change is measured against ACTB-targeting guide-only expression (including cargo) according to embodiments of the present teachings; 
         FIG.  41 C  shows top significantly upregulated and downregulated genes for Bxb1-only conditions, wherein genes are shown with their corresponding Z-scores of counts per million (cpm) for Bxb1 only expression, GFP-only expression, PASTE targeting ACTB for EGFP insertion, Prime targeting ACTB for EGFP expression without Bxb1, and guide/cargo only according to embodiments of the present teachings; 
         FIG.  42 A  shows a schematic of PASTE performance in the presence of cell cycle inhibition, wherein cells are transfected with plasmids for insertion with PASTE or Cas9-induced HDR and treated with aphidicolin to arrest cell division, and wherein the efficiency of PASTE and HDR are read out with ddPCR or amplicon sequencing respectively according to embodiments of the present teachings; 
         FIG.  42 B  shows the editing efficiency of single mutations by HDR at EMX1 locus with two Cas9 guides in the presence or absence of cell division read out with amplicon sequencing according to embodiments of the present teachings; 
         FIG.  42 C  shows the integration efficiency of various sized GFP inserts up to 13.3 kb at the ACTB locus with PASTE in the presence or absence of cell division according to embodiments of the present teachings; 
         FIG.  42 D  shows the PASTE editing efficiency with two vector (PE2 and Bxb1) and single vector (PE2-P2A-Bxb1) designs in K562 cells according to embodiments of the present teachings; 
         FIG.  42 E  shows the PASTE editing efficiency with single vector (PE2-P2A-Bxb1) designs in primary human T cells according to embodiments of the present teachings; 
         FIG.  42 F  shows the integration efficiency of therapeutically relevant genes at the ACTB locus according to embodiments of the present teachings; 
         FIG.  42 G  shows a schematic of protein production assay for PASTE-integrated transgene, wherein SERPINA1 and CPS1 transgenes are tagged with HIBIT luciferase for readout with both ddPCR and luminescence according to embodiments of the present teachings; 
         FIG.  42 H  shows the integration efficiency of SERPINA1 and CPS1 transgenes in HEK293FT cells at the ACTB locus according to embodiments of the present teachings; 
         FIG.  42 I  shows the integration efficiency of SERPINA1 and CPS1 transgenes in HepG2 cells at the ACTB locus according to embodiments of the present teachings; 
         FIG.  42 J  shows the intracellular levels of SERPINA1-HIBIT and CPS1-HIBIT in HepG2 cells according to embodiments of the present teachings; 
         FIG.  42 K  shows the secreted levels of SERPINA1-HIBIT and CPS1-HIBIT in HepG2 cells according to embodiments of the present teachings; 
         FIG.  43 A  shows the HDR mediated editing of the EMX1 locus that is significantly diminished in non-dividing HEK293FT cells blocked by 5 μM aphidicolin treatment according to embodiments of the present teachings; 
         FIG.  43 B  shows the effect of insert minicircle DNA amount on PASTE-mediated insertion at the ACTB locus in dividing and nondividing HEK293FT cells blocked by 5 μM aphidicolin treatment according to embodiments of the present teachings; 
         FIG.  43 C  shows the PASTE integration of GFP at the ACTB locus with the GFP template delivered via AAV, showing dose dependence of integration efficiency according to embodiments of the present teachings; 
         FIG.  44 A  shows the PASTE integration activity at three endogenous loci comparing the normal PASTE SV40 NLS to a c-Myc NLS/variable bi-partite SV40 NLS design according to embodiments of the present teachings; 
         FIG.  44 B  shows the PASTE integration activity at the ACTB locus with different GFP minicircle template amounts comparing the normal PASTE SV40 NLS to a c-Myc NLS/variable bi-partite SV40 NLS design according to embodiments of the present teachings; 
         FIG.  45    shows the improvement of the PASTE editing activity using a puromycin growth selection marker according to embodiments of the present teachings; 
         FIG.  46 A  shows the integration of SERPINA1 and CPS1 genes that are HIBIT tagged as measured by a protein expression luciferase assay according to embodiments of the present teachings; 
         FIG.  46 B  shows the integration of SERPINA1 and CPS1 genes that are HIBIT tagged as measured by a protein expression luciferase assay normalized to a standardized HIBIT ladder, enabling accurate quantification of protein levels according to embodiments of the present teachings; 
         FIG.  47 A  shows optimization of PASTE constructs with a panel of linkers and reverse transcriptase (RT) modifications for EGFP integration at the ACTB locus, according to embodiments of the present teachings; 
         FIG.  47 B  shows the effect of cargo size on PASTE insertion efficiency at the endogenous ACTB target. Cargos were transfected with fixed molar amounts, according to embodiments of the present teachings; 
         FIG.  48 A  shows prime editing efficiency for the insertion of different length BxbINT AttB sites at ACTB, according to embodiments of the present teachings; 
         FIG.  48 B  shows prime editing efficiency for the insertion of a BxbINT AttB site at ACTB with targeting and non-targeting guides, according to embodiments of the present teachings; 
         FIG.  48 C  shows prime editing efficiency for the insertion of different integrases&#39; (Bxb1, Tp9, and Bt1) AttB sites at ACTB. Both orientations of landing sites are profiled (F, forward; R, reverse), according to embodiments of the present teachings; 
         FIG.  48 D  shows PASTE editing efficiency for the insertion of EGFP at ACTB with and without a nicking guide, according to embodiments of the present teachings; and 
         FIG.  49 A  shows optimization of PASTE editing by dosage titration and protein optimization. PASTE integration efficiency of EGFP at ACTB measured with different doses of a single-vector delivery of components. 
         FIG.  49 B  PASTE integration efficiency of EGFP at ACTB measured with different ratios of a single-vector delivery of components to the EGFP template vector. 
         FIG.  49 C  PASTE integration efficiency of EGFP at ACTB with different RT domain fusions. 
         FIG.  49 D  PASTE integration efficiency of EGFP at ACTB with different RT domain fusions and linkers. 
         FIG.  49 E  PASTE integration efficiency of EGFP at ACTB with mutant RT domains. 
         FIG.  49 F  PASTE integration efficiency of EGFP at ACTB with mutated BxbINT domains. 
         FIG.  50 A  Insertion templates delivered via AAV transduction. PASTE editing machinery was delivered via transfection, and templates were co-delivered via AAV dosing at levels indicated. 
         FIG.  50 B  Schematic of AdV delivery of the complete PASTE system with three viral vectors. 
         FIG.  50 C  Integration efficiency of AdV delivery of integrase, guides, and cargo in HEK293FT and HepG2 cells. BxbINT and guide RNAs or cargo were delivered either via plasmid transfection (P1), AdV transduction (AdV), or omitted (−). SpCas9-RT was only delivered as plasmid or omitted. 
         FIG.  50 D  AdV delivery of all PASTE components in HEK293FT and HepG2 cells. 
         FIG.  50 E  Schematic of mRNA and synthetic guide delivery of PASTE components. 
         FIG.  50 F  Delivery of PASTE system components with mRNA and synthetic guides, paired with either AdV or plasmid cargo. 
         FIG.  50 G  Delivery of circular mRNA with synthetic guides and either AdV or plasmid cargo. 
         FIG.  50 H  PASTE editing efficiency with single vector designs in primary human T cells. 
         FIG.  50 I  PASTE editing efficiency with single vector designs in primary human hepatocytes. 
         FIG.  51 A  PASTE editing efficiency at the LMNB1 locus with 130 bp and 385 bp deletions of the first exon of LMNB1 with combined insertion of an attB sequence. 
         FIG.  51 B  PASTE editing efficiency with a 130 bp deletion of the first exon of LMNB1 with a combined insertion of a 967 bp cargo using the PASTE system. 
     
    
    
     DETAILED DESCRIPTION 
     It will be appreciated that for clarity, the following discussion will describe various aspects of embodiments of the applicant&#39;s teachings. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular feature, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. 
     General Definitions 
     Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &amp; Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley &amp; Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011). 
     As used herein, the singular forms “a”, “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells. 
     As used herein, the term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. 
     The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. 
     As used herein, the term “about” or “approximately” refers to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/−10% or less, +/−5% or less, +/−1% or less, +1-0.5% or less, and +1-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. 
     It is noted that all publications and references cited herein are expressly incorporated herein by reference in their entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. 
     Overview 
     The embodiments disclosed herein provide non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE). A schematic diagram illustrating the concept of PASTE is shown in  FIG.  1   . As discussed in more details below, PASTE comprises the addition of an integration site into a target genome followed by the insertion of one or more genes of interest or one or more nucleic acid sequences of interest at the site. This process can be done as one or more reactions in a cell. The addition of the integration site into the target genome is done using gene editing technologies that include for example, without limitation, prime editing, recombinant adeno-associated virus (rAAV)-mediated nucleic acid integration, transcription activator-like effector nucleases (TALENS), and zinc finger nucleases (ZFNs). The integration of the transgene at the integration site is done using integrase technologies that include for example, without limitation, integrases, recombinases and reverse transcriptases. The necessary components for the site-specific genetic engineering disclosed herein comprise at least one or more nucleases, one or more gRNA, one or more integration enzymes, and one or more sequences that are complementary or associated to the integration site and linked to the one or more genes of interest or one or more nucleic acid sequences of interest to be inserted into the cell genome. 
     An advantage of the non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering disclosed herein is programmable insertion of large elements without reliance on DNA damage responses. 
     Another advantage of the non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering disclosed herein is facile multiplexing, enabling programmable insertion at multiple sites. 
     Another advantage of the non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering disclosed herein is scalable production and delivery through minicircle templates. 
     Prime Editing 
     The present disclosure provides non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering using gene editing technologies, such as prime editing, to add an integration site into a target genome. Prime editing will be discussed in more details below. 
     Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site. A schematic diagram illustrating the concept of prime editing is shown in  FIG.  2   . See, Anzalone, A. V., et al. “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature 576, 149-157 (2019). Prime editing uses a catalytically-impaired Cas9 endonuclease that is fused to an engineered reverse transcriptase (RT) and programmed with a prime-editing guide RNA (pegRNA). The skilled person in the art would appreciate that the pegRNA both specifies the target site and encodes the desired edit. The catalytically-impaired Cas9 endonuclease also comprises a Cas9 nickase that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA. The reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process. 
     The prime editors refer to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a Cas9 H840A nickase. Fusing the RT to the C-terminus of the Cas9 nickase may result in higher editing efficiency. Such a complex is called PE1. The Cas9(H840A) can also be linked to a non-M-MLV reverse transcriptase such as a AMV-RT or XRT (Cas9(H840A)-AMV-RT or XRT). In some embodiments, Cas 9(H840A) can be replaced with Cas12a/b or Cas9(D10A). A Cas9 (wild type), Cas9(H840A), Cas9(D10A) or Cas 12a/b nickase fused to a pentamutant of M-MLV RT (D200N/L603W/T330P/T306K/W313F), having up to about 45-fold higher efficiency is called PE2. In some embodiments, the M-MLV RT comprise one or more of the mutations: Y8H, P51L, S56A, S67R, E69K, V129P, L139P, T197A, H204R, V223H, T246E, N249D, E286R, Q2911, E302K, E302R, F309N, M320L, P330E, L435G, L435R, N454K, D524A, D524G, D524N, E562Q, D583N, H594Q, E607K, D653N, and L671P. In some embodiments, the reverse transcriptase can also be a wild-type or modified transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), Feline Immunodeficiency Virus reverse transcriptase (FIV-RT), FeLV-RT (Feline leukemia virus reverse transcriptase), HIV-RT (Human Immunodeficiency Virus reverse transcriptase), or  Eubacterium  rectale maturase RT (MarathonRT). PE3 involves nicking the non-edited strand, potentially causing the cell to remake that strand using the edited strand as the template to induce HR. The nicking of the non-edited strand can involve the use of a nicking guide RNA (ngRNA). 
     Nicking the non-edited strand can increase editing efficiency. For example, nicking the non-edited strand can increase editing efficiency by about 1.1 fold, about 1.3 fold, about 1.5 fold, about 1.7 fold, about 1.9 fold, about 2.1 fold, about 2.3 fold, about 2.5 fold, about 2.7 fold, about 2.9 fold, about 3.1 fold, about 3.3 fold, about 3.5 fold, about 3.7 fold, about 3.9 fold, 4.1 fold, about 4.3 fold, about 4.5 fold, about 4.7 fold, about 4.9 fold, or any range that is formed from any two of those values as endpoints. 
     Although the optimal nicking position varies depending on the genomic site, nicks positioned 3′ of the edit about 40-90 bp from the pegRNA-induced nick can generally increase editing efficiency without excess indel formation. The prime editing practice allows starting with non-edited strand nicks about 50 bp from the pegRNA-mediated nick, and testing alternative nick locations if indel frequencies exceed acceptable levels. 
     As used herein, the term “guide RNA” (gRNA) and the like refer to a RNA that guide the insertion or deletion of one or more genes of interest or one or more nucleic acid sequences of interest into a target genome. The gRNA can also refer to a prime editing guide RNA (pegRNA), a nicking guide RNA (ngRNA), and a single guide RNA (sgRNA). In some embodiments, the term “gRNA molecule” refers to a nucleic acid encoding a gRNA. In some embodiments, the gRNA molecule is naturally occurring. In some embodiments, a gRNA molecule is non-naturally occurring. In some embodiments, a gRNA molecule is a synthetic gRNA molecule. A gRNA can target a nuclease or a nickase such as Cas9, Cas 12a/b, Cas9 (H840A) or Cas9 (D10A) molecule to a target nucleic acid or sequence in a genome. In some embodiments, the gRNA can bind to a DNA nickase bound to a reverse transcriptase domain. A “modified gRNA,” as used herein, refers to a gRNA molecule that has an improved half-life after being introduced into a cell as compared to a non-modified gRNA molecule after being introduced into a cell. In some embodiments, the guide RNA can facilitate the addition of the insertion site sequence for recognition by integrases, transposases, or recombinases. 
     As used herein, the term “prime-editing guide RNA” (pegRNA) and the like refer to an extended single guide RNA (sgRNA) comprising a primer binding site (PBS), a reverse transcriptase (RT) template sequence, and an integration site sequence that can be recognized by recombinases, integrases, or transposases. Exemplary design parameters for pegRNA are shown in  FIG.  24 A . For example, the PBS can have a length of at least about 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, or more nt. For example, the PBS can have a length of about 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, or any range that is formed from any two of those values as endpoints. For example, the RT template sequence can have a length of at least about 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, or more nt. For example, the RT template sequence can have a length of about 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, or any range that is formed from any two of those values as endpoints. 
     During genome editing, the primer binding site allows the 3′ end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information. The pegRNA is capable for instance, without limitation, of (i) identifying the target nucleotide sequence to be edited and (ii) encoding new genetic information that replaces the targeted sequence. In some embodiments, the pegRNA is capable of (i) identifying the target nucleotide sequence to be edited and (ii) encoding an integration site that replaces the targeted sequence. 
     As used herein, the term “nicking guide RNA” (ngRNA) and the like refer to an RNA sequence that can nick a strand such as an edited strand and a non-edited strand. Exemplary design parameters for ngRNA are shown in  FIG.  24 B . The ngRNA can induce nicks at about 1 or more nt away from the site of the gRNA-induced nick. For example, the ngRNA can nick at least at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or more nt away from the site of the gRNA induced nick. In some embodiments, the ngRNA comprises SEQ ID NO: 75 with guide sequence SEQ ID NO: 74. As used herein, the terms “reverse transcriptase” and “reverse transcriptase domain” refer to an enzyme or an enzymatically active domain that can reverse a RNA transcribe into a complementary DNA. The reverse transcriptase or reverse transcriptase domain is a RNA dependent DNA polymerase. Such reverse transcriptase domains encompass, but are not limited, to a M-MLV reverse transcriptase, or a modified reverse transcriptase such as, without limitation, Superscript® reverse transcriptase (Invitrogen; Carlsbad, Calif.), Superscript® VILO™ cDNA synthesis (Invitrogen; Carlsbad, Calif.), RTX, AMV-RT, and Quantiscript Reverse Transcriptase (Qiagen, Hilden, Germany). 
     The pegRNA-PE complex disclosed herein recognizes the target site in the genome and the Cas9 for example nicks a protospacer adjacent motif (PAM) strand. The primer binding site (PBS) in the pegRNA hybridizes to the PAM strand. The RT template operably linked to the PBS, containing the edit sequence, directs the reverse transcription of the RT template to DNA into the target site. Equilibration between the edited 3′ flap and the unedited 5′ flap, cellular 5′ flap cleavage and ligation, and DNA repair results in stably edited DNA. To optimize base editing, a Cas9 nickase can be used to nick the non-edited strand, thereby directing DNA repair to that strand, using the edited strand as a template. 
     Integrase Technologies 
     The present disclosure provides non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering using integrase technologies. Integrase technologies will be discussed in more details below. 
     The integrase technologies used herein comprise proteins or nucleic acids encoding the proteins that direct integration of a gene of interest or nucleic acid sequence of interest into an integration site via a nuclease such as a prime editing nuclease. The protein directing the integration can be an enzyme such as integration enzyme. The integration enzyme can be an integrase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by integration. The integration enzyme can be a recombinase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by recombination. The integration enzyme can be a reverse transcriptase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by reverse transcription. The integration enzyme can be a retrotransposase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by retrotransposition. 
     As used herein, the term “integration enzyme” refers to an enzyme or protein used to integrate a gene of interest or nucleic acid sequence of interest into a desired location or at the integration site, in the genome of a cell, in a single reaction or multiple reactions. Example of integration enzymes include for example, without limitation, Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, and retrotransposases encoded by R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos. In some embodiments, the term “integration enzyme” refers to a nucleic acid (DNA or RNA) encoding the above-mentioned enzymes. In some embodiments, the Cre recombinase is expressed from a Cre recombinase expression plasmid (SEQ ID NO: 71). 
     Mammalian expression plasmids can be found in Table 1 below. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Name 
                 Full Description 
                 SEQ ID NOS: 
               
               
                   
                   
               
             
            
               
                   
                 PE2-Bxb1 Single 
                 pCMV-PE2- 
                 (SEQ ID NO: 381) 
               
               
                   
                 Vector 
                 P2A- Bxb1 
                   
               
               
                   
                 PE2 prime editor 
                 pCMV-PE2/ 
                 (SEQ ID NO: 382) 
               
               
                   
                   
                 Addgene 
                   
               
               
                   
                   
                 #132775 
                   
               
               
                   
                 PE2*-Bxb1 Single 
                 New NLS 
                 (SEQ ID NO: 383) 
               
               
                   
                 Vector 
                 pCMV- PE2- 
                   
               
               
                   
                   
                 P2A-Bxb1 
                   
               
               
                   
                 PASTEv3 
                 pCMV-SpCas9- 
                 (SEQ ID NO: 384) 
               
               
                   
                   
                 XTEN-RT(1- 
                   
               
               
                   
                   
                 478)-Sto7d- 
                   
               
               
                   
                   
                 GGGGS- 
                   
               
               
                   
                   
                 BxbINT 
                   
               
               
                   
                 ACTB pegRNA 
                 ACTB N- 
                 (SEQ ID NO: 385) 
               
               
                   
                   
                 term PBS 13 
                   
               
               
                   
                   
                 RT 29 attB 46 
                   
               
               
                   
                   
                 pegRNA 
                   
               
               
                   
                 ACTB Nicking + 48 
                 ACTB N- 
                 (SEQ ID NO: 386) 
               
               
                   
                   
                 term Nicking 
                   
               
               
                   
                   
                 guide 1 + 48 
                   
               
               
                   
                   
                 guide 
                   
               
               
                   
                 Bxb1 integrase 
                 pCAG-NLS- 
                 (SEQ ID NO: 387) 
               
               
                   
                   
                 HA-Bxb1integrase/ 
                   
               
               
                   
                   
                 Addgene 
                   
               
               
                   
                   
                 #51271 
                   
               
               
                   
                 TP901-1 Integrase 
                 TP901-1 
                 (SEQ ID NO: 388) 
               
               
                   
                   
                 Integrase 
                   
               
               
                   
                 PhiBT Integrase 
                 PhiBT Integrase 
                 (SEQ ID NO: 389) 
               
               
                   
                 HDR sgRNA guide 
                 Minicircle U6- 
                 (SEQ ID NO: 390) 
               
               
                   
                   
                 sgRNA EFS- 
                   
               
               
                   
                   
                 SpCas9 
                   
               
               
                   
                 HDR EGFP cargo 
                 Cas9 HDR 
                 (SEQ ID NO: 391) 
               
               
                   
                   
                 template site 
                   
               
               
                   
                   
                 with EGFP 
                   
               
               
                   
                 AAV helper 
                 PDF6 AAV 
                 (SEQ ID NO: 392) 
               
               
                   
                 plasmid 
                 helper plasmid 
                   
               
               
                   
                 AAV EGFP donor 
                 GFP AAV donor 
                 (SEQ ID NO: 393) 
               
               
                   
                   
                 plasmid 
                   
               
               
                   
                 AAV2/8 
                 AAV2/8 capsid 
                 (SEQ ID NO: 394) 
               
               
                   
                   
                 protein 
               
               
                   
                   
               
            
           
         
       
     
     Minicircle cargo gene maps can be found in Table 2 below. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                 Full 
                   
               
               
                   
                 Name 
                 Description 
                 SEQ ID NOS: 
               
               
                   
                   
               
             
            
               
                   
                 Cargo EGFP 
                 Parent 
                 (SEQ ID NO: 76) 
               
               
                   
                   
                 minicircle 
                   
               
               
                   
                   
                 plasmid— 
                   
               
               
                   
                   
                 Cargo EGFP 
                   
               
               
                   
                   
                 with attP Bxb1 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                 Cargo 
                 Cargo EGFP 
                 (SEQ ID NO: 395) 
               
               
                   
                 EGFP 
                 with attP Bxb1 
                   
               
               
                   
                 post 
                 site—post 
                   
               
               
                   
                 cleavage 
                 minicircle 
                   
               
               
                   
                   
                 cleavage 
                   
               
               
                   
                 Cargo 
                 Parent 
                 (SEQ ID NO: 396) 
               
               
                   
                 EGFP 
                 minicircle 
                   
               
               
                   
                 for 
                 plasmid— 
                   
               
               
                   
                 fusion 
                 Cargo EGFP 
                   
               
               
                   
                   
                 with attP 
                   
               
               
                   
                   
                 Bxb1 site for 
                   
               
               
                   
                   
                 fusion 
                   
               
               
                   
                 mCherry 
                 Cargo 
                 (SEQ ID NO: 397) 
               
               
                   
                 Cargo post 
                 mCherry 
                   
               
               
                   
                 cleavage 
                 with attP 
                   
               
               
                   
                   
                 Bxb1 site— 
                   
               
               
                   
                   
                 post 
                   
               
               
                   
                   
                 minicircle 
                   
               
               
                   
                   
                 cleavage 
                   
               
               
                   
                 YFP 
                 Cargo YFP 
                 (SEQ ID NO: 398) 
               
               
                   
                 Cargo 
                 with attP Bxb1 
                   
               
               
                   
                 post 
                 site—post 
                   
               
               
                   
                 cleavage 
                 minicircle 
                   
               
               
                   
                   
                 cleavage 
                   
               
               
                   
                 SERPINA1 
                 Cargo 
                 (SEQ ID NO: 399) 
               
               
                   
                 Cargo 
                 SERPINA1 
                   
               
               
                   
                 post 
                 with attP 
                   
               
               
                   
                 cleavage 
                 Bxb1 site— 
                   
               
               
                   
                   
                 post 
                   
               
               
                   
                   
                 minicircle 
                   
               
               
                   
                   
                 cleavage 
                   
               
               
                   
                 CPS1 
                 Cargo CPS1 
                 (SEQ ID NO: 400) 
               
               
                   
                 Cargo 
                 with attP Bxb1 
                   
               
               
                   
                 post 
                 site—post 
                   
               
               
                   
                 cleavage 
                 minicircle 
                   
               
               
                   
                   
                 cleavage 
                   
               
               
                   
                 CFTR Cargo 
                 Parent 
                 (SEQ ID NO: 401) 
               
               
                   
                   
                 minicircle 
                   
               
               
                   
                   
                 plasmid— 
                   
               
               
                   
                   
                 Cargo CFTR 
                   
               
               
                   
                   
                 with attP Bxb1 
                   
               
               
                   
                   
                 site 
                   
               
               
                   
                 NYESO 
                 Cargo 
                 (SEQ ID NO: 402) 
               
               
                   
                 TCR Cargo 
                 NYESO 
                   
               
               
                   
                 post 
                 TCR with 
                   
               
               
                   
                 cleavage 
                 attP Bxb1 
                   
               
               
                   
                   
                 site—post 
                   
               
               
                   
                   
                 minicircle 
                   
               
               
                   
                   
                 cleavage 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, the serine integrase φC31 from φC31 phage is use as integration enzyme. The integrase φC31 in combination with a pegRNA can be used to insert the pseudo attP integration site (SEQ ID NO: 78). A DNA minicircle containing a gene or nucleic acid of interest and attB (SEQ ID NO: 3) site can be used to integrate the gene or nucleic acid of interest into the genome of a cell. This integration can be aided by a co-transfection of an expression vector having the φC31 integrase. 
     As used herein, the term “integrase” refers to a bacteriophage derived integrase, including wild-type integrase and any of a variety of mutant or modified integrases. As used herein, the term “integrase complex” may refer to a complex comprising integrase and integration host factor (IF). As used herein, the term “integrase complex” and the like may also refer to a complex comprising an integrase, an integration host factor, and a bacteriophage X-derived excisionase (Xis). 
     As used herein, the term “recombinase” and the like refer to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, without limitation, Hin, Gin, Tn3, β-six, CinH, ParA, γδ, Bxb1, φC31, TP901, TG1, φBT1, R1, R2, R3, R4, R5, φRV1, φFC1, MR11, A118, U153, and gp29. Examples of serine recombinases also include, without limitation, recombinases Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, and BxZ2 from Mycobacterial phages. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange. 
     Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications. See, e.g., Brown et al., “Serine recombinases as tools for genome engineering.” Methods, 2011; 53(4):372-9; Hirano et al., “Site-specific recombinases as tools for heterologous gene integration.” Appl. Microbiol. Biotechnol. 2011; 92(2):227-39; Chavez and Calos, “Therapeutic applications of the ΦC31 integrase system.” Curr. Gene Ther. 2011; 11(5):375-81; Turan and Bode, “Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.” FASEB J. 2011; 25(12):4088-107; Venken and Bellen, “Genome-wide manipulations of  Drosophila melanogaster  with transposons, Flp recombinase, and ΦC31 integrase.” Methods Mol. Biol. 2012; 859:203-28; Murphy, “Phage recombinases and their applications.” Adv. Virus Res. 2012; 83:367-414; Zhang et al., “Conditional gene manipulation: Creating a new biological era.” J. Zhejiang Univ. Sci. B. 2012; 13(7):511-24; Karpenshif and Bernstein, “From yeast to mammals: recent advances in genetic control of homologous recombination.” DNA Repair (Amst). 2012; 1; 11(10):781-8; the entire contents of each are hereby incorporated by reference in their entirety. 
     The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the disclosure. The methods and compositions of the disclosure can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities (See, e.g., Groth et al., “Phage integrases: biology and applications.” J. Mol. Biol. 2004; 335, 667-678; Gordley et al., “Synthesis of programmable integrases.” Proc. Natl. Acad. Sci. USA. 2009; 106, 5053-5058; the entire contents of each are hereby incorporated by reference in their entirety). 
     Other examples of recombinases that are useful in the systems, methods, and compositions described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the disclosure. 
     As used herein, the term “retrotransposase” and the like refer to an enzyme, or combination of one or more enzymes, wherein at least one enzyme has a reverse transcriptase domain. Retrotransposases are capable of inserting long sequences (e.g., over 3000 nucleotides) of heterologous nucleic acid into a genome. Examples of retrotransposases include for example, without limitation, retrotransposases encoded by elements such as R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), Minos, and any mutants thereof. 
     In some embodiments, the one or more genes of interest or one or more nucleic acid sequences of interest are inserted into a desired location in a genome using a RNA fragment, such as a retrotransposon, encoding the nucleic acid linked to a complementary or associated integration site. The insertion of the nucleic acid of interest into a location in the desired location in the genome using a retrotransposon is aided by a retrotransposase. 
     The gene and nucleic acid sequence of interest disclosed herein can be any gene and nucleic acid sequence that are known in the art. The gene and nucleic acid sequence of interest can be for therapeutic and/or diagnostic uses. Examples of genes of interest include, without limitation, GBA, BTK, ADA, CNGB3, CNGA3, ATF6, GNAT2, ABCA1, ABCA7, APOE, CETP, LIPC, MMP9, PLTP, VTN, ABCA4, MFSD8, TLR3, TLR4, ERCC6, HMCN1, HTRA1, MCDR4, MCDR5, ARMS2, C2, C3, CFB, CFH, JAG1, NOTCH2, CACNA1F, SERPINA1, TTR, GSN, B2M, APOA2, APOA1, OSMR, ELP4, PAX6, ARG, ASL, PITX2, FOXC1, BBS1, BBS10, BBS2, BBS9, MKKS, MKS1, BBS4, BBS7, TTC8, ARL6, BBS5, BBS12, TRIM32, CEP290, ADIPOR1, BBIP1, CEP19, IFT27, LZTFL1, DMD, BEST1, HBB, CYP4V2, AMACR, CYP7B1, HSD3B7, AKR1D1, OPN1SW, NR2F1, RLBP1, RGS9, RGS9BP, PROM1, PRPH2, GUCY2D, CACD, CHM, ALAD, ASS1, SLC25A13, OTC, ACADVL, ETFDH, TMEM67, CC2D2A, RPGRIP1L, KCNV2, CRX, GUCA1A, CERKL, CDHR1, PDE6C, TTLL5, RPGR, CEP78, C21orf2, C8ORF37, RPGRIP1, ADAMS, POC1B, PITPNM3, RAB28, CACNA2D4, AIPL1, UNC119, PDE6H, OPN1LW, RIMS1, CNNM4, IFT81, RAX2, RDH5, SEMA4A, CORD17, PDE6B, GRK1, SAG, RHO, CABP4, GNB3, SLC24A1, GNAT1, GRM6, TRPM1, LRIT3, TGFBI, TACSTD2, KRT12, OVOL2, CPS1, UGT1A1, UGT1A9, UGT1A8, UGT1A7, UGT1A6, UGT1A5, UGT1A4, CFTR, DLD, EFEMP1, ABCC2, ZNF408, LRP5, FZD4, TSPAN12, EVR3, APOB, SLC2A2, LOC106627981, GBA1, NR2E3, OAT, SLC40A1, F8, F9, UROD, CPDX, HFE, JH, LDLR, EPHX1, TJP2, BAAT, NBAS, LARS1, HAMP, HJV, RS1, ADAMTS18, LRAT, RPE65, LCA5, MERTK, GDF6, RD3, CCT2, CLUAP1, DTHD1, NMNAT1, SPATA7, IFT140, IMPDH1, OTX2, RDH12, TULP1, CRB1, MT-ND4, MT-ND1, MT-ND6, BCKDHA, BCKDHB, DBT, MMAB, ARSB, GUSB, NAGS, NPC1, NPC2, NDP, OPA1, OPA3, OPA4, OPA5, RTN4IP1, TMEM126A, OPA6, OPA8, ACO2, PAH, PRKCSH, SEC63, GAA, UROS, PPDX, HPX, HMOX1, HMBS, MIR223, CYP1B1, LTBP2, AGXT, ATP8B1, ABCB11, ABCB4, FECH, ALAS2, PRPF31, RP1, EYS, TOPORS, USH2A, CNGA1, C2ORF71, RP2, KLHL7, ORF1, RP6, RP24, RP34, ROM1, ADGRA3, AGBL5, AHR, ARHGEF18, CA4, CLCC1, DHDDS, EMC1, FAM161A, HGSNAT, HK1, IDH3B, KIAA1549, KIZ, MAK, NEUROD1, NRL, PDE6A, PDE6G, PRCD, PRPF3, PRPF4, PRPF6, PRPF8, RBP3, REEP6, SAMD11, SLC7A14, SNRNP200, SPP2, ZNF513, NEK2, NEK4, NXNL1, OFD1, RP1L1, RP22, RP29, RP32, RP63, RP9, RGR, POMGNT1, DHX38, ARL3, COL2A1, SLCO1B1, SLCO1B3, KCNJ13, TIMP3, ELOVL4, TFR2, FAH, HPD, MYO7A, CDH23, PCDH15, DFNB31, GPR98, USH1C, USH1G, CIB2, CLRN1, HARS, ABHD12, ADGRV1, ARSG, CEP250, IMPG1, IMPG2, VCAN, G6PC1, ATP7B and any derivatives thereof. 
     As used here, the terms “retrotransposons,” “jumping genes,” “jumping nucleic acids,” and the like refer to cellular movable genetic elements dependent on reverse transcription. The retrotransposons are of non-replication competent cellular origin, and are capable of carrying a foreign nucleic acid sequence. The retrotransposons can act as parasites of retroviruses, retaining certain classical hallmarks, such as long terminal repeats (LTR), retroviral primer binding sites, and the like. However, the naturally occurring retrotransposons usually do not contain functional retroviral structure genes, which would normally be capable of recombining to yield replication competent viruses. Some retrotransposons are examples of so-called “selfish DNA”, or genetic information, which encodes nothing except the ability to replicate itself. The retrotransposon may do so by utilizing the occasional presence of a retrovirus or a retrotransposase within the host cell, efficiently packaging itself within the viral particle, which transports it to the new host genome, where it is expressed again as RNA. The information encoded within that RNA is potentially transported with the jumping gene. A retrotransposon can be a DNA transposon or a retrotransposon, including a LTR retrotransposon or a non-LTR retrotransposon. 
     Non-long terminal repeat (LTR) retrotransposons are a type of mobile genetic elements that are widespread in eukaryotic genomes. They include two classes: the apurinic/apyrimidinic endonuclease (APE)-type and the restriction enzyme-like endonuclease (RLE)-type. The APE class retrotransposons are comprised of two functional domains: an endonuclease/DNA binding domain, and a reverse transcriptase domain. The RLE class are comprised of three functional domains: a DNA binding domain, a reverse transcription domain, and an endonuclease domain. The reverse transcriptase domain of non-LTR retrotransposon functions by binding an RNA sequence template and reverse transcribing it into the host genome&#39;s target DNA. The RNA sequence template has a 3′ untranslated region which is specifically bound to the transposase, and a variable 5′ region generally having Open Reading Frame(s) (“ORF”) encoding transposase proteins. The RNA sequence template may also comprise a 5′ untranslated region which specifically binds the retrotransposase. In some embodiments, a non-LTR transposons can include a LINE retrotransposon, such as L1, and a SINE retrotransposon, such as an Alu sequence. Other examples include for example, without limitation, R1, R2, R3, R4, and R5 retro-transposons (Moss, W. N. et al., RNA Biol. 2011, 8(5), 714-718; and Burke, W. D. et al., Molecular Biology and Evolution 2003, 20(8), 1260-1270). The transposon can be autonomous or non-autonomous. 
     LTR retrotransposons, which include retroviruses, make up a significant fraction of the typical mammalian genome, comprising about 8% of the human genome and 10% of the mouse genome. Lander et al., 2001, Nature 409, 860-921; Waterson et al., 2002, Nature 420, 520-562. LTR elements include retrotransposons, endogenous retroviruses (ERVs), and repeat elements with HERV origins, such as SINE-R. LTR retrotransposons include two LTR sequences that flank a region encoding two enzymes: integrase and retrotransposase. 
     ERVs include human endogenous retroviruses (HERVs), the remnants of ancient germ-cell infections. While most HERV proviruses have undergone extensive deletions and mutations, some have retained ORFS coding for functional proteins, including the glycosylated env protein. The env gene confers the potential for LTR elements to spread between cells and individuals. Indeed, all three open reading frames (pol, gag, and env) have been identified in humans, and evidence suggests that ERVs are active in the germline. See, e.g., Wang et al., 2010, Genome Res. 20, 19-27. Moreover, a few families, including the HERV-K (HML-2) group, have been shown to form viral particles, and an apparently intact provirus has recently been discovered in a small fraction of the human population. See, e.g., Bannert and Kurth, 2006, Proc. Natl. Acad. USA 101, 14572-14579. 
     LTR retrotransposons insert into new sites in the genome using the same steps of DNA cleavage and DNA strand-transfer observed in DNA transposons. In contrast to DNA transposons, however, recombination of LTR retrotransposons involves an RNA intermediate. LTR retrotransposons make up about 8% of the human genome. See, e.g., Lander et al., 2001, Nature 409, 860-921; Hua-Van et al., 2011, Biol. Dir. 6, 19. 
     Integration Site 
     The present disclosure provides non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering via the addition of an integration site into a target genome. The integration site will be discussed in more details below. 
     As used herein, the term “integration site” refers to the site within the target genome where one or more genes of interest or one or more nucleic acid sequences of interest are inserted. Examples of integration sites include for example, without limitation, a lox71 site (SEQ ID NO: 1), attB sites (SEQ ID NO: 3 and SEQ ID NO: 43), attP sites (SEQ ID NO: 4 and SEQ ID NO: 44), an attL site (SEQ ID NO: 67), an attR site (SEQ ID NO: 68), a Vox site (SEQ ID NO: 69), a FRT site (SEQ ID NO: 70), or a pseudo attP site (SEQ ID NO: 78). The integration site can be inserted into the genome or a fragment thereof of a cell using a nuclease, a gRNA, and/or an integration enzyme. The integration site can be inserted into the genome of a cell using a prime editor such as, without limitation, PE1, PE2, and PE3, wherein the integration site is carried on a pegRNA. The pegRNA can target any site that is known in the art. Examples of cites targeted by the pegRNA include, without limitation, ACTB, SUPT16H, SRRM2, NOLC1, DEPDC4, NES, LMNB1, AAVS1 locus, CC10, CFTR, SERPINA1, ABCA4, and any derivatives thereof. The complementary integration site may be operably linked to a gene of interest or nucleic acid sequence of interest in an exogenous DNA or RNA. In some embodiments, one integration site is added to a target genome. In some embodiments, more than one integration sites are added to a target genome. 
     To insert multiple genes or nucleic acids of interest, two or more integration sites are added to a desired location. Multiple DNA comprising nucleic acid sequences of interest are flanked orthogonal to the integration sequences, such as, without limitation, attB and attP. An integration site is “orthogonal” when it does not significantly recognize the recognition site or nucleotide sequence of a recombinase. Thus, one attB site of a recombinase can be orthogonal to an attB site of a different recombinase. In addition, one pair of attB and attP sites of a recombinase can be orthogonal to another pair of attB and attP sites recognized by the same recombinase. A pair of recombinases are considered orthogonal to each other, as defined herein, when there is recognition of each other&#39;s attB or attP site sequences. 
     The lack of recognition of integration sites or pairs of sites by the same recombinase or a different recombinase can be less than about 30%. In some embodiments, the lack of recognition of integration sites or pairs of sites by the same recombinase or a different recombinase can be less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, less than about 2%, about 1%, or any range that is formed from any two of those values as endpoints. The crosstalk can be less than about 30%. In some embodiments, the crosstalk is less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, less than about 2%, less than about 1%, or any range that is formed from any two of those values as endpoints. 
     In some embodiments, the attB and/or attP site sequences comprise a central dinucleotide sequence. It has been shown that, for example, the central dinucleotide can be changed to GA from GT and that only GA containing attB/attP sites interact and will not cross react with GT containing sequences. In some embodiments, the central dinucleotide is selected from the group consisting of AG, AC, TG, TC, CA, CT, GA, AA, TT, CC, GG, AT, TA, GC, CG and GT. 
     As used herein, the term “pair of an attB and attP site sequences” and the like refer to attB and attP site sequences that share the same central dinucleotide and can recombine. This means that in the presence of one serine integrase as many as six pairs of these orthogonal att sites can recombine (attPTT will specifically recombine with attBTT, attPTC will specifically recombine with attBTC, and so on). 
     In some embodiments, the central dinucleotide is nonpalindromic. In some embodiments, the central dinucleotide is palindromic. In some embodiments, a pair of an attB site sequence and an attP site sequence are used in different DNA encoding genes of interest or nucleic acid sequences of interest for inducing directional integration of two or more different nucleic acids. 
     The Table 3 below shows examples of pairs of attB site sequence and attP site sequence with different central dinucleotide (CD). 
     
       
         
           
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Pair 
                 attB 
                 attP 
                 CD 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 SEQ ID NO: 5 
                 SEQ ID NO: 6 
                 TT 
               
               
                 2 
                 SEQ ID NO: 7 
                 SEQ ID NO: 8 
                 AA 
               
               
                 3 
                 SEQ ID NO: 9 
                 SEQ ID NO: 10 
                 CC 
               
               
                 4 
                 SEQ ID NO: 11 
                 SEQ ID NO: 12 
                 GG 
               
               
                 5 
                 SEQ ID NO: 13 
                 SEQ ID NO: 14 
                 TG 
               
               
                 6 
                 SEQ ID NO: 15 
                 SEQ ID NO: 16 
                 GT 
               
               
                 7 
                 SEQ ID NO: 17 
                 SEQ ID NO: 18 
                 CT 
               
               
                 8 
                 SEQ ID NO: 19 
                 SEQ ID NO: 20 
                 CA 
               
               
                 9 
                 SEQ ID NO: 21 
                 SEQ ID NO: 22 
                 TC 
               
               
                 10 
                 SEQ ID NO: 23 
                 SEQ ID NO: 24 
                 GA 
               
               
                 11 
                 SEQ ID NO: 25 
                 SEQ ID NO: 26 
                 AG 
               
               
                 12 
                 SEQ ID NO: 27 
                 SEQ ID NO: 28 
                 AC 
               
               
                 13 
                 SEQ ID NO: 29 
                 SEQ ID NO: 30 
                 AT 
               
               
                 14 
                 SEQ ID NO: 31 
                 SEQ ID NO: 32 
                 GC 
               
               
                 15 
                 SEQ ID NO: 33 
                 SEQ ID NO: 34 
                 CG 
               
               
                 16 
                 SEQ ID NO: 35 
                 SEQ ID NO: 36 
                 TA 
               
               
                   
               
            
           
         
       
     
     PASTE 
     The present disclosure provides non-naturally occurring or engineered systems, methods, and compositions for site-specific genetic engineering using PASTE. PASTE will be discussed in more details below. 
     The site-specific genetic engineering disclosed herein is for the insertion of one or more genes of interest or one or more nucleic acid sequences of interest into a genome of a cell. In some embodiments, the gene of interest is a mutated gene implicated in a genetic disease such as, without limitation, a metabolic disease, cystic fibrosis, muscular dystrophy, hemochromatosis, Tay-Sachs, Huntington disease, Congenital Deafness, Sickle cell anemia, Familial hypercholesterolemia, adenosine deaminase (ADA) deficiency, X-linked SCID (X-SCID), and Wiskott-Aldrich syndrome (WAS). In some embodiments, the gene of interest or nucleic acid sequence of interest can be a reporter gene upstream or downstream of a gene for genetic analyses such as, without limitation, for determining the expression of a gene. In some embodiments, the reporter gene is a GFP template (SEQ ID NO: 76) or a  Gaussia  Luciferase (G-Luciferase) template (SEQ ID NO: 77) In some embodiments, the gene of interest or nucleic acid sequence of interest can be used in plant genetics to insert genes to enhance drought tolerance, weather hardiness, and increased yield and herbicide resistance in plants. In some embodiments, the gene of interest or nucleic acid sequence of interest can be used for site-specific insertion of a protein (e.g., a lysosomal enzyme), a blood factor (e.g., Factor I, II, V, VII, X, XI, XII or XIII), a membrane protein, an exon, an intracellular protein (e.g., a cytoplasmic protein, a nuclear protein, an organellar protein such as a mitochondrial protein or lysosomal protein), an extracellular protein, a structural protein, a signaling protein, a regulatory protein, a transport protein, a sensory protein, a motor protein, a defense protein, or a storage protein, an anti-inflammatory signaling molecules into cells for treatment of immune diseases, including but not limited to arthritis, psoriasis, lupus, coeliac disease, glomerulonephritis, hepatitis, and inflammatory bowel disease. 
     The size of the inserted gene or nucleic acid can vary from about 1 bp to about 50,000 bp. In some embodiments, the size of the inserted gene or nucleic acid can be about 1 bp, 10 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 600 bp, 800 bp, 1000 bp, 1200 bp, 1400 bp, 1600 bp, 1800 bp, 2000 bp, 2200 bp, 2400 bp, 2600 bp, 2800 bp, 3000 bp, 3200 bp, 3400 bp, 3600 bp, 3800 bp, 4000 bp, 4200 bp, 4400 bp, 4600 bp, 4800 bp, 5000 bp, 5200 bp, 5400 bp, 5600 bp, 5800 bp, 6000 bp, 6200, 6400 bp, 6600 bp, 6800 bp, 7000 bp, 7200 bp, 7400 bp, 7600 bp, 7800 bp, 8000 bp, 8200 bp, 8400 bp, 8600 bp, 8800 bp, 9000 bp, 9200 bp, 9400 bp, 9600 bp, 9800 bp, 10,000 bp, 10,200 bp, 10,400 bp, 10,600 bp, 10,800 bp, 11,000 bp, 11,200 bp, 11,400 bp, 11,600 bp, 11,800 bp, 12,000 bp, 14,000 bp, 16,000 bp, 18,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or any range that is formed from any two of those values as endpoints. 
     In some embodiments, the site-specific engineering using the gene of interest or nucleic acid sequence of interest disclosed herein is for the engineering of T cells and NKs for tumor targeting or allogeneic generation. These can involve the use of receptor or CAR for tumor specificity, anti-PD1 antibody, cytokines like IFN-gamma, TNF-alpha, IL-15, IL-12, IL-18, IL-21, and IL-10, and immune escape genes. 
     In the present disclosure, the site-specific insertion of the gene of interest or nucleic acid of interest is performed through Programmable Addition via Site-Specific Targeting Elements (PASTE). Components for inserting a gene of interest or a nucleic acid of interest using PASTE are for example, without limitation, a nuclease, a gRNA adding the integration site, a DNA or RNA strand comprising the gene or nucleic acid linked to a sequence that is complementary or associated to the integration site, and an integration enzyme. Components for inserting a gene of interest or a nucleic acid of interest using PASTE are for example, without limitation, a prime editor expression, pegRNA adding the integration site, nicking guide RNA, integration enzyme (Cre or serine recombinase), transgene vector comprising the gene of interest or nucleic acid sequence of interest with gene and integration signal. The nuclease and prime editor integrate the integration site into the genome. The integration enzyme integrates the gene of interest into the integration site. In some embodiments, the transgene vector comprising the gene or nucleic acid sequence of interest with gene and integration signal is a DNA minicircle devoid of bacterial DNA sequences. In some embodiments, the transgenic vector is a eukaryotic or prokaryotic vector. 
     As used herein, the term “vector” or “transgene vector” refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include for example, without limitation, a promoter, an operator (optional), a ribosome binding site, and/or other sequences. Eukaryotic cells are generally known to utilize promoters (constitutive, inducible or tissue specific), enhancers, and termination and polyadenylation signals, although some elements may be deleted and other elements added without sacrificing the necessary expression. The transgenic vector may encode the PE and the integration enzyme, linked to each other via a linker. The linker can be a cleavable linker. For example, transgenic vector encoding the PE and the integration enzyme, linked to each other via a linker is pCMV PE2 P2A Cre comprises SEQ ID NO: 73. In some embodiments, the linker can be a non-cleavable linker. In some embodiments the nuclease, prime editor, and/or integration enzyme can be encoded in different vectors. 
     A method of inserting multiple genes or nucleic acid sequences of interest into a single site according to embodiments of the present disclosure is illustrated in  FIG.  12   . In some embodiments, multiplexing involves inserting multiple genes of interest in multiple loci using unique pegRNA as illustrated in  FIG.  13    (Merrick, C. A. et al., ACS Synth. Biol. 2018, 7, 299-310). The insertion of multiple genes of interest or nucleic acids of interest into a cell genome, referred herein as “multiplexing,” is facilitated by incorporation of the complementary 5′ integration site to the 5′ end of the DNA or RNA comprising the first nucleic acid and 3′ integration site to the 3′ end of the DNA or RNA comprising the last nucleic acid. In some embodiments, the number of genome of interest or amino acid sequences of interest that are inserted into a cell genome using multiplexing can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or any range that is formed from any two of those values as endpoints. 
     In some embodiments, multiplexing allows integration of for example, signaling cascade, over-expression of a protein of interest with its cofactor, insertion of multiple genes mutated in a neoplastic condition, or insertion of multiple CARs for treatment of cancer. 
     In some embodiments, the integration sites may be inserted into the genome using non-prime editing methods such as rAAV mediated nucleic acid integration, TALENS and ZFNs. A number of unique properties make AAV a promising vector for human gene therapy (Muzyczka, CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, 158:97-129 (1992)). Unlike other viral vectors, AAVs have not been shown to be associated with any known human disease and are generally not considered pathogenic. Wild type AAV is capable of integrating into host chromosomes in a site-specific manner M. Kotin et al., PROC. NATL. ACAD. SCI, USA, 87:2211-2215 (1990); R. J. Samulski, EMBO 10(12):3941-3950 (1991)). Instead of creating a double-stranded DNA break, AAV stimulates endogenous homologous recombination to achieve the DNA modification. Further, transcription activator-like effector nucleases (TALENs) and Zinc-finger nucleases (ZFNs) for genome editing and introducing targeted DSBs. The specificity of TALENs arises from two polymorphic amino acids, the so-called repeat variable diresidues (RVDs) located at positions 12 and 13 of a repeated unit. TALENS are linked to FokI nucleases, which cleaves the DNA at the desired locations. ZFNs are artificial restriction enzymes for custom site-specific genome editing. Zinc fingers themselves are transcription factors, where each finger recognizes 3-4 bases. By mixing and matching these finger modules, researchers can customize which sequence to target. 
     As used herein, the terms “administration,” “introducing,” or “delivery” into a cell, a tissue, or an organ of a plasmid, nucleic acids, or proteins for modification of the host genome refers to the transport for such administration, introduction, or delivery that can occur in vivo, in vitro, or ex vivo. Plasmids, DNA, or RNA for genetic modification can be introduced into cells by transfection, which is typically accomplished by chemical means (e.g., calcium phosphate transfection, polyethyleneimine (PEI) Or lipofection), physical means (electroporation or microinjection), infection (this typically means the introduction of an infectious agent such as a virus (e.g., a baculovirus expressing the AAV Rep gene)), transduction (in microbiology, this refers to the stable infection of cells by viruses, or the transfer of genetic material from one microorganism to another by viral factors (e.g., bacteriophages)). Vectors for the expression of a recombinant polypeptide, protein or oligonucleotide may be obtained by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection) in a cell, a tissue, an organ or a subject. The vector can be delivered by preparing the vector in a pharmaceutically acceptable carrier for the in vitro, ex vivo, or in vivo delivery to the carrier. 
     As used herein, the term “transfection” refers to the uptake of an exogenous nucleic acid molecule by a cell. A cell is “transfected” when an exogenous nucleic acid has been introduced into the cell membrane. The transfection can be a single transfection, co-transfection, or multiple transfection. Numerous transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52: 456. Such techniques can be used to introduce one or more exogenous nucleic acid molecules into a suitable host cell. 
     In some embodiments, the exogenous nucleic acid molecule and/or other components for gene editing are combined and delivered in a single transfection. In other embodiments, the exogenous nucleic acid molecule and/or other components for gene editing are not combined and delivered in a single transfection. In some embodiments, exogenous nucleic acid molecule and/or other components for gene editing are combined and delivered in a single transfection to comprise for example, without limitation, a prime editing vector, a landing site such as a landing site containing pegRNA, a nicking guide such as a nicking guide for stimulating prime editing, an expression vector such as an expression vector for a corresponding integrase or recombinase, a minicircle DNA cargo such as a minicircle DNA cargo encoding for green fluorescent protein (GFP), any derivatives thereof, and any combinations thereof. In some embodiments, the gene of interest or amino acid sequence of interest can be introduced using liposomes. In some embodiments, the gene of interest or amino acid sequence of interest can be delivered using suitable vectors for instance, without limitation, plasmids and viral vectors. Examples of viral vectors include, without limitation, adeno-associated viruses (AAV), lentiviruses, adenoviruses, other viral vectors, derivatives thereof, or combinations thereof. The proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmids or viral vectors. In some embodiments, the delivery is via nanoparticles or exosomes. For example, exosomes can be particularly useful in delivery RNA. 
     In some embodiments, the prime editing inserts the landing site with efficiencies of at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%. In some embodiments, the prime editing inserts the landing site(s) with efficiencies of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, or any range that is formed from any two of those values as endpoints. 
     Sequences 
     Sequences of enzymes, guides, integration sites, and plasmids can be found in Table 4 below. 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 SEQ ID NO/ 
                   
               
               
                 DESCRIPTION/ 
                   
               
               
                 SOURCE 
                 SEQUENCE 
               
               
                   
               
             
            
               
                 SEQ ID NO: 1 
                 ATAACTTCGTATAATGTATGCTATACGAACGGTA 
               
               
                 Lox71 
                   
               
               
                 (Artificial sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 2 
                 TACCGTTCGTATAATGTATGCTATACGAAGTTAT 
               
               
                 Lox66 
                   
               
               
                 (Artificial sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 3 
                 GGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCCG 
               
               
                 attB 
                 G 
               
               
                 (Artificial sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 4 
                 CCGGATGATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGC 
               
               
                 attP 
                 C 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 5 
                 GGCTTGTCGACGACGGCGTTCTCCGTCGTCAGGATCAT 
               
               
                 attB-TT 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 6 
                 GTGGTTTGTCTGGTCAACCACCGCGTTCTCAGTGGTGTACGGTACA 
               
               
                 attP-TT 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 7 
                 GGCTTGTCGACGACGGCGAACTCCGTCGTCAGGATCAT 
               
               
                 attB-AA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 8 
                 GTGGTTTGTCTGGTCAACCACCGCGAACTCAGTGGTGTACGGTAC 
               
               
                 attP-AA 
                 AAACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 9 
                 GGCTTGTCGACGACGGCGCCCTCCGTCGTCAGGATCAT 
               
               
                 attB-CC 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 10 
                 GTGGTTTGTCTGGTCAACCACCGCGCCCTCAGTGGTGTACGGTACA 
               
               
                 attP-CC 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 11 
                 GGCTTGTCGACGACGGCGGGCTCCGTCGTCAGGATCAT 
               
               
                 attB-GG 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 12 
                 GTGGTTTGTCTGGTCAACCACCGCGGGCTCAGTGGTGTACGGTAC 
               
               
                 attP-GG 
                 AAACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 13 
                 GGCTTGTCGACGACGGCGTGCTCCGTCGTCAGGATCAT 
               
               
                 attB-TG 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 14 
                 GTGGTTTGTCTGGTCAACCACCGCGTGCTCAGTGGTGTACGGTACA 
               
               
                 attP-TG 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 15 
                 GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT 
               
               
                 attB-GT 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 16 
                 GTGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACA 
               
               
                 attP-GT 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 17 
                 GGCTTGTCGACGACGGCGCTCTCCGTCGTCAGGATCAT 
               
               
                 attB-CT 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 18 
                 GTGGTTTGTCTGGTCAACCACCGCGCTCTCAGTGGTGTACGGTACA 
               
               
                 attP-CT 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 19 
                 GGCTTGTCGACGACGGCGCACTCCGTCGTCAGGATCAT 
               
               
                 attB-CA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 20 
                 GTGGTTTGTCTGGTCAACCACCGCGCACTCAGTGGTGTACGGTACA 
               
               
                 attP-CA 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 21 
                 GGCTTGTCGACGACGGCGTCCTCCGTCGTCAGGATCAT 
               
               
                 attB-TC 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 22 
                 GTGGTTTGTCTGGTCAACCACCGCGTCCTCAGTGGTGTACGGTACA 
               
               
                 attP-TC 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 23 
                 GGCTTGTCGACGACGGCGGACTCCGTCGTCAGGATCAT 
               
               
                 attB-GA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 24 
                 GTGGTTTGTCTGGTCAACCACCGCGGACTCAGTGGTGTACGGTAC 
               
               
                 attP-GA 
                 AAACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 25 
                 GGCTTGTCGACGACGGCGAGCTCCGTCGTCAGGATCAT 
               
               
                 attB-AG 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 26 
                 GTGGTTTGTCTGGTCAACCACCGCGAGCTCAGTGGTGTACGGTAC 
               
               
                 attP-AG 
                 AAACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 27 
                 GGCTTGTCGACGACGGCGACCTCCGTCGTCAGGATCAT 
               
               
                 attB-AC 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 28 
                 GTGGTTTGTCTGGTCAACCACCGCGACCTCAGTGGTGTACGGTACA 
               
               
                 attP-AC 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 29 
                 GGCTTGTCGACGACGGCGATCTCCGTCGTCAGGATCAT 
               
               
                 attB-AT 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 30 
                 GTGGTTTGTCTGGTCAACCACCGCGATCTCAGTGGTGTACGGTACA 
               
               
                 attP-AT 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 31 
                 GGCTTGTCGACGACGGCGGCCTCCGTCGTCAGGATCAT 
               
               
                 attB-GC 
                   
               
               
                 (Artificial Sequence 
                   
               
               
                   
               
               
                 SEQ ID NO: 32 
                 GTGGTTTGTCTGGTCAACCACCGCGGCCTCAGTGGTGTACGGTACA 
               
               
                 attP-GC 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 33 
                 GGCTTGTCGACGACGGCGCGCTCCGTCGTCAGGATCAT 
               
               
                 attB-CG 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 34 
                 GTGGTTTGTCTGGTCAACCACCGCGCGCTCAGTGGTGTACGGTACA 
               
               
                 attP-CG 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 35 
                 GGCTTGTCGACGACGGCGTACTCCGTCGTCAGGATCAT 
               
               
                 attB-TA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 36 
                 GTGGTTTGTCTGGTCAACCACCGCGTACTCAGTGGTGTACGGTACA 
               
               
                 attP-TA 
                 AACCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 37 
                 TGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCC 
               
               
                 C31-attB 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 38 
                 GTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGG 
               
               
                 C31-attP 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 39 
                 GCGCCCAAGTTGCCCATGACCATGCCGAAGCAGTGGTAGAAGGGC 
               
               
                 R4-attB 
                 ACCGGCAGACAC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 40 
                 AGGCATGTTCCCCAAAGCGATACCACTTGAAGCAGTGGTACTGCT 
               
               
                 R4-attP 
                 TGTGGGTACACTCTGCGGGTGATGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 41 
                 GTCCTTGACCAGGTTTTTGACGAAAGTGATCCAGATGATCCAGCTC 
               
               
                 BT1-attB 
                 CACACCCCGAACGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 42 
                 GGTGCTGGGTTGTTGTCTCTGGACAGTGATCCATGGGAAACTACTC 
               
               
                 BT1-attP 
                 AGCACCACCAATGTTCC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 43 
                 TCGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCC 
               
               
                 Bxb-attB 
                 GGGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 44 
                 GTCGTGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGT 
               
               
                 Bxb-attP 
                 ACAAACCCCGAC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 45 
                 GATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGT 
               
               
                 TG1-attB 
                 C 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 46 
                 TCAACCCCGTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAG 
               
               
                 TG1-attP 
                 TTGGGCGGGATAGCCTGCCCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 47 
                 AACGATTTTCAAAGGATCACTGAATCAAAAGTATTGCTCATCCAC 
               
               
                 C1-attB 
                 GCGAAATTTTTC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 48 
                 AATATTTTAGGTATATGATTTTGTTTATTAGTGTAAATAACACTAT 
               
               
                 C1-attP 
                 GTACCTAAAAT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 49 
                 TGTAAAGGAGACTGATAATGGCATGTACAACTATACTCGTCGGTA 
               
               
                 C370-attB 
                 AAAAGGCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 50 
                 TAAAAAAATACAGCGTTTTTCATGTACAACTATACTAGTTGTAGTG 
               
               
                 C370-attP 
                 CCTAAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 51 
                 GAGCGCCGGATCAGGGAGTGGACGGCCTGGGAGCGCTACACGCT 
               
               
                 K38-attB 
                 GTGGCTGCGGTC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 52 
                 CCCTAATACGCAAGTCGATAACTCTCCTGGGAGCGTTGACAACTT 
               
               
                 K38-attP 
                 GCGCACCCTGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 53 
                 TCTCGTGGTGGTGGAAGGTGTTGGTGCGGGGTTGGCCGTGGTCGA 
               
               
                 RB-attB 
                 GGTGGGGTGGTGGTAGCCATTCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 54 
                 GCACAGGTGTAGTGTATCTCACAGGTCCACGGTTGGCCGTGGACT 
               
               
                 RV-attP 
                 GCTGAAGAACATTCCACGCCAGGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 55 
                 AGTGCAGCATGTCATTAATATCAGTACAGATAAAGCTGTATCTCCT 
               
               
                 SPBC-attB 
                 GTGAACACAATGGGTGCCA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 56 
                 AAAGTAGTAAGTATCTTAAAAAACAGATAAAGCTGTATATTAAGA 
               
               
                 SPBC-attP 
                 TACTTACTAC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 57 
                 TGATAATTGCCAACACAATTAACATCTCAATCAAGGTAAATGCTTT 
               
               
                 TP901-attB 
                 TTCGTTTT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 58 
                 AATTGCGAGTTTTTATTTCGTTTATTTCAATTAAGGTAACTAAAAA 
               
               
                 TP901-attP 
                 ACTCCTTT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 59 
                 AAGGTAGCGTCAACGATAGGTGTAACTGTCGTGTTTGTAACGGTA 
               
               
                 Wβ-attB 
                 CTTCCAACAGCTGGCGTTTCAGT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 60 
                 TAGTTTTAAAGTTGGTTATTAGTTACTGTGATATTTATCACGGTAC 
               
               
                 Wβ-attP 
                 CCAATAACCAATGAATATTTGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 61 
                 TGTAACTTTTTCGGATCAAGCTATGAAGGACGCAAAGAGGGAACT 
               
               
                 A118-attB 
                 AAACACTTAATT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 62 
                 TTGTTTAGTTCCTCGTTTTCTCTCGTTGGAAGAAGAAGAAACGAGA 
               
               
                 A118-attP 
                 AACTAAAATTA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 63 
                 CAACCTGTTGACATGTTTCCACAGACAACTCACGTGGAGGTAGTC 
               
               
                 BL3-attB 
                 ACGGCTTTTACGTTAGTT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 64 
                 GAGAATACTGTTGAACAATGAAAAACTAGGCATGTAGAAGTTGTT 
               
               
                 BL3-attP 
                 TGTGCACTAACTTTAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 65 
                 ACAGGTCAACACATCGCAGTTATCGAACAATCTTCGAAAATGTAT 
               
               
                 MR11-attB 
                 GGAGGCACTTGTATCAATATAGGATGTATACCTTCGAAGACACTT 
               
               
                 (Artificial Sequence) 
                 GTACATGATGGATTAGAAGGCAAATCCTTT 
               
               
                   
               
               
                 SEQ ID NO: 66 
                 CAAAATAAAAAACATTGATTTTTATTAACTTCTTTTGTGCGGAACT 
               
               
                 MR11-attP 
                 ACGAACAGTTCATTAATACGAAGTGTACAAACTTCCATACAAAAA 
               
               
                 (Artificial Sequence) 
                 TAACCACGACAATTAAGACGTGGTTTCTA 
               
               
                   
               
               
                 SEQ ID NO: 67 
                 ATTATTTCTCACCCTGA 
               
               
                 attL 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 68 
                 ATCATCTCCCACCCGGA 
               
               
                 attR 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 69 
                 AATAGGTCTG AGAACGCCCA TTCTCAGACG TATT 
               
               
                 Vox 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 70 
                 GAAGTTCCTATAC TTTCTAGA GAATAGGAACTTC 
               
               
                 FRT 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 71 
                 GGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGG 
               
               
                 Cre recombinase 
                 GGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACT 
               
               
                 expression plasmid 
                 TACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC 
               
               
                 (Artificial Sequence) 
                 ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG 
               
               
                   
                 GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCC 
               
               
                   
                 CACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA 
               
               
                   
                 TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT 
               
               
                   
                 ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATT 
               
               
                   
                 AGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTC 
               
               
                   
                 ACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATT 
               
               
                   
                 TATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG 
               
               
                   
                 GGCGCGCGCCAGGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGG 
               
               
                   
                 GGGGGGGCGGGGGGGGGCGGCGGCAGCCAATCAGAGCGGCGCGC 
               
               
                   
                 TCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCT 
               
               
                   
                 ATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGC 
               
               
                   
                 CTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCC 
               
               
                   
                 GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACG 
               
               
                   
                 GCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCT 
               
               
                   
                 TGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAG 
               
               
                   
                 GGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGT 
               
               
                   
                 GTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGC 
               
               
                   
                 TGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGT 
               
               
                   
                 GTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGG 
               
               
                   
                 GGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCG 
               
               
                   
                 TGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAA 
               
               
                   
                 CCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTT 
               
               
                   
                 CGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGC 
               
               
                   
                 CGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGG 
               
               
                   
                 CCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCC 
               
               
                   
                 CCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTG 
               
               
                   
                 CCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCC 
               
               
                   
                 CAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCC 
               
               
                   
                 TCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGA 
               
               
                   
                 AATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCT 
               
               
                   
                 TCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTT 
               
               
                   
                 CGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACC 
               
               
                   
                 GGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTT 
               
               
                   
                 CCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCAT 
               
               
                   
                 TTTGGCAAAGAATTCTGAGCCGCCACCATGGCCAATTTACTGACC 
               
               
                   
                 GTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGAT 
               
               
                   
                 GAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCG 
               
               
                   
                 TTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGT 
               
               
                   
                 GGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAG 
               
               
                   
                 AACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGG 
               
               
                   
                 TCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACAT 
               
               
                   
                 GCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGC 
               
               
                   
                 TGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGC 
               
               
                   
                 CGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTT 
               
               
                   
                 CGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGA 
               
               
                   
                 TATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTA 
               
               
                   
                 CGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGT 
               
               
                   
                 ACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACG 
               
               
                   
                 CTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTA 
               
               
                   
                 ACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATG 
               
               
                   
                 ATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTG 
               
               
                   
                 CCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAG 
               
               
                   
                 GGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATG 
               
               
                   
                 ACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTG 
               
               
                   
                 TCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGG 
               
               
                   
                 AGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGA 
               
               
                   
                 ACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCC 
               
               
                   
                 TGCTGGAAGATGGCGATGGACCGGTGGAACAAAAACTTATTTCTG 
               
               
                   
                 AAGAAGATCTGTGATAGCGGCCGCACTCCTCAGGTGCAGGCTGCC 
               
               
                   
                 TATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAA 
               
               
                   
                 TACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCA 
               
               
                   
                 TGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTAT 
               
               
                   
                 TTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAA 
               
               
                   
                 GGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATT 
               
               
                   
                 TGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAA 
               
               
                   
                 CAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCC 
               
               
                   
                 TGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAG 
               
               
                   
                 ATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTA 
               
               
                   
                 AAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTG 
               
               
                   
                 ACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTC 
               
               
                   
                 GACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCT 
               
               
                   
                 GTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCC 
               
               
                   
                 GGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA 
               
               
                   
                 CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA 
               
               
                   
                 ACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCAT 
               
               
                   
                 AGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGT 
               
               
                   
                 TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGC 
               
               
                   
                 AGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTG 
               
               
                   
                 AGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGT 
               
               
                   
                 TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAA 
               
               
                   
                 ATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTG 
               
               
                   
                 TCCAAACTCATCAATGTATCTTATCATGTCTGGATCCGCTGCATTA 
               
               
                   
                 ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG 
               
               
                   
                 CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG 
               
               
                   
                 CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA 
               
               
                   
                 TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA 
               
               
                   
                 AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG 
               
               
                   
                 CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG 
               
               
                   
                 ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT 
               
               
                   
                 ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC 
               
               
                   
                 GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA 
               
               
                   
                 AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGG 
               
               
                   
                 TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT 
               
               
                   
                 TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC 
               
               
                   
                 AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT 
               
               
                   
                 AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTC 
               
               
                   
                 TTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTT 
               
               
                   
                 GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTT 
               
               
                   
                 GGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT 
               
               
                   
                 TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT 
               
               
                   
                 CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA 
               
               
                   
                 ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA 
               
               
                   
                 GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC 
               
               
                   
                 AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG 
               
               
                   
                 CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA 
               
               
                   
                 TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG 
               
               
                   
                 AGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC 
               
               
                   
                 CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG 
               
               
                   
                 GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA 
               
               
                   
                 TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCC 
               
               
                   
                 AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG 
               
               
                   
                 GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC 
               
               
                   
                 AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAG 
               
               
                   
                 CGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC 
               
               
                   
                 CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT 
               
               
                   
                 ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT 
               
               
                   
                 CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT 
               
               
                   
                 CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAA 
               
               
                   
                 CTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAAC 
               
               
                   
                 TCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCAC 
               
               
                   
                 TCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT 
               
               
                   
                 CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA 
               
               
                   
                 ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC 
               
               
                   
                 AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATA 
               
               
                   
                 CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG 
               
               
                   
                 CACATTTCCCCGAAAAGTGCCACCTG 
               
               
                   
               
               
                 SEQ ID NO: 72 
                 AGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAA 
               
               
                 GFP-Lox66 Cre 
                 CAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGG 
               
               
                 expression plasmid 
                 CTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGAT 
               
               
                 (Artificial Sequence) 
                 GCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTG 
               
               
                   
                 TCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGG 
               
               
                   
                 CAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAG 
               
               
                   
                 CTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTAT 
               
               
                   
                 TGGGCGAAGTGCCGGGGCAGGATCTCCATGTCATCTACACCTTGC 
               
               
                   
                 TCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCT 
               
               
                   
                 GCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAA 
               
               
                   
                 ACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGT 
               
               
                   
                 CGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGC 
               
               
                   
                 CGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGA 
               
               
                   
                 TCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG 
               
               
                   
                 GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTG 
               
               
                   
                 TGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTG 
               
               
                   
                 CTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTA 
               
               
                   
                 CGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTT 
               
               
                   
                 CTTGACGAGTTCTTCTGAATTATTAACTCGAGATCCACTAGAGTGT 
               
               
                   
                 GGCGGCCGCATTCTTATAATCAGCATCATGATGTGGTACCACATCA 
               
               
                   
                 TGATGCTGATTACCCCCAACTGAGAGAACTCAAAGGTTACCCCAG 
               
               
                   
                 TTGGGGCGGGCCCACAAATAAAGCAATAGCATCACAAATTTCACA 
               
               
                   
                 AATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAAC 
               
               
                   
                 TCATCGAGCTCGAGATCTGGCGAAGGCGATGGGGGTCTTGAAGGC 
               
               
                   
                 GTGCTGGTACTCCACGATGCCCAGCTCGGTGTTGCTGTGCAGCTCC 
               
               
                   
                 TCCACGCGGCGGAAGGCGAACATGGGGCCCCCGTTCTGCAGGATG 
               
               
                   
                 CTGGGGTGGATGGCGCTCTTGAAGTGCATGTGGCTGTCCACCACG 
               
               
                   
                 AAGCTGTAGTAGCCGCCGTCGCGCAGGCTGAAGGTGCGGGCGAAG 
               
               
                   
                 CTGCCCACCAGCACGTTATCGCCCATGGGGTGCAGGTGCTCCACG 
               
               
                   
                 GTGGCGTTGCTGCGGATGATCTTGTCGGTGAAGATCACGCTGTCCT 
               
               
                   
                 CGGGGAAGCCGGTGCCCACCACCTTGAAGTCGCCGATCACGCGGC 
               
               
                   
                 CGGCCTCGTAGCGGTAGCTGAAGCTCACGTGCAGCACGCCGCCGT 
               
               
                   
                 CCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGAT 
               
               
                   
                 GGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAA 
               
               
                   
                 GTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCT 
               
               
                   
                 GAAGGTCAGGGCGCCTTTGGTGCTCTTCATCTTGTTGGTCATGCGG 
               
               
                   
                 CCCTGCTCGGGGGTGCCCTCTCCGCCGCCCACCAGCTCGAACTCCA 
               
               
                   
                 CGCCGTTCAGGGTGCCGGTGATGCGGCACTCGATCTTCATGGCGG 
               
               
                   
                 GCATGGTGGCGACCGGTAGCGCTAGCGGCTTCGGATAACTTCGTA 
               
               
                   
                 TAGCATACATTATACGAACGGTAAGCGCTACCGCCGGCATACCCA 
               
               
                   
                 AGTGAAGTTGCTCGCAGCTTATAGTCGCGCCCGGGGAGCCCAAGG 
               
               
                   
                 GCACGCCCTGGCACCGCGGCCGCTGAGTCTCGACCATCATCATCA 
               
               
                   
                 TCATCATTGAGTTTATCTGGGATAACAGGGTAATGTCATCTAGGGA 
               
               
                   
                 TAACAGGGTATGTCATCTGGGATAACAGGGTAATGTATCTAGGGA 
               
               
                   
                 TAACAGGGTAATGTCATCTGGGATAACAGGGTAATGTCATCTAGG 
               
               
                   
                 GATAACAGGGTATGTCATCTGGGATAACAGGGTAATGTATCTAGG 
               
               
                   
                 GATAACAGGGTAATGTCATCTGGGATAACAGGGTAATGTCATCTA 
               
               
                   
                 GGGATAACAGGGTATGTCATCTGGGATAACAGGGTAATGTATCTA 
               
               
                   
                 GGGATAACAGGGTAATGTCATCTGGGATAACAGGGTAATGTCATC 
               
               
                   
                 TAGGGATAACAGGGTATGTCATCTGGGATAACAGGGTAATGTATC 
               
               
                   
                 TAGGGATAACAGGGTAATGTCATCTGGGATAACAGGGTAATGTCA 
               
               
                   
                 TCTAGGGATAACAGGGTATGTCATCTGGGATAACAGGGTAATGTA 
               
               
                   
                 TCTAGGGATAACAGGGTAATGTCATCTGGGATAACAGGGTAATGT 
               
               
                   
                 CATCTAGGGATAACAGGGTATGTCATCTGGGATAACAGGGTAATG 
               
               
                   
                 TATCTAGGGATAACAGGGTAATGTCATCTGGGATAACAGGGTAAT 
               
               
                   
                 GTCATCTAGGGATAACAGGGTAAATGTCATCTAGGGATAACAGGG 
               
               
                   
                 TAATGTCATCTAGGGATAACAGGGTAATGTCATCTGGGATAACAG 
               
               
                   
                 GGTAATGTCATCTAGGGATAACAGGGTAATGTATCGCCAGCGTCG 
               
               
                   
                 CACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCG 
               
               
                   
                 CCAGCAGTTAGGGATTAGCGTCTTAAGCTGGCGCGAGGACCAACG 
               
               
                   
                 TATCAGCCAGGCGAAGCTGCTTTTGAGCACCACCCGGATGCCTAT 
               
               
                   
                 CGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTC 
               
               
                   
                 TCGCGGGTATTTAAAAAATGCACCGGGGCCAGCCCGAGCGAGTTC 
               
               
                   
                 CGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCCGTCAAGTTG 
               
               
                   
                 TCATAATTGGTAACGAATCAGACAATTGACGGCTTGACGGAGTAG 
               
               
                   
                 CATAGGGTTTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATT 
               
               
                   
                 ATGCATGCCGCTTCGCCTTCGCGCGCGAATTGATCTGCTGCCTCGC 
               
               
                   
                 GCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCC 
               
               
                   
                 GGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACA 
               
               
                   
                 AGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGC 
               
               
                   
                 AGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTT 
               
               
                   
                 AACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATG 
               
               
                   
                 CGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATC 
               
               
                   
                 AGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG 
               
               
                   
                 TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC 
               
               
                   
                 GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA 
               
               
                   
                 GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT 
               
               
                   
                 GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA 
               
               
                   
                 AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA 
               
               
                   
                 AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG 
               
               
                   
                 TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG 
               
               
                   
                 GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT 
               
               
                   
                 CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC 
               
               
                   
                 CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA 
               
               
                   
                 GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC 
               
               
                   
                 TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA 
               
               
                   
                 GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGT 
               
               
                   
                 ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA 
               
               
                   
                 GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT 
               
               
                   
                 GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGA 
               
               
                   
                 TCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT 
               
               
                   
                 GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGCGGATACA 
               
               
                   
                 TATTTGAATGTATTTAGAAAAATAAACAAAAGAGTTTGTAGAAAC 
               
               
                   
                 GCAAAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAATTTGATGCC 
               
               
                   
                 TGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTG 
               
               
                   
                 CTTCGCAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGG 
               
               
                   
                 AGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTC 
               
               
                   
                 TTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACT 
               
               
                   
                 CTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTC 
               
               
                   
                 ACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACTG 
               
               
                   
                 CCGCCAGGCAAATTCTGTTTTATCAGACCGCTTCTGCGTTCTGATT 
               
               
                   
                 TAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACAGCC 
               
               
                   
                 AAGCTGGAGACCGTTTGGCCCCCCTCGAGCACGTAGAAAGCCAGT 
               
               
                   
                 CCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCT 
               
               
                   
                 ATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGC 
               
               
                   
                 TTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATG 
               
               
                   
                 GACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAA 
               
               
                   
                 GGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCC 
               
               
                   
                 AAGGATCTGATGGCGCAGGGGATCA 
               
               
                   
               
               
                 SEQ ID NO: 73 
                 ACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA 
               
               
                 pCMV PE2 P2A Cre 
                 CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATA 
               
               
                 plasmid 
                 ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG 
               
               
                 (Artificial Sequence) 
                 CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA 
               
               
                   
                 GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT 
               
               
                   
                 GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC 
               
               
                   
                 CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC 
               
               
                   
                 AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT 
               
               
                   
                 ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATC 
               
               
                   
                 AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC 
               
               
                   
                 CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAAC 
               
               
                   
                 GGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAA 
               
               
                   
                 TGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTG 
               
               
                   
                 GTTTAGTGAACCGTCAGATCCGCTAGAGATCCGCGGCCGCTAATA 
               
               
                   
                 CGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACAGCCGAC 
               
               
                   
                 GGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCGACAA 
               
               
                   
                 GAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTG 
               
               
                   
                 GGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAA 
               
               
                   
                 GGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGAT 
               
               
                   
                 CGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCG 
               
               
                   
                 GCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACC 
               
               
                   
                 GGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGG 
               
               
                   
                 TGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGG 
               
               
                   
                 AAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCG 
               
               
                   
                 TGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACC 
               
               
                   
                 TGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGG 
               
               
                   
                 CTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACT 
               
               
                   
                 TCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACA 
               
               
                   
                 AGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGG 
               
               
                   
                 AAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGT 
               
               
                   
                 CTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCC 
               
               
                   
                 AGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTG 
               
               
                   
                 CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACC 
               
               
                   
                 TGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACG 
               
               
                   
                 ACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCG 
               
               
                   
                 ACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAG 
               
               
                   
                 CGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAG 
               
               
                   
                 CGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGAC 
               
               
                   
                 CCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAA 
               
               
                   
                 AGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACAT 
               
               
                   
                 TGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCC 
               
               
                   
                 CATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCT 
               
               
                   
                 GAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACG 
               
               
                   
                 GCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTC 
               
               
                   
                 TGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGG 
               
               
                   
                 AAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGG 
               
               
                   
                 GCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA 
               
               
                   
                 AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGG 
               
               
                   
                 ACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACT 
               
               
                   
                 TCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCC 
               
               
                   
                 TGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGA 
               
               
                   
                 AATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCG 
               
               
                   
                 AGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGA 
               
               
                   
                 AAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATC 
               
               
                   
                 GAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTC 
               
               
                   
                 AACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAG 
               
               
                   
                 GACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA 
               
               
                   
                 AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGAT 
               
               
                   
                 CGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGT 
               
               
                   
                 GATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGC 
               
               
                   
                 TGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCA 
               
               
                   
                 AGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAA 
               
               
                   
                 ACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGG 
               
               
                   
                 ACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACG 
               
               
                   
                 AGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCA 
               
               
                   
                 TCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGG 
               
               
                   
                 GCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAG 
               
               
                   
                 AACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAAT 
               
               
                   
                 GAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCC 
               
               
                   
                 TGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAG 
               
               
                   
                 CTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGAC 
               
               
                   
                 CAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCT 
               
               
                   
                 ATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAG 
               
               
                   
                 GTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGT 
               
               
                   
                 GCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGC 
               
               
                   
                 AGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATC 
               
               
                   
                 TGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCC 
               
               
                   
                 GGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAG 
               
               
                   
                 CACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGAC 
               
               
                   
                 GAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAA 
               
               
                   
                 GTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAA 
               
               
                   
                 GTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTG 
               
               
                   
                 AACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTG 
               
               
                   
                 GAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGG 
               
               
                   
                 AAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGC 
               
               
                   
                 CAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAG 
               
               
                   
                 ATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAG 
               
               
                   
                 ACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGA 
               
               
                   
                 TTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATAT 
               
               
                   
                 CGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGT 
               
               
                   
                 CTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGA 
               
               
                   
                 AGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG 
               
               
                   
                 TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT 
               
               
                   
                 CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCA 
               
               
                   
                 TGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAG 
               
               
                   
                 CCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTG 
               
               
                   
                 CCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATG 
               
               
                   
                 CTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTG 
               
               
                   
                 CCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGA 
               
               
                   
                 AGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTG 
               
               
                   
                 TGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA 
               
               
                   
                 GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACA 
               
               
                   
                 AAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAG 
               
               
                   
                 AGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGG 
               
               
                   
                 GAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGA 
               
               
                   
                 AGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCC 
               
               
                   
                 ACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC 
               
               
                   
                 AGCTGGGAGGTGACTCTGGAGGATCTAGCGGAGGATCCTCTGGCA 
               
               
                   
                 GCGAGACACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCAGT 
               
               
                   
                 GGCGGCAGCAGCGGCGGCAGCAGCACCCTAAATATAGAAGATGA 
               
               
                   
                 GTATCGGCTACATGAGACCTCAAAAGAGCCAGATGTTTCTCTAGG 
               
               
                   
                 GTCCACATGGCTGTCTGATTTTCCTCAGGCCTGGGCGGAAACCGG 
               
               
                   
                 GGGCATGGGACTGGCAGTTCGCCAAGCTCCTCTGATCATACCTCTG 
               
               
                   
                 AAAGCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCA 
               
               
                   
                 CAAGAAGCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTG 
               
               
                   
                 GACCAGGGAATACTGGTACCCTGCCAGTCCCCCTGGAACACGCCC 
               
               
                   
                 CTGCTACCCGTTAAGAAACCAGGGACTAATGATTATAGGCCTGTC 
               
               
                   
                 CAGGATCTGAGAGAAGTCAACAAGCGGGTGGAAGACATCCACCC 
               
               
                   
                 CACCGTGCCCAACCCTTACAACCTCTTGAGCGGGCTCCCACCGTCC 
               
               
                   
                 CACCAGTGGTACACTGTGCTTGATTTAAAGGATGCCTTTTTCTGCC 
               
               
                   
                 TGAGACTCCACCCCACCAGTCAGCCTCTCTTCGCCTTTGAGTGGAG 
               
               
                   
                 AGATCCAGAGATGGGAATCTCAGGACAATTGACCTGGACCAGACT 
               
               
                   
                 CCCACAGGGTTTCAAAAACAGTCCCACCCTGTTTAATGAGGCACT 
               
               
                   
                 GCACAGAGACCTAGCAGACTTCCGGATCCAGCACCCAGACTTGAT 
               
               
                   
                 CCTGCTACAGTACGTGGATGACTTACTGCTGGCCGCCACTTCTGAG 
               
               
                   
                 CTAGACTGCCAACAAGGTACTCGGGCCCTGTTACAAACCCTAGGG 
               
               
                   
                 AACCTCGGGTATCGOGCCTCGGCCAAGAAAGCCCAAATTTGCCAG 
               
               
                   
                 AAACAGGTCAAGTATCTGGGGTATCTTCTAAAAGAGGGTCAGAGA 
               
               
                   
                 TGGCTGACTGAGGCCAGAAAAGAGACTGTGATGGGGCAGCCTACT 
               
               
                   
                 CCGAAGACCCCTCGACAACTAAGGGAGTTCCTAGGGAAGGCAGGC 
               
               
                   
                 TTCTGTCGCCTCTTCATCCCTGGGTTTGCAGAAATGGCAGCCCCCC 
               
               
                   
                 TGTACCCTCTCACCAAACCGGGGACTCTGTTTAATTGGGGCCCAGA 
               
               
                   
                 CCAACAAAAGGCCTATCAAGAAATCAAGCAAGCTCTTCTAACTGC 
               
               
                   
                 CCCAGCCCTGGGGTTGCCAGATTTGACTAAGCCCTTTGAACTCTTT 
               
               
                   
                 GTCGACGAGAAGCAGGGCTACGCCAAAGGTGTCCTAACGCAAAA 
               
               
                   
                 ACTGGGACCTTGGCGTCGGCCGGTGGCCTACCTGTCCAAAAAGCT 
               
               
                   
                 AGACCCAGTAGCAGCTGGGTGGCCCCCTTGCCTACGGATGGTAGC 
               
               
                   
                 AGCCATTGCCGTACTGACAAAGGATGCAGGCAAGCTAACCATGGG 
               
               
                   
                 ACAGCCACTAGTCATTCTGGCCCCCCATGCAGTAGAGGCACTAGT 
               
               
                   
                 CAAACAACCCCCCGACCGCTGGCTTTCCAACGCCCGGATGACTCA 
               
               
                   
                 CTATCAGGCCTTGCTTTTGGACACGGACCGGGTCCAGTTCGGACCG 
               
               
                   
                 GTGGTAGCCCTGAACCCGGCTACGCTGCTCCCACTGCCTGAGGAA 
               
               
                   
                 GGGCTGCAACACAACTGCCTTGATATCCTGGCCGAAGCCCACGGA 
               
               
                   
                 ACCCGACCCGACCTAACGGACCAGCCGCTCCCAGACGCCGACCAC 
               
               
                   
                 ACCTGGTACACGGATGGAAGCAGTCTCTTACAAGAGGGACAGCGT 
               
               
                   
                 AAGGCGGGAGCTGCGGTGACCACCGAGACCGAGGTAATCTGGGCT 
               
               
                   
                 AAAGCCCTGCCAGCCGGGACATCCGCTCAGCGGGCTGAACTGATA 
               
               
                   
                 GCACTCACCCAGGCCCTAAAGATGGCAGAAGGTAAGAAGCTAAAT 
               
               
                   
                 GTTTATACTGATAGCCGTTATGCTTTTGCTACTGCCCATATCCATG 
               
               
                   
                 GAGAAATATACAGAAGGCGTGGGTGGCTCACATCAGAAGGCAAA 
               
               
                   
                 GAGATCAAAAATAAAGACGAGATCTTGGCCCTACTAAAAGCCCTC 
               
               
                   
                 TTTCTGCCCAAAAGACTTAGCATAATCCATTGTCCAGGACATCAAA 
               
               
                   
                 AGGGACACAGCGCCGAGGCTAGAGGCAACCGGATGGCTGACCAA 
               
               
                   
                 GCGGCCCGAAAGGCAGCCATCACAGAGACTCCAGACACCTCTACC 
               
               
                   
                 CTCCTCATAGAAAATTCATCACCCTCTGGCGGCTCAAAAAGAACC 
               
               
                   
                 GCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTCGG 
               
               
                   
                 AAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGT 
               
               
                   
                 GGAGGAGAACCCTGGACCTAATTTACTGACCGTACACCAAAATTT 
               
               
                   
                 GCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAA 
               
               
                   
                 CCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACC 
               
               
                   
                 TGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCA 
               
               
                   
                 AGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTC 
               
               
                   
                 GCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAAC 
               
               
                   
                 TATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCC 
               
               
                   
                 GGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATG 
               
               
                   
                 CGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAA 
               
               
                   
                 ACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCA 
               
               
                   
                 CTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCA 
               
               
                   
                 TTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTG 
               
               
                   
                 CCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAA 
               
               
                   
                 TGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAG 
               
               
                   
                 GTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGC 
               
               
                   
                 GATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCT 
               
               
                   
                 GTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCAC 
               
               
                   
                 CAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAAC 
               
               
                   
                 TCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATA 
               
               
                   
                 CCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGA 
               
               
                   
                 TATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGG 
               
               
                   
                 TGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTG 
               
               
                   
                 GATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGAT 
               
               
                   
                 TAATTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA 
               
               
                   
                 GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAA 
               
               
                   
                 GGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGAAAATTGCAT 
               
               
                   
                 CGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG 
               
               
                   
                 GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATG 
               
               
                   
                 CTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCA 
               
               
                   
                 GCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTAAT 
               
               
                   
                 CATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT 
               
               
                   
                 TCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTAGGG 
               
               
                   
                 TGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTG 
               
               
                   
                 CCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA 
               
               
                   
                 tcggccaacgcgcggggagaggcggtttgcgtattgggcgctctt 
               
               
                   
                 CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCG 
               
               
                   
                 GCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC 
               
               
                   
                 AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC 
               
               
                   
                 AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT 
               
               
                   
                 TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCT 
               
               
                   
                 CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG 
               
               
                   
                 GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCC 
               
               
                   
                 TGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT 
               
               
                   
                 GGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG 
               
               
                   
                 GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC 
               
               
                   
                 CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC 
               
               
                   
                 GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG 
               
               
                   
                 GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA 
               
               
                   
                 GTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT 
               
               
                   
                 CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAG 
               
               
                   
                 CTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT 
               
               
                   
                 GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA 
               
               
                   
                 AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA 
               
               
                   
                 AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC 
               
               
                   
                 TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT 
               
               
                   
                 AAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAAT 
               
               
                   
                 CAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATA 
               
               
                   
                 GTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC 
               
               
                   
                 TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGC 
               
               
                   
                 TCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGG 
               
               
                   
                 GCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT 
               
               
                   
                 CTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA 
               
               
                   
                 ATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC 
               
               
                   
                 ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGA 
               
               
                   
                 TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT 
               
               
                   
                 AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG 
               
               
                   
                 TGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGT 
               
               
                   
                 CATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC 
               
               
                   
                 AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC 
               
               
                   
                 CGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA 
               
               
                   
                 AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA 
               
               
                   
                 GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC 
               
               
                   
                 ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG 
               
               
                   
                 TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG 
               
               
                   
                 GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT 
               
               
                   
                 TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATAT 
               
               
                   
                 TTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACAT 
               
               
                   
                 TTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCGAT 
               
               
                   
                 CTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGC 
               
               
                   
                 CGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTC 
               
               
                   
                 GCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGC 
               
               
                   
                 TTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTG 
               
               
                   
                 CGCTGCTTCGCGATGTACGGGCCAGATAT 
               
               
                   
               
               
                 SEQ ID NO: 74 
                 GTCAACCAGTATCCCGGTGC 
               
               
                 +90 ngRNA guide 
                   
               
               
                 sequence 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 75 
                 GTCAACCAGTATCCCGGTGCGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 +90 ngRNA 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 (Artificial Sequence) 
                 CGGTGC 
               
               
                   
               
               
                 SEQ ID NO: 76 
                 TGATCCCCTGCGCCATCAGATCCTTGGCGGCGAGAAAGCCATCCA 
               
               
                 GFP minicircle 
                 GTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCT 
               
               
                 template (before 
                 GGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCT 
               
               
                 cleavage into a 
                 ATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCT 
               
               
                 minicircle) 
                 TGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGG 
               
               
                 (Artificial Sequence) 
                 GGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGCTCGAGGGGG 
               
               
                   
                 GCCAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGAT 
               
               
                   
                 TTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGAT 
               
               
                   
                 AAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGA 
               
               
                   
                 CCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAG 
               
               
                   
                 TGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAA 
               
               
                   
                 TAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCT 
               
               
                   
                 GTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGG 
               
               
                   
                 GAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGG 
               
               
                   
                 GCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG 
               
               
                   
                 GCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTT 
               
               
                   
                 TATTTTTCTAAATACATTCAAATATGTATCCGCTCATGACCAAAAT 
               
               
                   
                 CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA 
               
               
                   
                 AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCT 
               
               
                   
                 GCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT 
               
               
                   
                 TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT 
               
               
                   
                 CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTA 
               
               
                   
                 GTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC 
               
               
                   
                 GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT 
               
               
                   
                 CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG 
               
               
                   
                 CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT 
               
               
                   
                 TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC 
               
               
                   
                 TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG 
               
               
                   
                 TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA 
               
               
                   
                 GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT 
               
               
                   
                 CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG 
               
               
                   
                 GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT 
               
               
                   
                 TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTA 
               
               
                   
                 TCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTG 
               
               
                   
                 ATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGA 
               
               
                   
                 GCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC 
               
               
                   
                 ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAAT 
               
               
                   
                 CTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGC 
               
               
                   
                 TACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCG 
               
               
                   
                 CTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAG 
               
               
                   
                 ACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTC 
               
               
                   
                 ACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGC 
               
               
                   
                 GAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGC 
               
               
                   
                 AGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTC 
               
               
                   
                 TGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTT 
               
               
                   
                 TTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGC 
               
               
                   
                 ATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAA 
               
               
                   
                 CATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAA 
               
               
                   
                 GCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGAC 
               
               
                   
                 GCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGG 
               
               
                   
                 CGACAAGCAAACATGCTGTGCGACGCTGGCGATACATTACCCTGT 
               
               
                   
                 TATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCCTG 
               
               
                   
                 TTATCCCTAGATGACATTACCCTGTTATCCCTAGATGACATTTACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATAAACTCAA 
               
               
                   
                 TGATGATGATGATGATGGTCGAGACTCAGCGGCCGCGGTGCCAGG 
               
               
                   
                 GCGTGCCCTTGGGCTCCCCGGGCGCGACTATAAGCTGCGAGCAAC 
               
               
                   
                 TTCACTTGGGTATGCCGGCGGTAGCGCTTACCGTTCGTATAATGTA 
               
               
                   
                 TGCTATACGAAGTTATCCGAAGCCGCTAGCGGTGGTTTGTCTGGTC 
               
               
                   
                 AACCACCGCGGTCTCAGTGGTGTACGGTACAAACCCAGCTACCGG 
               
               
                   
                 TCGCCACCATGCCCGCCATGAAGATCGAGTGCCGCATCACCGGCA 
               
               
                   
                 CCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGC 
               
               
                   
                 ACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAA 
               
               
                   
                 AGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGG 
               
               
                   
                 CTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGAA 
               
               
                   
                 CCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCG 
               
               
                   
                 CATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAG 
               
               
                   
                 CTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGGT 
               
               
                   
                 GGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGAT 
               
               
                   
                 CATCCGCAGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGA 
               
               
                   
                 TAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGA 
               
               
                   
                 CGGCGGCTACTACAGCTTCGTGGTGGACAGCCACATGCACTTCAA 
               
               
                   
                 GAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTT 
               
               
                   
                 CGCCTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCTGGG 
               
               
                   
                 CATCGTGGAGTACCAGCACGCCTTCAAGACCCCCATCGCCTTCGCC 
               
               
                   
                 AGATCTCGAGCTCGATGAGTTTGGACAAACCACAACTAGAATGCA 
               
               
                   
                 GTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTA 
               
               
                   
                 TTTGTGGGCCCGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTT 
               
               
                   
                 GGGGGTAATCAGCATCATGATGTGGTACCACATCATGATGCTGAT 
               
               
                   
                 TATAAGAATGCGGCCGCCACACTCTAGTGGATCTCGAGTTAATAA 
               
               
                   
                 TTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCG 
               
               
                   
                 AATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCC 
               
               
                   
                 CATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCT 
               
               
                   
                 ATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATG 
               
               
                   
                 AATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAG 
               
               
                   
                 GCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTC 
               
               
                   
                 GCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGC 
               
               
                   
                 TCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAG 
               
               
                   
                 TACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCA 
               
               
                   
                 GGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCAT 
               
               
                   
                 GATGGATACTTTCTCGGCAGGAGCAAGGTGTAGATGACATGGAGA 
               
               
                   
                 TCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTT 
               
               
                   
                 CAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGG 
               
               
                   
                 CCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGC 
               
               
                   
                 ACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGC 
               
               
                   
                 TGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTG 
               
               
                   
                 TGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGA 
               
               
                   
                 ACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCAT 
               
               
                   
                 CCTGTCTCTTGATCAGAGCT 
               
               
                   
               
               
                 SEQ ID NO: 77 
                 TGATCCCCTGCGCCATCAGATCCTTGGCGGCGAGAAAGCCATCCA 
               
               
                   Gaussia  Luciferase 
                 GTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCT 
               
               
                 minicircle template 
                 GGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCT 
               
               
                 (Artificial Sequence) 
                 ATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCT 
               
               
                   
                 TGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGG 
               
               
                   
                 GGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGCTCGAGGGGG 
               
               
                   
                 GCCAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGAT 
               
               
                   
                 TTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGAT 
               
               
                   
                 AAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGA 
               
               
                   
                 CCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAG 
               
               
                   
                 TGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAA 
               
               
                   
                 TAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCT 
               
               
                   
                 GTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGG 
               
               
                   
                 GAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGG 
               
               
                   
                 GCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG 
               
               
                   
                 GCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTT 
               
               
                   
                 TATTTTTCTAAATACATTCAAATATGTATCCGCTCATGACCAAAAT 
               
               
                   
                 CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA 
               
               
                   
                 AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCT 
               
               
                   
                 GCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT 
               
               
                   
                 TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT 
               
               
                   
                 CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTA 
               
               
                   
                 GTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC 
               
               
                   
                 GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT 
               
               
                   
                 CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG 
               
               
                   
                 CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT 
               
               
                   
                 TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC 
               
               
                   
                 TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG 
               
               
                   
                 TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA 
               
               
                   
                 GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTT 
               
               
                   
                 CGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG 
               
               
                   
                 GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT 
               
               
                   
                 TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTA 
               
               
                   
                 TCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTG 
               
               
                   
                 ATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGA 
               
               
                   
                 GCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC 
               
               
                   
                 ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAAT 
               
               
                   
                 CTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGC 
               
               
                   
                 TACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCG 
               
               
                   
                 CTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAG 
               
               
                   
                 ACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTC 
               
               
                   
                 ACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGC 
               
               
                   
                 GAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGC 
               
               
                   
                 AGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTC 
               
               
                   
                 TGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTT 
               
               
                   
                 TTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGC 
               
               
                   
                 ATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAA 
               
               
                   
                 CATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAA 
               
               
                   
                 GCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGAC 
               
               
                   
                 GCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGG 
               
               
                   
                 CGACAAGCAAACATGCTGTGCGACGCTGGCGATACATTACCCTGT 
               
               
                   
                 TATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTACCCTG 
               
               
                   
                 TTATCCCTAGATGACATTACCCTGTTATCCCTAGATGACATTTACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATGACATTAC 
               
               
                   
                 CCTGTTATCCCTAGATACATTACCCTGTTATCCCAGATGACATACC 
               
               
                   
                 CTGTTATCCCTAGATGACATTACCCTGTTATCCCAGATAAACTCAA 
               
               
                   
                 TGATGATGATGATGATGGTCGAGACTCAGCGGCCGCGGTGCCAGG 
               
               
                   
                 GCGTGCCCTTGGGCTCCCCGGGCGCGACTATAAGCTGCGAGCAAC 
               
               
                   
                 TTCACTTGGGTATGCCGGCGGTAGCGCTTACCGTTCGTATAATGTA 
               
               
                   
                 TGCTATACGAAGTTATCCGAAGCCGCTAGCGGTGGTTTGTCTGGTC 
               
               
                   
                 AACCACCGCGGTCTCAGTGGTGTACGGTACAAACCCACTACCGGT 
               
               
                   
                 CGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCT 
               
               
                   
                 GTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATC 
               
               
                   
                 GTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGAC 
               
               
                   
                 CGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAA 
               
               
                   
                 GAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGT 
               
               
                   
                 CTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAG 
               
               
                   
                 TTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCC 
               
               
                   
                 GCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATT 
               
               
                   
                 CCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAG 
               
               
                   
                 GTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTT 
               
               
                   
                 GCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAA 
               
               
                   
                 CGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAG 
               
               
                   
                 ATCAAGGGGGCCGGTGGTGACTAAGCGGAGCTCGATGAGTTTGGA 
               
               
                   
                 CAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAA 
               
               
                   
                 ATTTGTGATGCTATTGCTTTATTTGTGGGCCCGCCCCAACTGGGGT 
               
               
                   
                 AACCTTTGAGTTCTCTCAGTTGGGGGTAATCAGCATCATGATGTGG 
               
               
                   
                 TACCACATCATGATGCTGATTATAAGAATGCGGCCGCCACACTCT 
               
               
                   
                 AGTGGATCTCGAGTTAATAATTCAGAAGAACTCGTCAAGAAGGCG 
               
               
                   
                 ATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAA 
               
               
                   
                 GCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAA 
               
               
                   
                 TATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACAC 
               
               
                   
                 CCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCA 
               
               
                   
                 CCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGAT 
               
               
                   
                 CCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGG 
               
               
                   
                 CTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGAC 
               
               
                   
                 AAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTC 
               
               
                   
                 GCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGC 
               
               
                   
                 CGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCA 
               
               
                   
                 AGGTGTAGATGACATGGAGATCCTGCCCCGGCACTTCGCCCAATA 
               
               
                   
                 GCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTG 
               
               
                   
                 CGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCT 
               
               
                   
                 CGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAA 
               
               
                   
                 AAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCA 
               
               
                   
                 TCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGC 
               
               
                   
                 CTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTT 
               
               
                   
                 CAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCT 
               
               
                   
               
               
                 SEQ ID NO: 78 
                 CCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGG 
               
               
                 pseudo attP site 
                   
               
               
                 (Artificial sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 79 
                 GACTGAAACTTCACAGAATAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 Albumin-pegRNA- 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 SERPIN 
                 CGGTGCTTGGGATAGTTATGAATTCAATCTTCAACCCTATCCGGAT 
               
               
                 (Artificial Sequence) 
                 GATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTCTGT 
               
               
                   
                 GAAGTTTCAGTCA 
               
               
                   
               
               
                 SEQ ID NO: 80 
                 GACTGAAACTTCACAGAATAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 Albumin-pegRNA- 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 CPS1 
                 CGGTGCTTGGGATAGTTATGAATTCAATCTTCAACCCTATCCGGAT 
               
               
                 (Artificial Sequence) 
                 GATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTCTGT 
               
               
                   
                 GAAGTTTC 
               
               
                   
               
               
                 SEQ ID NO: 81 
                 GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 34 bp lox71 pegRNA 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 (Artificial Sequence) 
                 CGGTGCTGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCATACCGT 
               
               
                   
                 TCGTATAGCATACATTATACGAAGTTATCGTGCTCAGTCTG 
               
               
                   
               
               
                 SEQ ID NO: 82 
                 GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 34 bp lox66 pegRNA 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 (Artificial Sequence) 
                 CGGTGCTGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCAATAACT 
               
               
                   
                 TCGTATAGCATACATTATACGAACGGTACGTGCTCAGTCTG 
               
               
                   
               
               
                 SEQ ID NO: 83 
                 GGCCCAGACTGAGCACGTGA 
               
               
                 gRNA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 84 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 46 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCCGGATGATCC 
               
               
                 (original length) 
                 TGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGA 
               
               
                 pegRNA 
                 GAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 85 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 PBS_13_RT_29_with 
                 TCGGTGCGAGTCGGTGCGACGAGCGCGGCGATATCATCATCCAT 
               
               
                 TP901-1 minimal 
                 GGCACAATTAACATCTCAATCAAGGTAAATGCTTGAGCTGCGAG 
               
               
                 attB f pegRNA 
                 AA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 86 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 PBS_13_RT_29_with 
                 TCGGTGCGAGTCGGTGCGACGAGCGCGGCGATATCATCATCCAT 
               
               
                 TP901-1 minimal 
                 GGAGCATTTACCTTGATTGAGATGTTAATTGTGTGAGCTGCGAGA 
               
               
                 attB rc pegRNA 
                 A 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 87 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 PBS_13_RT_29_with 
                 TCGGTGCGAGTCGGTGCGACGAGCGCGGCGATATCATCATCCAT 
               
               
                 PhiBT1 minimal 
                 GGCAGGTTTTTGACGAAAGTGATCCAGATGATCCAGTGAGCTGC 
               
               
                 attB f pegRNA 
                 GAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 88 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 PBS 13 RT_29_with 
                 TCGGTGCGAGTCGGTGCGACGAGCGCGGCGATATCATCATCCAT 
               
               
                 PhiBT1 minimal 
                 GGCTGGATCATCTGGATCACTTTCGTCAAAAACCTGTGAGCTGCG 
               
               
                 attB rc pegRNA 
                 AGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 89 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 ACTB N-term 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 Nicking guide 1 +48 
                 GTCGGTGC 
               
               
                 guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 90 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 ACTB N-term 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 PBS_18_RT_16_with_ 
                 GTCGGTGCATATCATCATCCATGGTACCGTTCGTATAGCATACAT 
               
               
                 Lox71_Cre 
                 TATACGAAGTTATTGAGCTGCGAGAATAGCC 
               
               
                 pegRNA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 91 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 ACTB N-term 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 PBS_13_RT_29_with_ 
                 GTCGGTGCGACGAGCGCGGCGATATCATCATCCATGGTACCGTT 
               
               
                 Lox71_Cre 
                 CGTATAGCATACATTATACGAAGTTATTGAGCTGCGAGAA 
               
               
                 pegRNA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 92 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 34 pegRNA 
                 GGTGCTCGACGACGAGCGCGGCGATATCATCATCCATGGCCGGAT 
               
               
                 (Artificial Sequence) 
                 GATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGAGC 
               
               
                   
                 TGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 93 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 26 pegRNA 
                 GGTGCGAGCGCGGCGATATCATCATCCATGGCCGGATGATCCTGA 
               
               
                 (Artificial Sequence) 
                 CGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 94 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 23 pegRNA 
                 GGTGCCGCGGCGATATCATCATCCATGGCCGGATGATCCTGACGAC 
               
               
                 (Artificial Sequence) 
                 GGAGACCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 95 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 20 pegRNA 
                 GGTGCGGCGATATCATCATCCATGGCCGGATGATCCTGACGACGG 
               
               
                 (Artificial Sequence) 
                 AGACCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 96 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 16 pegRNA 
                 GGTGCATATCATCATCCATGGCCGGATGATCCTGACGACGGAGAC 
               
               
                 (Artificial Sequence) 
                 CGCCGTCGTCGACAAGCCGGCCTGAGCTGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 97 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 18 RT 34 pegRNA 
                 GGTGCTCGACGACGAGCGCGGCGATATCATCATCCATGGCCGGAT 
               
               
                 (Artificial Sequence) 
                 GATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGAGC 
               
               
                   
                 TGCGAGAATAGCC 
               
               
                   
               
               
                 SEQ ID NO: 98 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 18 RT 29 pegRNA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCCGGATGATCC 
               
               
                 (Artificial Sequence) 
                 TGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGA 
               
               
                   
                 GAATAGCC 
               
               
                   
               
               
                 SEQ ID NO: 99 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 18 RT 16 pegRNA 
                 GGTGCATATCATCATCCATGGCCGGATGATCCTGACGACGGAGAC 
               
               
                 (Artificial Sequence) 
                 CGCCGTCGTCGACAAGCCGGCCTGAGCTGCGAGAATAGCC 
               
               
                   
               
               
                 SEQ ID NO: 100 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 39 pegRNA 
                 TCGGTGCCTGCCCATCCGCGGCGGCACGGGGGTCGCAGTCGCCA 
               
               
                 (Artificial Sequence) 
                 TGCCGGATGATCCTGACGACGGAGACCGCCGTCGTCGACAAGCC 
               
               
                   
                 GGCCCGGGCGGCGGAGA 
               
               
                   
               
               
                 SEQ ID NO: 101 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 34 pegRNA 
                 TCGGTGCCATCCGCGGCGGCACGGGGGTCGCAGTCGCCATGCCG 
               
               
                 (Artificial Sequence) 
                 GATGATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCC 
               
               
                   
                 GGGCGGCGGAGA 
               
               
                   
               
               
                 SEQ ID NO: 102 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 29 pegRNA 
                 TCGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGCCGGATGA 
               
               
                 (Artificial Sequence) 
                 TCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCCGGGCG 
               
               
                   
                 GCGGAGA 
               
               
                   
               
               
                 SEQ ID NO: 103 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 24 pegRNA 
                 TCGGTGCGGCACGGGGGTCGCAGTCGCCATGCCGGATGATCCTG 
               
               
                 (Artificial Sequence) 
                 ACGACGGAGACCGCCGTCGTCGACAAGCCGGCCCGGGCGGCGGA 
               
               
                   
                 GA 
               
               
                   
               
               
                 SEQ ID NO: 104 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 19 pegRNA 
                 TCGGTGCGGGGGTCGCAGTCGCCATGCCGGATGATCCTGACGAC 
               
               
                 (Artificial Sequence) 
                 GGAGACCGCCGTCGTCGACAAGCCGGCCCGGGCGGCGGAGA 
               
               
                   
               
               
                 SEQ ID NO: 105 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 18 RT 39 pegRNA 
                 TCGGTGCCTGCCCATCCGCGGCGGCACGGGGGTCGCAGTCGCCA 
               
               
                 (Artificial Sequence) 
                 TGCCGGATGATCCTGACGACGGAGACCGCCGTCGTCGACAAGCC 
               
               
                   
                 GGCCCGGGCGGCGGAGACAGCG 
               
               
                   
               
               
                 SEQ ID NO: 106 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 18 RT 34 pegRNA 
                 TCGGTGCCATCCGCGGCGGCACGGGGGTCGCAGTCGCCATGCCG 
               
               
                 (Artificial Sequence) 
                 GATGATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCC 
               
               
                   
                 GGGCGGCGGAGACAGCG 
               
               
                   
               
               
                 SEQ ID NO: 107 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 18 RT 29 pegRNA 
                 CGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGCCGGATGATC 
               
               
                 (Artificial Sequence) 
                 CTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCCGGGCGGCG 
               
               
                   
                 GAGACAGCG 
               
               
                   
               
               
                 SEQ ID NO: 108 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 18 RT 24 pegRNA 
                 TCGGTGCGGCACGGGGGTCGCAGTCGCCATGCCGGATGATCCTG 
               
               
                 (Artificial Sequence) 
                 ACGACGGAGACCGCCGTCGTCGACAAGCCGGCCCGGGCGGCGGA 
               
               
                   
                 GACAGCG 
               
               
                   
               
               
                 SEQ ID NO: 109 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 18 RT 19 pegRNA 
                 TCGGTGCGGGGGTCGCAGTCGCCATGCCGGATGATCCTGACGAC 
               
               
                 (Artificial Sequence) 
                 GGAGACCGCCGTCGTCGACAAGCCGGCCCGGGCGGCGGAGACAG 
               
               
                   
                 CG 
               
               
                   
               
               
                 SEQ ID NO: 110 
                 GCGTGGTGGGGCCGCCAGCGGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 LMNB1 N-term 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 Nicking guide 1 +46 
                 GTCGGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 111 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 42 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGGGATGATCCTG 
               
               
                 pegRNA 
                 ACGACGGAGACCGCCGTCGTCGACAAGCCGGTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 112 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 40 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGGATGATCCTGA 
               
               
                 pegRNA 
                 CGACGGAGACCGCCGTCGTCGACAAGCCGTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 113 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 38 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGATGATCCTGAC 
               
               
                 pegRNA 
                 GACGGAGACCGCCGTCGTCGACAAGCCTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 114 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 36 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGTGATCCTGACG 
               
               
                 pegRNA 
                 ACGGAGACCGCCGTCGTCGACAAGCTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 115 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 13 RT 29 attB 44 
                 CGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGCGGATGATCC 
               
               
                 pegRNA v2 
                 TGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCGGGCGGCGG 
               
               
                 (Artificial Sequence) 
                 AGA 
               
               
                   
               
               
                 SEQ ID NO: 116 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 13 RT 29 attB 42 
                 CGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGGGATGATCCT 
               
               
                 pegRNA v2 
                 GACGACGGAGACCGCCGTCGTCGACAAGCCGGCGGGCGGCGGAG 
               
               
                 (Artificial Sequence) 
                 A 
               
               
                   
               
               
                 SEQ ID NO: 117 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 13 RT 29 attB 40 
                 CGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGGATGATCCTG 
               
               
                 pegRNA v2 
                 ACGACGGAGACCGCCGTCGTCGACAAGCCGCGGGCGGCGGAGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 118 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 13 RT 29 attB 38 
                 CGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGATGATCCTGA 
               
               
                 pegRNA v2 
                 CGACGGAGACCGCCGTCGTCGACAAGCCCGGGCGGCGGAGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 119 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 NOLC1 N-term PBS 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 18 RT 29 attB 46 
                 GTCGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCCCGGATG 
               
               
                 pegRNA 
                 ATCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTCCTC 
               
               
                 (Artificial Sequence) 
                 CAGGCAATACGCG 
               
               
                   
               
               
                 SEQ ID NO: 120 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 NOLC1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 13 RT 29 attB 46 
                 CGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCCCGGATGATC 
               
               
                 pegRNA 
                 CTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTCCTCCAGG 
               
               
                 (Artificial Sequence) 
                 CAAT 
               
               
                   
               
               
                 SEQ ID NO: 121 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 NOLC1 N-term PBS 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 13 RT 29 attB 44 
                 GTCGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCCGGATGA 
               
               
                 pegRNA 
                 TCCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCTCCTCCA 
               
               
                 (Artificial Sequence) 
                 GGCAAT 
               
               
                   
               
               
                 SEQ ID NO: 122 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 NOLC1 N-term PBS 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 13 RT 29 attB 42 
                 GTCGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCGGATGAT 
               
               
                 pegRNA 
                 CCTGACGACGGAGACCGCCGTCGTCGACAAGCCGGTCCTCCAGG 
               
               
                 (Artificial Sequence) 
                 CAAT 
               
               
                   
               
               
                 SEQ ID NO: 123 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 NOLC1 N-term PBS 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 13 RT 29 attB 40 
                 GTCGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCGATGATC 
               
               
                 pegRNA 
                 CTGACGACGGAGACCGCCGTCGTCGACAAGCCGTCCTCCAGGCA 
               
               
                 (Artificial Sequence) 
                 AT 
               
               
                   
               
               
                 SEQ ID NO: 124 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 NOLC1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 29 attB 38 
                 TCGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCATGATCCT 
               
               
                 pegRNA 
                 GACGACGGAGACCGCCGTCGTCGACAAGCCTCCTCCAGGCAAT 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 125 
                 GAGCCGAGCACGAGGGGATACGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 NOLC1 nicking 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 guide-43 
                 TCGGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 126 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 20 attB 38 
                 GGTGCGGCGATATCATCATCCATGGATGATCCTGACGACGGAGAC 
               
               
                 pegRNA 
                 CGCCGTCGTCGACAAGCCTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 127 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 15 attB 38 
                 GGTGCTATCATCATCCATGGATGATCCTGACGACGGAGACCGCCG 
               
               
                 pegRNA 
                 TCGTCGACAAGCCTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 128 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 10 attB 38 
                 GGTGCTCATCCATGGATGATCCTGACGACGGAGACCGCCGTCGTC 
               
               
                 pegRNA 
                 GACAAGCCTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 129 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term PBS 9 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 RT 20 attB 38 
                 TCGGTGCGGCGATATCATCATCCATGGATGATCCTGACGACGGAG 
               
               
                 pegRNA 
                 ACCGCCGTCGTCGACAAGCCTGAGCTGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 130 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 9 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 RT 15 attB 38 
                 GGTGCTATCATCATCCATGGATGATCCTGACGACGGAGACCGCCG 
               
               
                 pegRNA 
                 TCGTCGACAAGCCTGAGCTGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 131 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 9 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 RT 10 attB 38 
                 GGTGCTCATCCATGGATGATCCTGACGACGGAGACCGCCGTCGTC 
               
               
                 pegRNA 
                 GACAAGCCTGAGCTGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 132 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 20 attB 38 
                 TCGGTGCCGGGGGTCGCAGTCGCCATGATGATCCTGACGACGGA 
               
               
                 pegRNA 
                 GACCGCCGTCGTCGACAAGCCCGGGCGGCGGAGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 133 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 15 attB 38 
                 TCGGTGCGTCGCAGTCGCCATGATGATCCTGACGACGGAGACCG 
               
               
                 pegRNA 
                 CCGTCGTCGACAAGCCCGGGCGGCGGAGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 134 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 13 RT 10 attB 38 
                 TCGGTGCAGTCGCCATGATGATCCTGACGACGGAGACCGCCGTC 
               
               
                 pegRNA 
                 GTCGACAAGCCCGGGCGGCGGAGA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 135 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 9 RT 20 attB 38 
                 CGGTGCCGGGGGTCGCAGTCGCCATGATGATCCTGACGACGGAGA 
               
               
                 pegRNA 
                 CCGCCGTCGTCGACAAGCCCGGGCGGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 136 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 9 RT 15 attB 38 
                 TCGGTGCGTCGCAGTCGCCATGATGATCCTGACGACGGAGACCG 
               
               
                 pegRNA 
                 CCGTCGTCGACAAGCCCGGGCGGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 137 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 9 RT 10 attB 38 
                 CGGTGCAGTCGCCATGATGATCCTGACGACGGAGACCGCCGTCGT 
               
               
                 pegRNA 
                 CGACAAGCCCGGGCGGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 138 
                 GAGAAGCGGCGTCCGGGGCTAGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 SUPT16H N-term 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 PBS 13 RT 24 Bxb1- 
                 TCGGTGCTCTTTGTCCAGAGTCACAGCCATACCGGATGATCCTGAC 
               
               
                 GT_Initial length 
                 GACGGAGACCGCCGTCGTCGACAAGCCGGCCCCCCGGACGCCGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 139 
                 GGGCACGGGGCCATGTACAAGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 SRRM2 N-term PBS 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA 
               
               
                 13 RT 24 Bxb1 
                 GTCGGTGCGGCGTCGGCAGCCCGATCCCGTTGCCGGATGATCCT 
               
               
                 Initial length 
                 GACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTACATGGCCC 
               
               
                 (Artificial Sequence) 
                 CGT 
               
               
                   
               
               
                 SEQ ID NO: 140 
                 GTGTCAGGTGGGGCGGGGCTAGTTTTAGAGCTAGAAATAGCAAG 
               
               
                 DEPDC4 N-term 
                 TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG 
               
               
                 PBS 18 RT 24 Bxb1 
                 AGTCGGTGCGCTGGCTCCTCCCCTGGCACCATACCGGATGATCCT 
               
               
                 Initial length 
                 GACGACGGAGACCGCCGTCGTCGACAAGCCGGCCCCCCGCCCCA 
               
               
                 (Artificial Sequence) 
                 CCTGACAC 
               
               
                   
               
               
                 SEQ ID NO: 141 
                 GAGTGGGTCAGACGAGCAGGAGTTTTAGAGCTAGAAATAGCAAGT 
               
               
                 NES N-term PBS 13 
                 TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 RT 29 Bxb1 Initial 
                 TCGGTGCGATGGAGGGCTGCATGGGGGAGGAGTCGCCGGATGATC 
               
               
                 length 
                 CTGACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGCTCGTCT 
               
               
                 (Artificial Sequence) 
                 GACC 
               
               
                   
               
               
                 SEQ ID NO: 142 
                 GCAGCCACCCGCTCTCGGCCCGTTTTAGAGCTAGAAATAGCAAG 
               
               
                 SUPT16H nicking 
                 TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG 
               
               
                 guide-53 
                 AGTCGGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 143 
                 GTGTAGTCAGGCCGCTCACCCGTTTTAGAGCTAGAAATAGCAAG 
               
               
                 SRRM2 N-term 
                 TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG 
               
               
                 nicking guide 1 +87 
                 AGTCGGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 144 
                 GCTGACAAGTCTACGGAACCTGTTTTAGAGCTAGAAATAGCAAG 
               
               
                 DEPDC4 N-term 
                 TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG 
               
               
                 Nicking guide 1 +59 
                 AGTCGGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 145 
                 GCTCCTCCAGCGCCTTGACCGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 NES N-term Nicking 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 guide 2 + 9 
                 GGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 146 
                 GCTATTCTCGCAGCTCACCA 
               
               
                 HITI_ACTB_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 147 
                 AGAAGCGGCGTCCGGGGCTA 
               
               
                 HITI_SUPTH16_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 148 
                 GGGCACGGGGCCATGTACAA 
               
               
                 HITI_SRRM2_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 149 
                 GCGTATTGCCTGGAGGATGG 
               
               
                 HITI_NOLCl_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 150 
                 TGTCAGGTGGGGCGGGGCTA 
               
               
                 HITI_DEPDC4_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 151 
                 AGTGGGTCAGACGAGCAGGA 
               
               
                 HITI_NES_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 152 
                 GCTGTCTCCGCCGCCCGCCA 
               
               
                 HITI_LMNB1_guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 153 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 HDR Cas9 ACTB 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG 
               
               
                 guide 
                 TCGGTGC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 154 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCCGGATGATCC 
               
               
                 original length 
                 TGACGACGGAG XX CGCCGTCGTCGACAAGCCGGCCTGAGCTGCGA 
               
               
                 pegRNAs for 
                 GAA 
               
               
                 dinucleotides 
                   XX : CG, GC, AT, TA, GG, TT, GA, AG, CC, TC, CT, 
               
               
                 (Artificial Sequence) 
                 AA, TG, GT, CA, or AC 
               
               
                   
               
               
                 SEQ ID NO: 155 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 pegRNA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGCCGGATGATCCT 
               
               
                 with attB 46 GT for 
                 GACGACGGAGACCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGAG 
               
               
                 fusion 
                 AA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 156 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 pegRNA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGCCGGATGATCCT 
               
               
                 with attB 46 CT for 
                 GACGACGGAGAGCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGA 
               
               
                 multiplexing 
                 GAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 157 
                 GCGTATTGCCTGGAGGATGGGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 NOLC1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 18 RT 29 pegRNA 
                 CGGTGCGAACCACGCGGCGAATGCCGGCGTCCGCCCCGGATGATC 
               
               
                 with attB 46 GA for 
                 CTGACGACGGAGTCCGCCGTCGTCGACAAGCCGGCCTCCTCCAGG 
               
               
                 multiplexing 
                 CAATACGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 158 
                 GCTGTCTCCGCCGCCCGCCAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term PBS 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 18 RT 29 pegRNA 
                 CGGTGCGCGGCGGCACGGGGGTCGCAGTCGCCATGCCGGATGATC 
               
               
                 with attB 46 AG for 
                 CTGACGACGGAGCTCGCCGTCGTCGACAAGCCGGCCCGGGCGGCG 
               
               
                 multiplexing 
                 GAGACAGCG 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 159 
                 GTCACCTCCAATGACTAGGG 
               
               
                 EMX1 Cas9 guide 1 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 160 
                 GGGCAACCACAAACCCACGA 
               
               
                 EMX1 Cas9 guide 2 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 161 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 56 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCTATGCCGGAT 
               
               
                 pegRNA 
                 GATCCTGACGACGGAGTCCGCCGTCGTCGACAAGCCGGCCCTAGC 
               
               
                 (Artificial Sequence) 
                 TGAGCTGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 162 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 51 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGTGCCGGATGAT 
               
               
                 pegRNA 
                 CCTGACGACGGAGTCCGCCGTCGTCGACAAGCCGGCCCTATGAGC 
               
               
                 (Artificial Sequence) 
                 TGCGAGAA 
               
               
                   
               
               
                 SEQ ID NO: 163 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 46 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCCGGATGATCC 
               
               
                 pegRNA 
                 TGACGACGGAGTCCGCCGTCGTCGACAAGCCGGCCTGAGCTGCGA 
               
               
                 (Artificial Sequence) 
                 GAA 
               
               
                   
               
               
                 SEQ ID NO: 164 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 41 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGGGATGATCCTG 
               
               
                 pegRNA 
                 ACGACGGAGTCCGCCGTCGTCGACAAGCCGTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 165 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 36 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGTGATCCTGACG 
               
               
                 pegRNA 
                 ACGGAGTCCGCCGTCGTCGACAAGCTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 166 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 31 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGATCCTGACGAC 
               
               
                 pegRNA 
                 GGAGTCCGCCGTCGTCGACATGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 167 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 26 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCCTGACGACGG 
               
               
                 pegRNA 
                 AGTCCGCCGTCGTCGTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 168 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 21 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGTGACGACGGAG 
               
               
                 pegRNA 
                 TCCGCCGTCGTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 169 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 16 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGACGACGGAGTC 
               
               
                 pegRNA 
                 CGCCGTGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 170 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 11 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGGACGGAGTCCG 
               
               
                 pegRNA 
                 TGAGCTGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 171 
                 GCTATTCTCGCAGCTCACCAGTTTTAGAGCTAGAAATAGCAAGTTA 
               
               
                 ACTB N-term PBS 
                 AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC 
               
               
                 13 RT 29 attB 6 GA 
                 GGTGCGACGAGCGCGGCGATATCATCATCCATGGCGGAGTTGAGC 
               
               
                 pegRNA 
                 TGCGAGAA 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 172 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 PBS_18_RT_34_with_ 
                 CGGTGCTCGACGACGAGCGCGGCGATATCATCATCCATGGTACCG 
               
               
                 Lox71_Cre 
                 TTCGTATAGCATACATTATACGAAGTTATTGAGCTGCGAGAATAG 
               
               
                 pegRNA 
                 CC 
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 173 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 PBS_18_RT_29_with_ 
                 CGGTGCGACGAGCGCGGCGATATCATCATCCATGGTACCGTTCGT 
               
               
                 Lox71_Cre 
                 ATAGCATACATTATACGAAGTTATTGAGCTGCGAGAATAGCC 
               
               
                 pegRNA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 174 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 PBS_13_RT_34_with_ 
                 CGGTGCTCGACGACGAGCGCGGCGATATCATCATCCATGGTACCG 
               
               
                 Lox71_Cre 
                 TTCGTATAGCATACATTATACGAAGTTATTGAGCTGCGAGAA 
               
               
                 pegRNA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 175 
                 GAAGCCGGCCTTGCACATGCGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 PBS_13_RT_16_with_ 
                 CGGTGCATATCATCATCCATGGTACCGTTCGTATAGCATACATTAT 
               
               
                 Lox71_Cre 
                 ACGAAGTTATTGAGCTGCGAGAA 
               
               
                 pegRNA 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 176 
                 CCCCACGATGGAGGGGAAGAGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 ACTB N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 Nicking guide 2 +93 
                 CGGTGC 
               
               
                 guide 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 177 
                 CCTTCTCCTGGAGCCGCGACGTTTTAGAGCTAGAAATAGCAAGTT 
               
               
                 LMNB1 N-term 
                 AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT 
               
               
                 Nicking guide 2 +87 
                 CGGTGC 
               
               
                 guide 
                   
               
               
                 (Artificial Sequence) 
               
               
                   
               
            
           
         
       
     
     Sequences of insertion sites can be found in Table 4 below. 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   
                 FORWARD SEQUENCE (5′-3′) 
                 REVERSE SEQUENCE (5′-3′) 
               
            
           
           
               
               
               
               
               
            
               
                 DESCRIPTION/ 
                 SEQ ID 
                   
                 SEQ ID 
                   
               
               
                 SOURCE 
                 NO 
                 Sequence 
                 NO 
                 Sequence 
               
               
                   
               
               
                 Bxb1_attP_GT_ 
                 178 
                 GTGGTTTGTCTGGTC 
                 179 
                 TGGGTTTGTACCGTA 
               
               
                 original_site 
                   
                 AACCACCGCGGTCT 
                   
                 CACCACTGAGACCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_CG_ 
                 180 
                 GTGGTTTGTCTGGTC 
                 181 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGCGCT 
                   
                 CACCACTGAGCGCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_GC_ 
                 182 
                 GTGGTTTGTCTGGTC 
                 183 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGGCCT 
                   
                 CACCACTGAGGCCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_AT_ 
                 184 
                 GTGGTTTGTCTGGTC 
                 185 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGATCT 
                   
                 CACCACTGAGATCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_TA_ 
                 186 
                 GTGGTTTGTCTGGTC 
                 187 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGTACT 
                   
                 CACCACTGAGTACG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_GG_ 
                 188 
                 GTGGTTTGTCTGGTC 
                 189 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGGGCT 
                   
                 CACCACTGAGCCCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_TT_ 
                 190 
                 GTGGTTTGTCTGGTC 
                 191 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGTTCTC 
                   
                 CACCACTGAGAACG 
               
               
                 (Artificial 
                   
                 AGTGGTGTACGGTA 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 CAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_GA_ 
                 192 
                 GTGGTTTGTCTGGTC 
                 193 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGGACT 
                   
                 CACCACTGAGTCCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_AG_ 
                 194 
                 GTGGTTTGTCTGGTC 
                 195 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGAGCT 
                   
                 CACCACTGAGCTCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_CC_ 
                 196 
                 GTGGTTTGTCTGGTC 
                 197 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGCCCT 
                   
                 CACCACTGAGGGCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_TC_ 
                 198 
                 GTGGTTTGTCTGGTC 
                 199 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGTCCTC 
                   
                 CACCACTGAGGACG 
               
               
                 (Artificial 
                   
                 AGTGGTGTACGGTA 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 CAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_CT_ 
                 200 
                 GTGGTTTGTCTGGTC 
                 201 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGCTCTC 
                   
                 CACCACTGAGAGCG 
               
               
                 (Artificial 
                   
                 AGTGGTGTACGGTA 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 CAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_AA_ 
                 202 
                 GTGGTTTGTCTGGTC 
                 203 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGAACT 
                   
                 CACCACTGAGTTCGC 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 GGTGGTTGACCAGA 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 CAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_C 
                 204 
                 GTGGTTTGTCTGGTC 
                 205 
                 TGGGTTTGTACCGTA 
               
               
                 A_site 
                   
                 AACCACCGCGCACT 
                   
                 CACCACTGAGTGCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_AC_ 
                 206 
                 GTGGTTTGTCTGGTC 
                 207 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGACCT 
                   
                 CACCACTGAGGTCG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attP_TG_ 
                 208 
                 GTGGTTTGTCTGGTC 
                 209 
                 TGGGTTTGTACCGTA 
               
               
                 site 
                   
                 AACCACCGCGTGCT 
                   
                 CACCACTGAGCACG 
               
               
                 (Artificial 
                   
                 CAGTGGTGTACGGT 
                   
                 CGGTGGTTGACCAG 
               
               
                 Sequence) 
                   
                 ACAAACCCA 
                   
                 ACAAACCAC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 210 
                 GGCCGGCTTGTCGA 
                 211 
                 CCGGATGATCCTGA 
               
               
                 GT_original_ 
                   
                 CGACGGCGGTCTCC 
                   
                 CGACGGAGACCGCC 
               
               
                 site 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 (Artificial 
                   
                 CGG 
                   
                 GGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 212 
                 GGCCGGCTTGTCGA 
                 213 
                 CCGGATGATCCTGA 
               
               
                 AA_site 
                   
                 CGACGGCGAACTCC 
                   
                 CGACGGAGTTCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 214 
                 GGCCGGCTTGTCGA 
                 215 
                 CCGGATGATCCTGA 
               
               
                 GA_site 
                   
                 CGACGGCGGACTCC 
                   
                 CGACGGAGTCCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 216 
                 GGCCGGCTTGTCGA 
                 217 
                 CCGGATGATCCTGA 
               
               
                 CA_site 
                   
                 CGACGGCGCACTCC 
                   
                 CGACGGAGTGCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 218 
                 GGCCGGCTTGTCGA 
                 219 
                 CCGGATGATCCTGA 
               
               
                 TA_site 
                   
                 CGACGGCGTACTCC 
                   
                 CGACGGAGTACGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 220 
                 GGCCGGCTTGTCGA 
                 221 
                 CCGGATGATCCTGA 
               
               
                 AG_site 
                   
                 CGACGGCGAGCTCC 
                   
                 CGACGGAGCTCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 222 
                 GGCCGGCTTGTCGA 
                 223 
                 CCGGATGATCCTGA 
               
               
                 GG_site 
                   
                 CGACGGCGGGCTCC 
                   
                 CGACGGAGCCCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 224 
                 GGCCGGCTTGTCGA 
                 225 
                 CCGGATGATCCTGA 
               
               
                 CG_site 
                   
                 CGACGGCGCGCTCC 
                   
                 CGACGGAGCGCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 226 
                 GGCCGGCTTGTCGA 
                 227 
                 CCGGATGATCCTGA 
               
               
                 TG_site 
                   
                 CGACGGCGTGCTCC 
                   
                 CGACGGAGCACGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 228 
                 GGCCGGCTTGTCGA  
                 229 
                 CCGGATGATCCTGA 
               
               
                 AC_site 
                   
                 CGACGGCGACCTCC 
                   
                 CGACGGAGGTCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 230 
                 GGCCGGCTTGTCGA 
                 231 
                 CCGGATGATCCTGA 
               
               
                 GC_site 
                   
                 CGACGGCGGCCTCC 
                   
                 CGACGGAGGCCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 232 
                 GGCCGGCTTGTCGA 
                 233 
                 CCGGATGATCCTGA 
               
               
                 CC_site 
                   
                 CGACGGCGCCCTCC 
                   
                 CGACGGAGGGCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 234 
                 GGCCGGCTTGTCGA 
                 235 
                 CCGGATGATCCTGA 
               
               
                 TC_site 
                   
                 CGACGGCGTCCTCC 
                   
                 CGACGGAGGACGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 236 
                 GGCCGGCTTGTCGA 
                 237 
                 CCGGATGATCCTGA 
               
               
                 AT_site 
                   
                 CGACGGCGATCTCC 
                   
                 CGACGGAGATCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 238 
                 GGCCGGCTTGTCGA 
                 239 
                 CCGGATGATCCTGA 
               
               
                 CT_site 
                   
                 CGACGGCGCTCTCC 
                   
                 CGACGGAGAGCGCC 
               
               
                 (Artificial 
                   
                 GTCGTCAGGATCATC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 CGG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_46_ 
                 240 
                 GGCCGGCTTGTCGA 
                 241 
                 CCGGATGATCCTGA 
               
               
                 TT_site 
                   
                 CGACGGCGTTCTCCG 
                   
                 CGACGGAGAACGCC 
               
               
                 (Artificial 
                   
                 TCGTCAGGATCATCC 
                   
                 GTCGTCGACAAGCC 
               
               
                 Sequence) 
                   
                 GG 
                   
                 GGCC 
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 242 
                 GGCTTGTCGACGAC 
                 243 
                 ATGATCCTGACGAC 
               
               
                 GT_site 
                   
                 GGCGGTCTCCGTCGT 
                   
                 GGAGACCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 244 
                 GGCTTGTCGACGAC 
                 245 
                 ATGATCCTGACGAC 
               
               
                 AA_site 
                   
                 GGCGAACTCCGTCG 
                   
                 GGAGTTCGCCGTCGT 
               
               
                 (Artificial 
                   
                 TCAGGATCAT 
                   
                 CGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 246 
                 GGCTTGTCGACGAC 
                 247 
                 ATGATCCTGACGAC 
               
               
                 GA_site 
                   
                 GGCGGACTCCGTCG 
                   
                 GGAGTCCGCCGTCG 
               
               
                 (Artificial 
                   
                 TCAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 248 
                 GGCTTGTCGACGAC 
                 249 
                 ATGATCCTGACGAC 
               
               
                 CA_site 
                   
                 GGCGCACTCCGTCGT 
                   
                 GGAGTGCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 250 
                 GGCTTGTCGACGAC 
                 251 
                 ATGATCCTGACGAC 
               
               
                 TA_site 
                   
                 GGCGTACTCCGTCGT 
                   
                 GGAGTACGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 252 
                 GGCTTGTCGACGAC 
                 253 
                 ATGATCCTGACGAC 
               
               
                 AG_site 
                   
                 GGCGAGCTCCGTCG 
                   
                 GGAGCTCGCCGTCG 
               
               
                 (Artificial 
                   
                 TCAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 254 
                 GGCTTGTCGACGAC 
                 255 
                 ATGATCCTGACGAC 
               
               
                 GG_site 
                   
                 GGCGGGCTCCGTCG 
                   
                 GGAGCCCGCCGTCG 
               
               
                 (Artificial 
                   
                 TCAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 256 
                 GGCTTGTCGACGAC 
                 257 
                 ATGATCCTGACGAC 
               
               
                 CG_site 
                   
                 GGCGCGCTCCGTCGT 
                   
                 GGAGCGCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 258 
                 GGCTTGTCGACGAC 
                 259 
                 ATGATCCTGACGAC 
               
               
                 TG_site 
                   
                 GGCGTGCTCCGTCGT 
                   
                 GGAGCACGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 260 
                 GGCTTGTCGACGAC 
                 261 
                 ATGATCCTGACGAC 
               
               
                 AC_site 
                   
                 GGCGACCTCCGTCGT 
                   
                 GGAGGTCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 262 
                 GGCTTGTCGACGAC 
                 263 
                 ATGATCCTGACGAC 
               
               
                 GC_site 
                   
                 GGCGGCCTCCGTCGT 
                   
                 GGAGGCCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 264 
                 GGCTTGTCGACGAC 
                 265 
                 ATGATCCTGACGAC 
               
               
                 CC_site 
                   
                 GGCGCCCTCCGTCGT 
                   
                 GGAGGGCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 266 
                 GGCTTGTCGACGAC 
                 267 
                 ATGATCCTGACGAC 
               
               
                 TC_site 
                   
                 GGCGTCCTCCGTCGT 
                   
                 GGAGGACGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 268 
                 GGCTTGTCGACGAC 
                 269 
                 ATGATCCTGACGAC 
               
               
                 AT_site 
                   
                 GGCGATCTCCGTCGT 
                   
                 GGAGATCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 270 
                 GGCTTGTCGACGAC 
                 271 
                 ATGATCCTGACGAC 
               
               
                 CT_site 
                   
                 GGCGCTCTCCGTCGT 
                   
                 GGAGAGCGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Bxb1_attB_38_ 
                 272 
                 GGCTTGTCGACGAC 
                 273 
                 ATGATCCTGACGAC 
               
               
                 TT_site 
                   
                 GGCGTTCTCCGTCGT 
                   
                 GGAGAACGCCGTCG 
               
               
                 (Artificial 
                   
                 CAGGATCAT 
                   
                 TCGACAAGCC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Cre Lox 66 
                 274 
                 TACCGTTCGTATAAT 
                 275 
                 ATAACTTCGTATAGC 
               
               
                 site 
                   
                 GTATGCTATACGAA 
                   
                 ATACATTATACGAA 
               
               
                 (Artificial 
                   
                 GTTAT 
                   
                 CGGTA 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 Cre Lox 71 
                 276 
                 ATAACTTCGTATAAT 
                 277 
                 TACCGTTCGTATAGC 
               
               
                 site 
                   
                 GTATGCTATACGAA 
                   
                 ATACATTATACGAA 
               
               
                 (Artificial 
                   
                 CGGTA 
                   
                 GTTAT 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 TP901-1 
                 278 
                 TTTACCTTGATTGAG 
                 279 
                 CACAATTAACATCTC 
               
               
                 minimal attB 
                   
                 ATGTTAATTGTG 
                   
                 AATCAAGGTAAA 
               
               
                 site 
                   
                   
                   
                   
               
               
                 (Artificial 
                   
                   
                   
                   
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 TP901-1 
                 280 
                 GCGAGTTTTTATTTC 
                 281 
                 AAAGGAGTTTTTTAG 
               
               
                 minimal attP 
                   
                 GTTTATTTCAATTAA 
                   
                 TTACCTTAATTGAAA 
               
               
                 site 
                   
                 GGTAACTAAAAAAC 
                   
                 TAAACGAAATAAAA 
               
               
                 (Artificial 
                   
                 TCCTTT 
                   
                 ACTCGC 
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 PhiBT1 
                 282 
                 CTGGATCATCTGGAT 
                 283 
                 CAGGTTTTTGACGAA 
               
               
                 minimal attB 
                   
                 CACTTTCGTCAAAAA 
                   
                 AGTGATCCAGATGA 
               
               
                 site 
                   
                 CCTG 
                   
                 TCCAG 
               
               
                 (Artificial 
                   
                   
                   
                   
               
               
                 Sequence) 
                   
                   
                   
                   
               
               
                   
               
               
                 PhiBT1 
                 284 
                 TTCGGGTGCTGGGTT 
                 285 
                 TGGTGCTGAGTAGTT 
               
               
                 minimal attP 
                   
                 GTTGTCTCTGGACAG 
                   
                 TCCCATGGATCACTG 
               
               
                 site 
                   
                 TGATCCATGGGAAA 
                   
                 TCCAGAGACAACAA 
               
               
                 (Artificial 
                   
                 CTACTCAGCACCA 
                   
                 CCCAGCACCCGAA 
               
               
                 Sequence) 
               
               
                   
               
            
           
         
       
     
     Sequences of Bxb1 and RT mutants can be found in Table 6 below. 
     
       
         
           
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 SEQ ID NO/ 
                   
               
               
                 DESCRIPTION/ 
                   
               
               
                 SOURCE 
                 FORWARD SEQUENCE (5′-3′) 
               
               
                   
               
             
            
               
                 SEQ ID NO: 286 
                 AAAAGTGTGGGCTGCAGGATCTGA 
               
               
                 Bxb1_mut_V368A 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 287 
                 GGAGCTGGCAGCTGTCAATGCC 
               
               
                 Bxb1_mut_E379A 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 288 
                 AGTCAATGCCGCTCTCGTGGA 
               
               
                 Bxb1_mut_E383A 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 403 
                 TTGAGCGGGCCCCCACCGT 
               
               
                 RT_mut_L139P 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 289 
                 CAGCGGGCTCAGCTGATAGCA 
               
               
                 RT_mut_E562Q 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 290 
                 CGGATGGCTAACCAAGCGGCC 
               
               
                 RT_mut_D653N 
                   
               
               
                 (Artificial Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 404 
                 atgactcactatcaggccttgcttaggacacggaccgggtccagttcggaccggtggtagccctgaaccc 
               
               
                 RT(1-478)_Sto7d 
                 ggctacgctgctcccactgcctgaggaagggctgcaacacaactgccttgatGGGACAGGTGG 
               
               
                 fusion 
                 CGGTGGTGTCACCGTCAAGTTCAAGTACAAGGGTGAGGAACTT 
               
               
                   
                 GAAGTTGATATTAGCAAAATCAAGAAGGTTTGGCGCGTTGGTA 
               
               
                   
                 AAATGATATCTTTTACTTATGACGACAACGGCAAGACAGGTAG 
               
               
                   
                 AGGGGCAGTGTCTGAGAAAGACGCCCCCAAGGAGCTGTTGCAA 
               
               
                   
                 ATGTTGGAAAAGTCTGGGAAAAAGtctggcggctcaaaaagaaccgccgacgg 
               
               
                   
                 cagcgaattcgagcccaagaagaagaggaaagtc 
               
               
                   
               
            
           
         
       
     
     Sequences of primers, probes and restriction enzymes used in ddPCR readout can be found in Table 7 below. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 SEQ 
                 Forward 
                 SEQ 
                 Reverse 
                   
                 SEQ 
                 Restriction 
               
               
                 Locus 
                 Cargo 
                 ID NO: 
                 Primer 
                 ID NO: 
                 Primer 
                 Probe 
                 ID NO: 
                 Enzymes 
               
               
                   
               
             
            
               
                 ACTB 
                 GFP 
                 291 
                 CCCG 
                 292 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                   
                 (pDY0186) 
                   
                 GCTTC 
                   
                 TCCAC 
                 FAM/C 
                   
                 HindIII 
               
               
                   
                   
                   
                 CTTTG 
                   
                 GCCG 
                 C GGC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 TTCA 
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 C GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3IAB 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 kFQ/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 TP90-1 
                 293 
                 CCCG 
                 294 
                 AACC 
                 /56- 
                 406 
                 None 
               
               
                   
                 GFP 
                   
                 GCTTC 
                   
                 ACAA 
                 FAM/T 
                   
                   
               
               
                   
                 (pDY0333) 
                   
                 CTTTG 
                   
                 CTAG 
                 G CTA 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 AATG 
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 CAGT 
                 C/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GA 
                 T TTA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 TTT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 /3IABk 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 FQ/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 TP90-1 
                 295 
                 CCCG 
                 296 
                 GAAC 
                 /56- 
                 407 
                 None 
               
               
                   
                 rc GFP 
                   
                 GCTTC 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 (pDY0334) 
                   
                 CTTTG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 PhiBT1 
                 297 
                 CCCG 
                 298 
                 AACC 
                 /56- 
                 406 
                 None 
               
               
                   
                 GFP 
                   
                 GCTTC 
                   
                 ACAA 
                 FAM/T 
                   
                   
               
               
                   
                 (pDY0367) 
                   
                 CTTTG 
                   
                 CTAG 
                 G CTA 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 AATG 
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 CAGT 
                 C/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GA 
                 T TTA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 TTT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 /3IABk 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 FQ/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 PhiBT1 
                 299 
                 CCCG 
                 300 
                 GAAC 
                 /56- 
                 407 
                 None 
               
               
                   
                 rc GFP 
                   
                 GCTTC 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 (pDY0368) 
                   
                 CTTTG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 LMNB1 
                 GFP 
                 301 
                 TCCTT 
                 302 
                 GAAC 
                 /56- 
                 407 
                 Eco91I, 
               
               
                   
                 (pDY0186) 
                   
                 ATCA 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 CGGT 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 CCCG 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                   
                   
                 CTCG 
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NOLC1 
                 GFP 
                 303 
                 CGTC 
                 304 
                 GAAC 
                 /56- 
                 407 
                 Eco91I, 
               
               
                   
                 (pDY0186) 
                   
                 GACA 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 ACGG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 TAGT 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                   
                   
                 G 
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 SUPT1 
                 GFP 
                 305 
                 TCGC 
                 306 
                 GAAC 
                 /56- 
                 407 
                 Eco91I, 
               
               
                 6 H 
                 pDY0186) 
                   
                 GTGA 
                   
                 TCCA 
                 FAM/C 
                   
                 HindIII 
               
               
                   
                   
                   
                 TTCTC 
                   
                 CGCC 
                 C ATG 
                   
                   
               
               
                   
                   
                   
                 GGAA 
                   
                 GTTC 
                 AAG 
                   
                   
               
               
                   
                   
                   
                 C 
                   
                 A 
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/31A 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 BkFQ/ 
                   
                   
               
               
                   
               
               
                 SRRM2 
                 GFP 
                 307 
                 GGGC 
                 308 
                 GAAC 
                 /56- 
                 407 
                 Eco91I, 
               
               
                   
                 (pDY0186) 
                   
                 GGTA 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 AGTG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 GTTA 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                   
                   
                 GTTT 
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 DEPDC4 
                 GFP 
                 309 
                 AAGA 
                 310 
                 GAAC 
                 /56- 
                 407 
                 Eco91I, 
               
               
                   
                 (pDY0186) 
                   
                 GGCG 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 GAGC 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 CAGT 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NES 
                 GFP 
                 311 
                 CTCCC 
                 312 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                   
                 (pDY0186) 
                   
                 TTCTC 
                   
                 TCCAC 
                 FAM/C 
                   
                 HindIII 
               
               
                   
                   
                   
                 CCGG 
                   
                 GCCG 
                 C GGC 
                   
                   
               
               
                   
                   
                   
                 TGCCC 
                   
                 TTCA 
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 C GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3IAB 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 kFQ/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 ACTB 
                 313 
                 CCCG 
                 314 
                 GAAC 
                 /56- 
                 407 
                 Eco91I 
               
               
                   
                 HITI 
                   
                 GCTTC 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 CTTTG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 TCC 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                 (pDY0219) 
                   
                   
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/ZE 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 N/T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 SRRM2 
                 SRRM2 
                 315 
                 GGGC 
                 316 
                 GAAC 
                 /56- 
                 407 
                 Eco91I 
               
               
                   
                 HITI 
                   
                 GGTA 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 AGTG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 GTTA 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                 (aRY0182_ 
                   
                 GTTT 
                   
                   
                 AAG 
                   
                   
               
               
                   
                 A2) 
                   
                   
                   
                   
                 A/ZE 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 N/T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NOLC1 
                 NOLC1 
                 317 
                 CGTC 
                 318 
                 GAAC 
                 /56- 
                 407 
                 Eco91I 
               
               
                   
                 HITI 
                   
                 GACA 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 ACGG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 TAGT 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                 (aRY0182_ 
                   
                 G 
                   
                   
                 AAG 
                   
                   
               
               
                   
                 A3) 
                   
                   
                   
                   
                 A/ZE 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 N/T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 DEPDC4 
                 DEPDC4 
                 319 
                 AAGA 
                 320 
                 GAAC 
                 /56- 
                 407 
                 Eco91I 
               
               
                   
                 HITI 
                   
                 GGCG 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 GAGC 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 CAGT 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                 (aRY0182_ 
                   
                 A 
                   
                   
                 AAG 
                   
                   
               
               
                   
                 A5) 
                   
                   
                   
                   
                 A/ZE 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 N/T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NES 
                 NES 
                 321 
                 CTCCC 
                 322 
                 GAAC 
                 /56- 
                 407 
                 Eco91I 
               
               
                   
                 HITI 
                   
                 TTCTC 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 CCGG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 TGCCC 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                 (aRY0182_ 
                   
                   
                   
                   
                 AAG 
                   
                   
               
               
                   
                 A7) 
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 LMNB1 
                 LMNB1 
                 323 
                 TCCTT 
                 324 
                 GAAC 
                 /56- 
                 407 
                 Eco91I 
               
               
                   
                 HITI 
                   
                 ATCA 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 CGGT 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 CCCG 
                   
                 TTCA 
                 ATG 
                   
                   
               
               
                   
                 (aRY0182_ 
                   
                 CTCG 
                   
                   
                 AAG 
                   
                   
               
               
                   
                 A4) 
                   
                   
                   
                   
                 A/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CCG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAT 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CA/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 SERPINA 
                 325 
                 CCCG 
                 326 
                 GGCC 
                 /56- 
                 405 
                 EcoRI, 
               
               
                   
                 (pDY0298) 
                   
                 GCTTC 
                   
                 TGCC 
                 FAM/ 
                   
                 XhoI, 
               
               
                   
                   
                   
                 CTTTG 
                   
                 AGCA 
                 CC 
                   
                 HindIII 
               
               
                   
                   
                   
                 TCC 
                   
                 GGAG 
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GA 
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 CPS1 
                 327 
                 CCCG 
                 328 
                 GGTG 
                 /56- 
                 408 
                 XhoI, 
               
               
                   
                 (pDY299) 
                   
                 GCTTC 
                   
                 TGCA 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 CTTTG 
                   
                 GTCA 
                 AC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 CATTG 
                 AGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GTAA 
                 TTT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AGCC 
                 C/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 TGG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 TGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/3IA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 BkFQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 / 
                   
                   
               
               
                   
               
               
                 ACTB 
                 CFTR 
                 329 
                 CCCG 
                 330 
                 GATG 
                 /56- 
                 409 
                 Eco91I, 
               
               
                   
                 (pDY0373) 
                   
                 GCTTC 
                   
                 GGTCT 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 CTTTG 
                   
                 AGTC 
                 TAC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 CAGC 
                 GGT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TAAA 
                 ACA/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 G 
                 ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ACC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 NYESO 
                 331 
                 CCCG 
                 332 
                 GAGA 
                 /56- 
                 409 
                 Eco47III, 
               
               
                   
                 TRAC 
                   
                 GCTTC 
                   
                 GACA 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                 (pDY0318) 
                   
                 CTTTG 
                   
                 AGGC 
                 TAC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 TGCA 
                 GGT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 CA 
                 ACA/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ACC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 A/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NC_ 
                 GFP 
                 333 
                 CCAG 
                 334 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                 000003 
                 (pDY0186) 
                   
                 GTGA 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 GAGT 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 CAGG 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                   
                   
                 GTAG 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                 TGTTC 
                   
                   
                 T/ZEN/ 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                 C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NC_ 
                 GFP 
                 335 
                 AGGG 
                 336 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                 000002 
                 (pDY0186) 
                   
                 ACCTT 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 TGCCT 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 GTGT 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                   
                   
                 GAGT 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                 C 
                   
                   
                 T/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 NC_ 
                 GFP 
                 337 
                 TCAG 
                 338 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                 000009 
                 (pDY0186) 
                   
                 CTCTG 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 TGCTG 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 AGGC 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                   
                   
                 GAA 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/ZEN/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 chr6: 
                 GFP 
                 339 
                 AAGC 
                 340 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                 149045959 
                 (pDY0186) 
                   
                 CATCT 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 CCCA 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 GAAT 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                   
                   
                 ATCTG 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                 CTTAG 
                   
                   
                 T/ZE 
                   
                   
               
               
                   
                   
                   
                 AAAT 
                   
                   
                 N/C 
                   
                   
               
               
                   
                   
                   
                 G 
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 chr16: 
                 GFP 
                 341 
                 GAGA 
                 342 
                 GAAC 
                 /56- 
                 405 
                 Eco91I, 
               
               
                 18607730 
                 (pDY0186) 
                   
                 GGAG 
                   
                 TCCAC 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                   
                   
                 CAAC 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                   
                   
                 AGTG 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                   
                   
                 AGCA 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                 TGAT 
                   
                   
                 T/ZE 
                   
                   
               
               
                   
                   
                   
                 G 
                   
                   
                 N/C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 chr6: 
                 ACTB 
                 343 
                 AAGC 
                 344 
                 GAAC 
                 /56- 
                 405 
                 Eco91I 
               
               
                 149045959 
                 HITI 
                   
                 CATCT 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 CCCA 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 GAAT 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                 (pDY0219) 
                   
                 ATCTG 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                 CTTAG 
                   
                   
                 T/ZE 
                   
                   
               
               
                   
                   
                   
                 AAAT 
                   
                   
                 N/C 
                   
                   
               
               
                   
                   
                   
                 G 
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 chr16: 
                 ACTB 
                 345 
                 GAGA 
                 346 
                 GAAC 
                 /56- 
                 405 
                 Eco91I 
               
               
                 18607730 
                 HITI 
                   
                 GGAG 
                   
                 TCCAC 
                 FAM/ 
                   
                   
               
               
                   
                 template 
                   
                 CAAC 
                   
                 GCCG 
                 CC 
                   
                   
               
               
                   
                 GFP 
                   
                 AGTG 
                   
                 TTCA 
                 GGC 
                   
                   
               
               
                   
                 (pDY0219) 
                   
                 AGCA 
                   
                   
                 TTG 
                   
                   
               
               
                   
                   
                   
                 TGAT 
                   
                   
                 T/ZE 
                   
                   
               
               
                   
                   
                   
                 G 
                   
                   
                 N/C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 CAG_ 
                 347 
                 CCCG 
                 348 
                 GGCT 
                 /56- 
                 405 
                 Eco91I, 
               
               
                   
                 Kozak_ 
                   
                 GCTTC 
                   
                 ATGA 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                 bGH_ 
                   
                 CTTTG 
                   
                 ACTA 
                 CC 
                   
                   
               
               
                   
                 thera- 
                   
                 TCC 
                   
                 ATGA 
                 GGC 
                   
                   
               
               
                   
                 peutic_ 
                   
                   
                   
                 CCCC 
                 TTG 
                   
                   
               
               
                   
                 genes 
                   
                   
                   
                 GT 
                 T/ZE 
                   
                   
               
               
                   
                 generic 
                   
                   
                   
                   
                 N/C 
                   
                   
               
               
                   
                 minicircle 
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 Hibit- 
                 349 
                 CCCG 
                 350 
                 GGCC 
                 /56- 
                 405 
                 EcoRI, 
               
               
                   
                 SERPINA 
                   
                 GCTTC 
                   
                 TGCC 
                 FAM/ 
                   
                 XhoI, 
               
               
                   
                 (pDY045) 
                   
                 CTTTG 
                   
                 AGCA 
                 CC 
                   
                 HindIII 
               
               
                   
                   
                   
                 TCC 
                   
                 GGAG 
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GA 
                 TTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/ZE 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 N/C 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 G/3I 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 ABkF 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Q/ 
                   
                   
               
               
                   
               
               
                 ACTB 
                 Hibit- 
                 351 
                 CCCG 
                 352 
                 GGTG 
                 /56- 
                 408 
                 XhoI, 
               
               
                   
                 CPS1 
                   
                 GCTTC 
                   
                 TGCA 
                 FAM/ 
                   
                 HindIII 
               
               
                   
                 (pDY406) 
                   
                 CTTTG 
                   
                 GTCA 
                 AC 
                   
                   
               
               
                   
                   
                   
                 TCC 
                   
                 CATTG 
                 AGC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GTAA 
                 TTT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AGCC 
                 C/ZE 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 N/A 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 TGG 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 TGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 GGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 CAC 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 T/3IA 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 BkFQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 / 
               
               
                   
               
            
           
         
       
     
     Sequences of primers used for NGS readout can be found in Table 8 below. 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 SEQ ID NO/ 
                   
                   
               
               
                 DESCRIPTION/ 
                   
                   
               
               
                 SOURCE 
                 ID 
                 SEQUENCE (5′-3′) 
               
               
                   
               
             
            
               
                 SEQ ID NO: 353 
                 PD0966 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGAC 
               
               
                 N-term ACTB Tn5 
                   
                 CTCGGC TCACAGCG 
               
               
                 readout F 1 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 354 
                 PD0967 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACCGA 
               
               
                 N-term ACTB Tn5 
                   
                 CCTCGG CTCACAGCG 
               
               
                 readout F 2 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 355 
                 PD0968 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGACCG 
               
               
                 N-term ACTB Tn5 
                   
                 ACCTCG GCTCACAGCG 
               
               
                 readout F 3 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 356 
                 PD0969 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACC 
               
               
                 N-term ACTB Tn5 
                   
                 GACCTC GGCTCACAGCG 
               
               
                 readout F 4 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 357 
                 PD0970 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGAC 
               
               
                 N-term ACTB Tn5 
                   
                 CGACCT CGGCTCACAGCG 
               
               
                 readout F 5 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 358 
                 PD0971 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGA 
               
               
                 N-term ACTB Tn5 
                   
                 CCGACC TCGGCTCACAGCG 
               
               
                 readout F 6 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 359 
                 PD0972 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACTG 
               
               
                 N-term ACTB Tn5 
                   
                 ACCGAC CTCGGCTCACAGCG 
               
               
                 readout F 7 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 360 
                 PD0973 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACT 
               
               
                 N-term ACTB Tn5 
                   
                 GACCGA CCTCGGCTCACAGCG 
               
               
                 readout F 8 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 361 
                 FP0952 
                 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAC 
               
               
                 ACTB N-term NGS 
                   
                 CCAGCC AGCTCCC 
               
               
                 R for Cas14 indels 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 362 
                 PD0313 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGGT 
               
               
                 NGS EMX1 
                   
                 GGCGCAT TGCCAC 
               
               
                 Forward 1 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 363 
                 PD0314 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACCGG 
               
               
                 NGS EMX1 
                   
                 TGGCGCA TTGCCAC 
               
               
                 Forward 2 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 364 
                 PD0315 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGACCG 
               
               
                 NGS EMX1 
                   
                 GTGGCGC ATTGCCAC 
               
               
                 Forward 3 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 365 
                 PD0316 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACC 
               
               
                 NGS EMX1 
                   
                 GGTGGCG CATTGCCAC 
               
               
                 Forward 4 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 366 
                 PD0317 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGAC 
               
               
                 NGS EMX1 
                   
                 CGGTGGC GCATTGCCAC 
               
               
                 Forward 5 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 367 
                 PD0318 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGA 
               
               
                 NGS EMX1 
                   
                 CCGGTGG CGCATTGCCAC 
               
               
                 Forward 6 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 368 
                 PD0319 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACTG 
               
               
                 NGS EMX1 
                   
                 ACCGGTG GCGCATTGCCAC 
               
               
                 Forward 7 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 369 
                 PD0320 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACT 
               
               
                 NGS EMX1 
                   
                 GACCGG GGCGCATTGCCAC 
               
               
                 Forward 8 
                   
                   
               
               
                 (Artificial Sequence) 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 370 
                 PD0321 
                 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGA 
               
               
                 NGS EMX1 Reverse 
                   
                 GTCCAGC TTGGGCCCA 
               
               
                 (Artificial Sequence) 
               
               
                   
               
            
           
         
       
     
     Sequences of off-target sites can be found in Table 9 below. 
     
       
         
           
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 SEQ ID NO/ 
                   
               
               
                 DESCRIPTION/ 
                   
               
               
                 SOURCE 
                 SEQUENCE (5′-3′) 
               
               
                   
               
             
            
               
                 SEQ ID NO: 371 
                 GATATTTTCCCAGCTCACCA 
               
               
                 Cas9_chr6:149045959 
                   
               
               
                 (Artificial 
                   
               
               
                 Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 372 
                 TCTATTCTCCCAGCTCCCCA 
               
               
                 Cas9_chr16:18607730 
                   
               
               
                 (Artificial 
                   
               
               
                 Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 373 
                 AGCGGCTTCTGTCTCTGTGAGTGAGCTGGCGGTCTCCGTC 
               
               
                 Bxb1_NC_000002 
                   
               
               
                 (Artificial 
                   
               
               
                 Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 374 
                 GACTAGCCCACGCTCCGGTTCTGAGCCGCGACGGCGGTCTCCG 
               
               
                 Bxb1_NC_000003 
                   
               
               
                 (Artificial 
                   
               
               
                 Sequence) 
                   
               
               
                   
               
               
                 SEQ ID NO: 375 
                 CCCAGGGTCCCATGCGCTCCCCGGCCCTGACGGCGGTCTCC 
               
               
                 Bxb1_NC_000009 
                   
               
               
                 (Artificial 
                   
               
               
                 Sequence) 
               
               
                   
               
            
           
         
       
     
     Linker sequences in Table 10 below. 
     
       
         
           
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Description 
                 Sequence (5′-3′) 
                 Amino acid sequence 
               
               
                   
               
             
            
               
                 A-P2A 
                 GGAAGCGGAGCTACTAACTTCAGCCT 
                 GSGATNFSLLKQAGDVEENPGP (SEQ ID 
               
               
                   
                 GCTGAAGCAGGCTGGCGACGTGGAGG 
                 NO: 418) 
               
               
                   
                 AGAACCCTGGACCT (SEQ ID NO: 410) 
                   
               
               
                   
               
               
                 B-(GGGS)3 
                 GGGGGAGGAGGTTCTGGAGGCGGAGG 
                 GGGGSGGGGSGGGGS (SEQ ID NO: 419) 
               
               
                   
                 CTCCGGAGGCGGAGGGTCA (SEQ ID 
                   
               
               
                   
                 NO: 411) 
                   
               
               
                   
               
               
                 C-GGGGS 
                 GGAGGTGGCGGGAGC (SEQ ID NO: 
                 GGGGS (SEQ ID NO: 420) 
               
               
                   
                 412) 
                   
               
               
                   
               
               
                 D-PAPAP 
                 CCCGCACCAGCGCCT (SEQ ID NO: 
                 PAPAP (SEQ ID NO: 421) 
               
               
                   
                 413) 
                   
               
               
                   
               
               
                 E-(EAAAK)3 
                 GAGGCAGCTGCCAAGGAAGCCGCT 
                 EAAAKEAAAKEAAAK (SEQ ID NO: 
               
               
                   
                 GCCAAGGAGGCGGCCGCAAAG 
                 422) 
               
               
                   
                 (SEQ ID NO: 414) 
                   
               
               
                   
               
               
                 F-XTEN 
                 AGTGGGAGCGAGACCCCTGGGACT 
                 SGSETPGTSESATPES (SEQ ID NO: 423) 
               
               
                   
                 AGCGAGTCAGCTACACCCGAAAGC 
                   
               
               
                   
                 (SEQ ID NO: 415) 
                   
               
               
                   
               
               
                 G-(GGS)6 
                 GGGGGGTCAGGTGGATCCGGCGG 
                 GGSGGSGGSGGSGGSGGS (SEQ ID NO 
               
               
                   
                 AAGTGGCGGATCCGGTGGATCTGG 
                 424) 
               
               
                   
                 CGGCAGT (SEQ ID NO: 416) 
                   
               
               
                   
               
               
                 H-EAAAK 
                 GAAGCTGCTGCTAAG (SEQ ID NO: 
                 EAAAK (SEQ ID NO: 425) 
               
               
                   
                 417) 
               
               
                   
               
            
           
         
       
     
     Exemplary fusion sequences in Table 11 below. 
     
       
         
           
               
               
             
               
                   
               
               
                 Description 
                 Sequence 
               
               
                   
               
             
            
               
                 SpCas9-XTEN- 
                 MKRTADGSEFESPKKKRKV   DKKYSIGLDIGTNSVGWAVITDEYKVPS     
               
               
                 RT(1-478)-Sto7d- 
                 
                   
                     KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR 
                   
                 
               
               
                 GGGGS-BxbINT 
                 
                   
                     KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                   
                 
               
               
                 Amino acid 
                 
                   
                     VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF 
                   
                 
               
               
                 SEQ ID NO: 376 
                 
                   
                     LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR 
                   
                 
               
               
                   
                 
                   
                     LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA 
                   
                 
               
               
                   
                 
                   
                     KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN 
                   
                 
               
               
                   
                 
                   
                     TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS 
                   
                 
               
               
                   
                 
                   
                     KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK 
                   
                 
               
               
                   
                 
                   
                     QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP 
                   
                 
               
               
                   
                 
                   
                     YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER 
                   
                 
               
               
                   
                 
                   
                     MTNFDKNLPNEKVLPKIISLLYEYFTVYNELTKVKYVTEGMRKPAF 
                   
                 
               
               
                   
                 
                   
                     LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
                 
               
               
                   
                 
                   
                     RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE 
                   
                 
               
               
                   
                 
                   
                     ERLKTYAHLFDDKVMKQLICRRRYTGWGRLSRKLINGIRDKQSGK 
                   
                 
               
               
                   
                 
                   
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI 
                   
                 
               
               
                   
                 
                   
                     ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT 
                   
                 
               
               
                   
                 
                   
                     QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL 
                   
                 
               
               
                   
                 
                   
                     QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK 
                   
                 
               
               
                   
                 
                   
                     NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                   
                 
               
               
                   
                 
                   
                     GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE 
                   
                 
               
               
                   
                 
                   
                     VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI 
                   
                 
               
               
                   
                 
                   
                     KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN 
                   
                 
               
               
                   
                 
                   
                     FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP 
                   
                 
               
               
                   
                 
                   
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDS 
                   
                 
               
               
                   
                 
                   
                     PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE 
                   
                 
               
               
                   
                 
                   
                     AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 
                   
                 
               
               
                   
                 
                   
                     PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEHEQISE 
                   
                 
               
               
                   
                 
                   
                     FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
                   
                 
               
               
                   
                     AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD   S 
               
               
                   
                 GGSSGGSSGSETPGTSESATPESSGSETPGTSESATPESSGSETPGTSESAT 
               
               
                   
                 PESSGGSSGGSST   LNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAET     
               
               
                   
                 
                   
                     GGMGLAVRQAPLIIPLKATSTPVSIKQVPMSQEARLGIKPHIQRLLD 
                   
                 
               
               
                   
                 
                   
                     QGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTV 
                   
                 
               
               
                   
                 
                   
                     PNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP 
                   
                 
               
               
                   
                 
                   
                     EMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQ 
                   
                 
               
               
                   
                 
                   
                     YVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQKQKQV 
                   
                 
               
               
                   
                 
                   
                     KYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFC 
                   
                 
               
               
                   
                 
                   
                     RLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPA 
                   
                 
               
               
                   
                 
                   
                     LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP 
                   
                 
               
               
                   
                 
                   
                     VAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVK 
                   
                 
               
               
                   
                 
                   
                     QPPDRWLSNARNITHYQALLLDTDRVQFGPVVALNPATLLPLPEEG 
                   
                 
               
               
                   
                 
                   
                     LQIINCLDGTGGGGVTVKFKYKGEELEVDISKIKKVWRVGKNIISFT 
                   
                 
               
               
                   
                     YDDNGKTGRGAVSEKDAPKELLQMLEKSGKKSGGSKRTADGS   EFE 
               
               
                   
                 PKKKRKVGGGGSPKKKRKVYPYDVPDYAG SRALVVIRLSRVTDATTS   
               
               
                   
                 
                   PERQLESCQQLCAQRGWDVVGVAEDLDVSGAVDPFDRKRRPNLAR 
                 
               
               
                   
                 
                   WLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVVSAT 
                 
               
               
                   
                 
                   EAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKY 
                 
               
               
                   
                 
                   RGSLPPWGYLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLH 
                 
               
               
                   
                 
                   LVAHDLNRRGVLSPKDYFAQLQGREPQGREWSATALKRSMISEAM 
                 
               
               
                   
                 
                   LGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAELVKTSRAKP 
                 
               
               
                   
                 
                   AVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHC 
                 
               
               
                   
                 
                   GNGTVAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAE 
                 
               
               
                   
                 
                   VNAELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEAR 
                 
               
               
                   
                 
                   PSGWEWRETGQRFGDWWREQDTAAKNTWLRSMNVRLTFDVRGG 
                 
               
               
                   
                 
                   LTRTIDFGDLQEYEQHLRLGSVVERLHTGMS 
                 
               
               
                   
               
               
                 SpCas9-XTEN- 
                 ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAG 
               
               
                 RT(1-478)-Sto7d- 
                 AAGCGGAAAGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCA 
               
               
                 GGGGS-BxbINT 
                 CCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCC 
               
               
                 Nucleic acid 
                 CAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATC 
               
               
                 SEQ ID NO: 377 
                 AAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAG 
               
               
                   
                 CCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCA 
               
               
                   
                 GACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGA 
               
               
                   
                 GATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCC 
               
               
                   
                 TTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCG 
               
               
                   
                 GCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCAT 
               
               
                   
                 CTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC 
               
               
                   
                 CTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGG 
               
               
                   
                 CCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTG 
               
               
                   
                 GACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA 
               
               
                   
                 GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTG 
               
               
                   
                 TCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCC 
               
               
                   
                 AGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGC 
               
               
                   
                 CCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGG 
               
               
                   
                 CCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGA 
               
               
                   
                 CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTG 
               
               
                   
                 TTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACAT 
               
               
                   
                 CCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCT 
               
               
                   
                 ATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGA 
               
               
                   
                 AAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTT 
               
               
                   
                 CTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGA 
               
               
                   
                 GCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAA 
               
               
                   
                 AGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGG 
               
               
                   
                 ACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCA 
               
               
                   
                 CCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAA 
               
               
                   
                 GATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGA 
               
               
                   
                 TCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGA 
               
               
                   
                 AACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCA 
               
               
                   
                 CCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCA 
               
               
                   
                 GAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAAC 
               
               
                   
                 GAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCG 
               
               
                   
                 TGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAG 
               
               
                   
                 AAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC 
               
               
                   
                 CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAG 
               
               
                   
                 AGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCC 
               
               
                   
                 GGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATC 
               
               
                   
                 TGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA 
               
               
                   
                 CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGG 
               
               
                   
                 ACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTT 
               
               
                   
                 CGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGG 
               
               
                   
                 CTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAG 
               
               
                   
                 CAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGC 
               
               
                   
                 CAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTT 
               
               
                   
                 AAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC 
               
               
                   
                 TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAA 
               
               
                   
                 GGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTG 
               
               
                   
                 ATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAG 
               
               
                   
                 AGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAA 
               
               
                   
                 TGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCT 
               
               
                   
                 GAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCT 
               
               
                   
                 GTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAG 
               
               
                   
                 GAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCG 
               
               
                   
                 TGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCT 
               
               
                   
                 GACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTC 
               
               
                   
                 CGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTG 
               
               
                   
                 AACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGG 
               
               
                   
                 CCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAA 
               
               
                   
                 GAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACA 
               
               
                   
                 GATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAG 
               
               
                   
                 CTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT 
               
               
                   
                 CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAAC 
               
               
                   
                 AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAA 
               
               
                   
                 CCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTA 
               
               
                   
                 CGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGC 
               
               
                   
                 GAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCA 
               
               
                   
                 ACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGA 
               
               
                   
                 GATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGA 
               
               
                   
                 GATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTG 
               
               
                   
                 CTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGA 
               
               
                   
                 CAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGA 
               
               
                   
                 TAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGC 
               
               
                   
                 GGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAA 
               
               
                   
                 AGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCT 
               
               
                   
                 GCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCC 
               
               
                   
                 ATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACC 
               
               
                   
                 TGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGC 
               
               
                   
                 CGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACG 
               
               
                   
                 AACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGC 
               
               
                   
                 CACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAAC 
               
               
                   
                 AGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGA 
               
               
                   
                 GCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAAT 
               
               
                   
                 CTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCA 
               
               
                   
                 TCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAAT 
               
               
                   
                 CTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCG 
               
               
                   
                 GAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATC 
               
               
                   
                 CACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC 
               
               
                   
                 AGCTGGGAGGTGACTCTGGAGGATCTAGCGGAGGATCCTCTGGCAG 
               
               
                   
                 CGAGACACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCTCTGGT 
               
               
                   
                 AGCGAGACACCCGGTACCAGTGAAAGCGCCACGCCAGAAAGCAGT 
               
               
                   
                 GGGAGTGAGACTCCGGGTACATCTGAATCAGCGACACCGGAATCAA 
               
               
                   
                 GTGGCGGCAGCAGCGGCGGCAGCAGCACCCTAAATATAGAAGATGA 
               
               
                   
                 GTATCGGCTACATGAGACCTCAAAAGAGCCAGATGTTTCTCTAGGGT 
               
               
                   
                 CCACATGGCTGTCTGATTTTCCTCAGGCCTGGGCGGAAACCGGGGGC 
               
               
                   
                 ATGGGACTGGCAGTTCGCCAAGCTCCTCTGATCATACCTCTGAAAGC 
               
               
                   
                 AACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCACAAGAA 
               
               
                   
                 GCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCAGG 
               
               
                   
                 GAATACTGGTACCCTGCCAGTCCCCCTGGAACACGCCCCTGCTACCC 
               
               
                   
                 GTTAAGAAACCAGGGACTAATGATTATAGGCCTGTCCAGGATCTGA 
               
               
                   
                 GAGAAGTCAACAAGCGGGTGGAAGACATCCACCCCACCGTGCCCAA 
               
               
                   
                 CCCTTACAACCTCTTGAGCGGGCCCCCACCGTCCCACCAGTGGTACA 
               
               
                   
                 CTGTGCTTGATTTAAAGGATGCCTTTTTCTGCCTGAGACTCCACCCC 
               
               
                   
                 ACCAGTCAGCCTCTCTTCGCCTTTGAGTGGAGAGATCCAGAGATGGG 
               
               
                   
                 AATCTCAGGACAATTGACCTGGACCAGACTCCCACAGGGTTTCAAA 
               
               
                   
                 AACAGTCCCACCCTGTTTAATGAGGCACTGCACAGAGACCTAGCAG 
               
               
                   
                 ACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACGTGGAT 
               
               
                   
                 GACTTACTGCTGGCCGCCACTTCTGAGCTAGACTGCCAACAAGGTAC 
               
               
                   
                 TCGGGCCCTGTTACAAACCCTAGGGAACCTCGGGTATCGGGCCTCG 
               
               
                   
                 GCCAAGAAAGCCCAAATTTGCCAGAAACAGGTCAAGTATCTGGGGT 
               
               
                   
                 ATCTTCTAAAAGAGGGTCAGAGATGGCTGACTGAGGCCAGAAAAGA 
               
               
                   
                 GACTGTGATGGGGCAGCCTACTCCGAAGACCCCTCGACAACTAAGG 
               
               
                   
                 GAGTTCCTAGGGAAGGCAGGCTTCTGTCGCCTCTTCATCCCTGGGTT 
               
               
                   
                 TGCAGAAATGGCAGCCCCCCTGTACCCTCTCACCAAACCGGGGACT 
               
               
                   
                 CTGTTTAATTGGGGCCCAGACCAACAAAAGGCCTATCAAGAAATCA 
               
               
                   
                 AGCAAGCTCTTCTAACTGCCCCAGCCCTGGGGTTGCCAGATTTGACT 
               
               
                   
                 AAGCCCTTTGAACTCTTTGTCGACGAGAAGCAGGGCTACGCCAAAG 
               
               
                   
                 GTGTCCTAACGCAAAAACTGGGACCTTGGCGTCGGCCGGTGGCCTA 
               
               
                   
                 CCTGTCCAAAAAGCTAGACCCAGTAGCAGCTGGGTGGCCCCCTTGC 
               
               
                   
                 CTACGGATGGTAGCAGCCATTGCCGTACTGACAAAGGATGCAGGCA 
               
               
                   
                 AGCTAACCATGGGACAGCCACTAGTCATTCTGGCCCCCCATGCAGTA 
               
               
                   
                 GAGGCACTAGTCAAACAACCCCCCGACCGCTGGCTTTCCAACGCCC 
               
               
                   
                 GGATGACTCACTATCAGGCCTTGCTTTTGGACACGGACCGGGTCCAG 
               
               
                   
                 TTCGGACCGGTGGTAGCCCTGAACCCGGCTACGCTGCTCCCACTGCC 
               
               
                   
                 TGAGGAAGGGCTGCAACACAACTGCCTTGATGGGACAGGTGGCGGT 
               
               
                   
                 GGTGTCACCGTCAAGTTCAAGTACAAGGGTGAGGAACTTGAAGTTG 
               
               
                   
                 ATATTAGCAAAATCAAGAAGGTTTGGCGCGTTGGTAAAATGATATC 
               
               
                   
                 TTTTACTTATGACGACAACGGCAAGACAGGTAGAGGGGCAGTGTCT 
               
               
                   
                 GAGAAAGACGCCCCCAAGGAGCTGTTGCAAATGTTGGAAAAGTCTG 
               
               
                   
                 GGAAAAAGTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATT 
               
               
                   
                 CGAGCCCAAGAAGAAGAGGAAAGTCGGAGGTGGCGGGAGCCCAAA 
               
               
                   
                 AAAGAAAAGAAAAGTGTATCCCTATGATGTCCCCGATTATGCCGGT 
               
               
                   
                 TCAAGAGCCCTGGTCGTGATTAGACTGAGCCGAGTGACAGACGCCA 
               
               
                   
                 CCACAAGTCCCGAGAGACAGCTGGAATCATGCCAGCAGCTCTGTGC 
               
               
                   
                 TCAGCGGGGTTGGGATGTGGTCGGCGTGGCAGAGGATCTGGACGTG 
               
               
                   
                 AGCGGGGCCGTCGATCCATTCGACAGAAAGAGGAGGCCCAACCTGG 
               
               
                   
                 CAAGATGGCTCGCTTTCGAGGAACAGCCCTTTGATGTGATCGTCGCC 
               
               
                   
                 TACAGAGTGGACCGGCTGACCCGCTCAATTCGACATCTCCAGCAGCT 
               
               
                   
                 GGTGCATTGGGCTGAGGACCACAAGAAACTGGTGGTCAGCGCAACA 
               
               
                   
                 GAAGCCCACTTCGATACTACCACACCTTTTGCCGCTGTGGTCATCGC 
               
               
                   
                 ACTGATGGGCACTGTGGCCCAGATGGAGCTCGAAGCTATCAAGGAG 
               
               
                   
                 CGAAACAGGAGCGCAGCCCATTTCAATATTAGGGCCGGTAAATACA 
               
               
                   
                 GAGGCTCCCTGCCCCCTTGGGGATATCTCCCTACCAGGGTGGATGGG 
               
               
                   
                 GAGTGGAGACTGGTGCCAGACCCCGTCCAGAGAGAGCGGATTCTGG 
               
               
                   
                 AAGTGTACCACAGAGTGGTCGATAACCACGAACCACTCCATCTGGT 
               
               
                   
                 GGCACACGACCTGAATAGACGCGGCGTGCTCTCTCCAAAGGATTAT 
               
               
                   
                 TTTGCTCAGCTGCAGGGAAGAGAGCCACAGGGAAGAGAATGGAGTG 
               
               
                   
                 CTACTGCACTGAAGAGATCTATGATCAGTGAGGCTATGCTGGGTTAC 
               
               
                   
                 GCAACACTCAATGGCAAAACTGTCCGGGACGATGACGGAGCCCCTC 
               
               
                   
                 TGGTGAGGGCTGAGCCTATTCTCACCAGAGAGCAGCTCGAAGCTCT 
               
               
                   
                 GCGGGCAGAACTGGTCAAGACTAGTCGCGCCAAACCTGCCGTGAGC 
               
               
                   
                 ACCCCAAGCCTGCTCCTGAGGGTGCTGTTCTGCGCCGTCTGTGGAGA 
               
               
                   
                 GCCAGCATACAAGTTTGCCGGCGGAGGGCGCAAACATCCCCGCTAT 
               
               
                   
                 CGATGCAGGAGCATGGGGTTCCCTAAGCACTGTGGAAACGGGACAG 
               
               
                   
                 TGGCCATGGCTGAGTGGGACGCCTTTTGCGAGGAACAGGTGCTGGA 
               
               
                   
                 TCTCCTGGGTGACGCTGAGCGGCTGGAAAAAGTGTGGGTGGCAGGA 
               
               
                   
                 TCTGACTCCGCTGTGGAGCTGGCAGAAGTCAATGCCGAGCTCGTGG 
               
               
                   
                 ATCTGACTTCCCTCATCGGATCTCCTGCATATAGAGCTGGGTCCCCA 
               
               
                   
                 CAGAGAGAAGCTCTGGACGCACGAATTGCTGCACTCGCTGCTAGAC 
               
               
                   
                 AGGAGGAACTGGAGGGCCTGGAGGCCAGGCCCTCTGGATGGGAGTG 
               
               
                   
                 GCGAGAAACCGGACAGAGGTTTGGGGATTGGTGGAGGGAGCAGGA 
               
               
                   
                 CACCGCAGCCAAGAACACATGGCTGAGATCCATGAATGTCCGGCTC 
               
               
                   
                 ACATTCGACGTGCGCGGTGGCCTGACTCGAACCATCGATTTTGGCGA 
               
               
                   
                 CCTGCAGGAGTATGAACAGCACCTGAGACTGGGGTCCGTGGTCGAA 
               
               
                   
                 AGACTGCACACTGGGATGTCC 
               
               
                   
               
               
                 SpCas9 
                 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                 Amino acid 
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH 
               
               
                 SEQ ID NO: 378 
                 RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 
               
               
                   
                 ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
               
               
                   
                 ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG 
               
               
                   
                 LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN 
               
               
                   
                 LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE 
               
               
                   
                 KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN 
               
               
                   
                 REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILT 
               
               
                   
                 FRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER 
               
               
                   
                 MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG 
               
               
                   
                 EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL 
               
               
                   
                 GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH 
               
               
                   
                 LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
               
               
                   
                 NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGIL 
               
               
                   
                 QTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE 
               
               
                   
                 EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL 
               
               
                   
                 SDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN 
               
               
                   
                 YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK 
               
               
                   
                 HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI 
               
               
                   
                 NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS 
               
               
                   
                 EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWD 
               
               
                   
                 KGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK 
               
               
                   
                 DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERS 
               
               
                   
                 SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ 
               
               
                   
                 KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII 
               
               
                   
                 EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 
               
               
                   
                 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 RT(1-478)-Sto7d 
                 LNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII 
               
               
                 Amino acid 
                 PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP 
               
               
                 SEQ ID NO: 379 
                 VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTV 
               
               
                   
                 LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT 
               
               
                   
                 LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT 
               
               
                   
                 LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPT 
               
               
                   
                 PKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQK 
               
               
                   
                 AYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRR 
               
               
                   
                 PVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAP 
               
               
                   
                 HAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL 
               
               
                   
                 PLPEEGLQHNCLDGTGGGGVTVKFKYKGEELEVDISKIKKVWRVGKMI 
               
               
                   
                 SFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKKSGGSKRTADGS 
               
               
                   
               
               
                 BxbINT 
                 SRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDVSG 
               
               
                 Amino acid 
                 AVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHW 
               
               
                 SEQ ID NO: 380 
                 AEDHKKLVVSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSA 
               
               
                   
                 AHFNIRAGKYRGSLPPWGYLPTRVDGEWRLVPDPVQRERILEVYHRVV 
               
               
                   
                 DNHEPLHLVAHDLNRRGVLSPKDYFAQLQGREPQGREWSATALKRSM 
               
               
                   
                 ISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAELVKTSRA 
               
               
                   
                 KPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCG 
               
               
                   
                 NGTVAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNA 
               
               
                   
                 ELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEARPSGWE 
               
               
                   
                 WRETGQRFGDWWREQDTAAKNTWLRSMNVRLTFDVRGGLTRTIDFG 
               
               
                   
                 DLQEYEQHLRLGSVVERLHTGMS 
               
               
                   
               
            
           
         
       
     
     EXAMPLES 
     While several experimental Examples are contemplated, these Examples are intended to be non-limiting. 
     Example 1 
     CRE Integration Efficiency 
     The efficiency of the CRE integration was tested. In order to test the efficacy of PASTE with GFP using lox71/lox66/Cre recombinase system, a clonal HEK293FT cell line with lox71 sequence (SEQ ID NO: 1) integrated into the genome using lentivirus was developed. The integration of GFP was tested by transfection of modified HEK293FT cell line with: (1) plus/minus SEQ ID NO: 71 comprising a Cre recombinase expression plasmid, and (2) SEQ ID NO: 72 comprising a GFP template and a lox 66 Cre site of SEQ ID NO: 2. After 72 hours, the percent integration of GFP into the lox71 site was probed.  FIG.  3    shows the percent integration of GFP in the lentiviral integrated lox71 site in HEK293FT cell line in the presence of various plasmids. It was observed that pCMV PE2 P2A Cre (SEQ ID NO: 73), a mammalian expression vector with prime editing complex and Cre recombinase linked to PE2 via a cleavable linker or a non-cleavable linker, shows integration of GFP. 
     Example 2 
     Programmable Addition Via Site-Specific Targeting Elements (PASTE) with Cre Recombinase—Addition of Lox Site 
     The lox71 (SEQ ID NO: 1) or lox66 (SEQ ID NO: 2) sequence was inserted into the HEK293FT cell genome using prime editing to test integration of GFP into the HEK293FT genome. In order to insert lox71 or lox66 sequence into HEK293FT cell genome, a pegRNA with PBS length of 13 base pairs operably linked to RT region of varying lengths was used. The following plasmids were used in the transfection of HEK293FT cells. The cells were transfected with (1) prime editing construct (PE2) or PE2 with conditional Cre expression, (2) Lox71 or Lox66 pegRNA targeting the HEK3 locus, and (3) plus/minus+90 HEK3 nicking second guide RNA targeting the HEK3 locus (+90 ngRNA). After 72 hours, the percent editing of the HEK293FT genome at the HEK3 locus was probed for incorporation of various lengths of lox71 or lox66 (see  FIG.  4   ). It was observed that 34 base pair lox71 (HEK3 locus guide, SEQ ID NO: 83; and Lox71 pegRNA with RT 34 and PBS 13, SEQ ID NO: 81) with +90 ngRNA (SEQ ID NO: 75) and 34 base pair lox66 (HEK3 locus guide, SEQ ID NO: 83; and Lox66 pegRNA with RT 34 and PBS 13, SEQ ID NO: 82) with +90 ngRNA (SEQ ID NO: 75) had the highest percent editing. 
     Example 3 
     PASTE with Cre Recombinase—Integration of Gene 
     The lox71 or lox66 pegRNAs having PBS length of 13 base pairs and insert length of 34 base pairs were used to probe integration of GFP in the HEK293F genome. The PE and Cre were delivered in an inducible expression vectors and induced at day 2. The HEK293FT cells were transfected with the following plasmids: (1) prime editing construct (PE2 or PE2 with conditional Cre expression); (2) Lox71 pegRNA; (3) plus/minus+90 HEK3 nicking guide RNA; and (4) EGFP template with Lox66 site. After 72 hours, the percent editing of lox71 site and percent integration of GFP was probed with or without lox66 site in the presence of various PE/Cre constructs.  FIG.  5 A  summarizes the percent editing of lox71 site with different PE/Cre vectors.  FIG.  5 B  summarizes the percent integration of GFP at the lox71 site in HEK293FT cell genome. It was observed that although the lox71 site was edited in the presence of inducible or non-inducible PE/Cre expression system, there was no GFP integration. 
     Example 4 
     Bxb1 Integration Data  Lenti  Reporter 
     The integration system was switched to an integrase system that could result in an integration of target genes into a genome with higher efficiency. Serine integrase Bxb1 has been shown to be more active than Cre recombinase and highly efficient in bacteria and mammalian cells for irreversible integration of target genes.  FIG.  6    shows a schematic of PASTE methodology using Bxb1 (Merrick, C. A. et al., ACS Synth. Biol. 2018, 7, 299-310). 
     To probe the efficiency of the Bxb1 integration system, a clonal HEK293FT cell line with attB Bxb1 site (SEQ ID NO: 3) integrated using lentivirus was developed. The modified HEK293FT cell line was then transferred with the following plasmids: (1) plus/minus Bxb1 expression plasmid and (2) plus/minus GFP (SEQ ID NO: 76) or G-Luc (SEQ ID NO: 77) minicircle template with attP Bxb1 site. After 72 hours, the integration of GFP or Gluc into the attB site in the HEK293FT genome was probed. The percent integrations of GFP or Gluc into the attB locus are shown in  FIG.  7   . It was observed that GFP and Gluc showed efficient integration into the attB site in HEK293FT cells. 
     Example 5 
     Addition of Bxb1 Site to Human Genome Using PRIME 
     The maximum length of attB that can be integrated into a HEK293FT cell line with the best efficiency was probed. To probe the best length of attB (SEQ ID NO: 3) or its reverse complement attP (SEQ ID NO: 4) for prime editing, pegRNAs having PBS length of 13 nt with varying RT homology length were used. The following plasmids were transfected in HEK293FT: (1) prime expression plasmid; (2) HEK3 targeting pegRNA design; and (3) HEK3 +90 nicking guide. After 72 hours, the percent integration of each of the attB construct was probed.  FIG.  8    shows the percent editing in each HEK3 targeting pegRNA. It was observed that attB with 44, 34 and 26 base pairs and attB reverse complement with 34 and 26 base pairs showed the highest percent editing. 
     Integration PASTE was then tested with tagging cell-organelle marker proteins with GFP in HEK29FT cells. PASTE was used to tag SUPT16H, SRRM2, LAMNB1, NOLC1 and DEPDC4 with GFP in different cell-culture wells and to test the usefulness of PASTE in tracking protein localization within the cells using microscopy.  FIGS.  9 A- 9 G  shows the fluorescent microscopy results for each of the organelles. SUPT16H-GFP was observed to be enriched in the nucleus, SRRM2-GFP was observed to be enriched in the nuclear speckles, LAMNB1-GFP was observed to be enriched in the nuclear membrane, NOLC1-GFP was observed to be enriched in the fibrillar center, and DEPDC4-GFP was observed to be enriched in the aggresome. 
     The transfection of the plasmids can be achieved using electroporation as illustrated in  FIGS.  10 A- 10 B . 
     Example 6 
     Programmable Integration of Genes with PASTE 
     The efficiency of gene integration of Gluc or EGFP with PASTE was tested. To enable gene integration with PASTE, the following HEK3 targeting pegRNAs were used: (1) 44 pegRNA: PBS of 13nt and RT homology of 44nt; (2) 34 pegRNA: PBS of 13nt and RT homology of 34nt; and (3) 26 pegRNA: PBS of 13nt and RT homology of 26nt. 
     A HEK293 cell line was transfected with following plasmids HEK293FT: (1) Prime expression plasmid; (2) Bxb1 expression plasmid; (3) HEK3 targeting pegRNA design; (4) HEK3 +90 nicking guide; and (5) EGFP or Gluc minicircle. After 72 hours, the percent integration of Gluc or EGFP was observed.  FIG.  11    shows integration of EGFP and Gluc with each of the tested HEK3 targeting pegRNAs. It was observed that EGFP and Gluc were efficiently integrated using PASTE. 
     Example 7 
     PASTE for Integration of Multiple Genes 
     The PASTE technique for site-specific integration of multiple genes into a cell is facilitated with the use of orthogonal attB and attP sites. Central dinucleotide can be changed to GA from GT, and only GA containing attB/attP sites can interact and do not cross react with GT containing sequences. A screen of dinucleotide combinations to find orthogonal attB/attP pairs for multiplexed PASTE editing can be performed. It has been shown that many orthogonal dinucleotide combinations can be found using a Bxb1 reporter system. 
     To test this, attB GT  and attB GA  dinucleotides for Bxb1 was added at a ACTB site by prime editing. A EGFP—attP GT  DNA minicircle and a mCherry—attP GA  DNA minicircle was introduced to test the percent EGFP and mCherry editing in the presence or absence of Bxb1. The results of EGFP and mCherry editing are shown in  FIGS.  14 A- 14 B . 
     Orthogonal editing with the right GT-EGFP and GA-mCherry pairs was achieved demonstrating the ability for multiplexed PASTE editing in cells. 
     Two genes were introduced in the same cell using multiplexed PASTE to tag two different genes in a single reaction. EGFP and mCherry were tagged into the loci of ACTB and NOLC1 in a x cell line, in a single reaction. Further, EGFP and mCherry were tagged into the loci of ACTB and LAMNB1. The cells were visualized using fluorescence microscopy.  FIGS.  15 A- 15 B  show the results of fluorescent microscopy for multiplexed PASTE. 
     The ability of multiplexing with 9-different attB and attP central dinucleotides—AA, GA, CA, AG, AC, CC, GT, CT and TT (SEQ ID NOs: 7, 8, 23, 24, 19, 20, 25, 26, 27, 28, 9, 10, 15, 16, 17, 18, 5 and 6)— in a 9×9 cross of attB and attP was tested. The edits were probed using next-generation sequencing. The results of the 9×9 cross of attB and attP central dinucleotides— AA, GA, CA, AG, AC, CC, GT, CT and TT— are shown in  FIG.  16 A . Only orthogonal pairs of attB and attP show the highest edit percentage. This result is also shown in the heat-map of  FIG.  16 B . 
     Example 8 
     Integration of Albumin and CPS1 Into Albumin Locus 
     12 pegRNAs with albumin guide were linked to PBS and reverse transcriptase sequence of variable length, and different nicking guide RNAs were used to transfect HEK293FT cells. The percent editing in the albumin was probed using next-generation sequencing. The results of prime editing at the albumin locus are shown in  FIG.  17   . It was observed that SEQ ID NO: 79 showed the highest percent edits with SERPINA1 and SEQ ID NO: 80 showed the highest percent edits with CPS1. 
     Example 9 
     Engineering T-cells 
     In order to engineer CD8+ T-cells, the efficiency of PASTE delivery and editing in T-cells can be evaluated ( FIG.  18   ). ACTB targeting pegRNA can be used to insert an integration site with an EGFP insertion template. To deliver the PASTE components to CD8+ T-cells, electroporation can be used along with an optimized electroporation protocol for unstimulated T-cells. As multiple plasmids may reduce the efficiency of electroporation, the consolidated PASTE components that use fewer vectors can be applied. 
     Five vectors, three vectors, and two vectors PASTE systems show that robust T-cell editing can be achieved with maximal editing using the three-vector approach ( FIG.  19   ). Further, expanded sets of electroporation conditions, including the overall plasmid amounts, cell numbers, and voltage/amperage protocol can be tested. In addition, stimulation of T-cells may influence the efficiency of transduction and PASTE efficiency. Further, CD4+/CD8+ T cell mixtures stimulated with T-Activator CD3/CD28 ligands can have higher PASTE editing efficiency versus unstimulated cells. In order to separate efficiency of PASTE from the overall delivery rate, an mCherry expression cassette on PASTE vectors can be evaluated in order to sort successfully transfected T cells. Once optimized parameters are achieved, a panel of 10 insertion sites with PASTE in T cells, including the TRAC, IL2Ra, and PDCD1 loci, can be evaluated, using different insertions (e.g. EGFP, BFP, and YFP), both in single and multiplexed editing contexts. A tested subset of relevant sites in HEK293FT achieved greater than 40% editing for EGFP insertion ( FIG.  20   ). The PASTE efficiency at TRAC locus with different TCR and CAR constructs can be evaluated. The T-cells can successfully be transfected to achieve insertion of CARs or TCRs. 
     Example 10 
     PASTE for CFTR 
     PASTE for the CFTR locus can be tested in HEK293FT cells to identify top performing pegRNA and nicking designs for human cells. Neuro-2A cells can also be tested to identify top performing pegRNA and nicking designs for mouse cells. The best constructs can be applied for testing in mouse air lung interface (ALI) organoids in vitro or for delivery in pre-clinical models of cystic fibrosis in mice. Table 12 shows the pegRNA, nicking guide and minicircle DNA characteristics for the CFTR gene modulation. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                 Variables 
                 Characteristics 
               
               
                   
                   
               
             
            
               
                   
                 pegRNA 
                 38 bp shortened minimal attB and normal 
               
               
                   
                   
                 46 bp attB sequence with: 
               
               
                   
                   
                 a. PBS of 17, 13, and 9 nt length, and 
               
               
                   
                   
                 b. RT of 20, 15, and 10 nt in length 
               
               
                   
                 Nicking guides 
                 Nicking guide 1 +64 bp Nicking guide 2 +23 bp 
               
               
                   
                   
                 Nicking guide 3 −60 bp Nicking guide 4 −78 bp 
               
               
                   
                   
                 (distance is calculated from cut site of pegRNA) 
               
               
                   
                 Minicircle 
                 A. CFTR coding sequence alone 
               
               
                   
                 template 
                 (~4,454 pb in size) 
               
               
                   
                   
                 B. CFTR coding sequence plus 5′ and 3′ UTRs 
               
               
                   
                   
                 (~6,011 bp in size) 
               
               
                   
                   
                 (Both minicircles have attP site on them for 
               
               
                   
                   
                 integration by Bxb1 and a bGH poly A signal) 
               
               
                   
                   
               
            
           
         
       
     
     Example 11 
     AttB and EGPF Integration Using PASTE 
     The efficiency of the integration of attB and EGPF at the ACTB locus was evaluated ( FIGS.  21 A- 21 C ). To investigate whether Bxb1 can add an EGFP template into this site, a delivery approach using a 5 plasmid system expressing each of the following component was deployed: 1) pegRNA expression, 2) nicking guide expression, 3) Prime expression (Cas9-RT), 4) Bxb1 expression and 5) the insertion template (in this case EGFP). This approach was found to yield editing efficiency of the attB site up to 24% and integration of EGFP ˜10% in HEK293FT cells as measured by sequencing ( FIGS.  21 A- 21 B ). Optimal activity is achieved in 3-4 days and can be performed as a single step transfection or electroporation of all components. Because the EGFP plasmid is designed as a minicircle, allowing removal of all undesired bacterial components, only the desired gene is inserted along with minimal scars from the Bxb1 recombined sites. 
     To make the tool simpler to use, the Bxb1 can be linked to Prime via a P2A linker to the Cas9-RT fusion, allowing for only a single plasmid to be used for PASTE protein expression rather than two. This optimization can maintain the same level of editing, making it easier to use the tool and deliver it ( FIG.  21 C ). 
     Example 12 
     Programmable EGFP Integrations in Different Cell Types 
     The programmable EGFP integration in liver hepatocellular carcinoma cell line HEPG2 ( FIG.  22 A ) and chronic myelogenous leukemia cell line K562 ( FIG.  22 B ) was evaluated. EGFP integration at the ACTB locus in K562 and HEPG2 cells of about 15% was observed, demonstrating robustness of the platform across cell types. 
     Example 13 
     Mutagenesis of Bxb1 for Enhanced PASTE Activity 
     The mutagenesis of Bxb1 for enhanced PASTE activity was evaluated ( FIGS.  23 A- 23 C ). Two levers for optimizing PASTE activity exist: 1) improving the activity of the integrase and 2) enhancing the Prime addition of the integration sequence. As illustrated in  FIGS.  23 A- 23 B , Bxb1 activity can be improved as only about 30% of Bxb1 attB sites that are added by PASTE are integrated into by Bxb1. This illustrates that if the Bxb1 efficiency can be improved, the PASTE can be improved. Furthermore, catalytic residues in the Bxb1 integrase were identified via conservation and structural analyses and Bxb1 mutants were generated to test as part of PASTE. As illustrated in  FIG.  23 B , the mutations can improve integration by about 20-30%. 
     Example 14 
     Effect of the pegRNA PBS and RT Lengths on the Prime Editing Integration Efficiency 
     The effect of the pegRNA PBS and RT lengths on the prime editing integration efficiency was evaluated ( FIGS.  25 A- 25 F ). It was found that PASTE can be optimized by tuning the PBS and RT lengths at the ACTB locus to achieve editing rates up to about 20% ( FIG.  25 A ). It was found that shortening the attB site can help improve PASTE function as Prime is better at inserting shorter sequences. Further optimization of PBS, RT, and attB lengths showed that optimal designs can be found for insertion upstream of the LMNB1, NOLC1, and GRSF1 loci ( FIGS.  25 B,  25 C, and  25 D ). Lengths as short as 36nt for attB were found to be still functional for integration into a reporter plasmid ( FIGS.  25 B and  25 C ). It was found that the reverse complemented version of the attB sequence was better integrated via Prime editing, suggesting that the sequence of what Prime is inserting matters. EGFP integrations with attP site mutants showed that certain mutants can improve integration efficiency significantly ( FIG.  25 E ). PASTE was also performed with a large panel of genes, inserting EGFP at the N-terminus of ACTB, LMNB1, SUPT16H, SRRM2, NOLC1, KLHL15, GRSF1, DEPDC4, NES, PGM1, CLTA, BASP1, and DNAJC18 ( FIG.  25 F ). Editing rates that are about 5%-40% were found using digital droplet PCR (ddPCR). 
     Example 15 
     Comparison of PASTE and HITI On-target and Off-target Activities 
     The PASTE and HITI on-target and off-target activities were compared ( FIGS.  26 A- 26 F ). PASTE and HITI were found to have about 22% and 5% integration efficiencies respectively when using the same guide sequence ( FIGS.  26 A and  26 B ). PASTE was found to outperform HITI at most sites when analyzing the editing of 14 genes ( FIG.  26 C ). Using a ddPCR based approach, it was found that PASTE was very specific with minimal off-target activity for Bxb1 off-targets integrations ( FIG.  26 D ) and Cas9 off-targets integrations ( FIG.  26 E ). The analysis of inserts of different sizes showed that PASTE can reliably insert sequences 1 kb-10 kb in size ( FIG.  26 F ), revealing the wide range of sequence sizes PASTE is capable of working with. A decrease in insertion efficiency at larger sizes was also observed, which was likely due to the reduction in plasmid delivery to HEK293FT cells at larger plasmid sizes. 
     Example 16 
     Multiplexing with PASTE and Orthogonal Di-nucleotide attB and attP Sites 
     Multiplexing with PASTE and orthogonal di-nucleotide attB and attP sites was evaluated ( FIGS.  28 A- 28 C ). Multiple orthogonal combinations were found for mutants of the central di-nucleotide motif ( FIGS.  28 A and  28 B ). As illustrated in  FIG.  28 C , programmable multiplexed gene insertion can be achieved by using these orthogonal combinations with PASTE only delivering different pegRNAs and gene inserts while keeping the protein components the same ( FIG.  8 C ). 
     Example 17 
     PASTE Multiplexed Integrations at Endogenous Sites 
     PASTE multiplexed integrations at endogenous sites were evaluated ( FIGS.  28 A- 28 G ). A reading frame for the attR scar that is left post-integration by Bxb1 that is ideal for a protein linker due to the enrichment of glycines, serines, and prolines in the sequence (GLSGQPPRSPSSGSSG (SEQ ID NO: 426)) was identified. PegRNAs were designed using this linker frame for the resolution of the attR for tagging a number of genes at the N-terminus with EGFP (ACTB, NOLC1, LMNB1, SUPT16H, SRRM2, and DEPDC4). As these genes all have distinct protein localization appearances, microscopy can be used for ascertaining proper gene tagging. PASTE was found to be capable of high-efficiency gene tagging with protein localizations that match the reference images and expected localization of the proteins in the cells ( FIGS.  28 A- 28 C ). Genes were also tagged in multiplexed fashion to demonstrate the orthogonality of the engineered integration sites. ACTB, LMNB1, NOLC1, and GRSF were targeted with orthogonal pegRNAs carrying GT, TG, AC, and CA, respectively in HEK293FT in groups of single, dual-plexing, and triple-plexing ( FIGS.  28 D- 28 E ). These dinucleotides were paired with templates carrying EGFP, BFP, and mCherry to allow for multicolor imaging of these labeled genes. The efficiencies of integration for these multiplexing experiments were found to range from about 5%-32%, revealing efficient multiplex integration with PASTE. Using confocal microscopy of these multiplexed integration experiments, cells were found with simultaneous labeling of these different proteins ( FIGS.  28 F- 28 G ). 
     Example 18 
     Combination of CRISPR-based Genome Editing and Site-specific Integration 
     The combination of CRISPR-based genome editing and site-specific integration was evaluated. 
     PegRNAs containing different attB length truncations were assessed ( FIG.  29 A ). Prime editing was found to be capable of inserting sequences up to 56 bp at the beta-actin (ACTB) gene locus, with higher efficiency at lengths below 31 bp ( FIGS.  29 A-B ) The integration of cognate landing sites was tested for multiple insertion enzymes: Bxb1, TP901, and phiBT1 phage serine integrases and Cre recombinase. Prime editing successfully inserted all landing sites tested, with efficiencies between 10-30% ( FIGS.  29 C-D ). To test the complete system, all components were combined and delivered in a single transfection: the prime editing vector, the landing site containing pegRNA, a nicking guide for stimulating prime editing, a mammalian expression vector for the corresponding integrase or recombinase and a 969 bp minicircle DNA cargo encoding green fluorescent protein (GFP) ( FIG.  29 E ). GFP integration rates among the four integrases and recombinases were compared and Bxb1 integrase was found to have the highest integration rate (˜20%) at the targeted ACTB locus and require the prime editing nicking guide for optimal performance ( FIGS.  29 F-H ). Finally, to reduce the number of transfected components, Bxb1 was co-expressed with the SpCas9-M-MLV reverse transcriptase (PE2) fusion protein via a P2A protein cleavage site. This combination maintained high GFP insertion efficiency, up to 30% ( FIG.  29 E ). The complete system, PASTE, achieved precise integration of templates as large as 9,500 bp with greater than 10% integration efficiency ( FIGS.  29 J-K  and  26 E), with complete integration of the full-length cargo confirmed by Sanger sequencing ( FIG.  30 A-E ). 
     Example 19 
     Impact of Prime Editing and Integrase Parameters on PRIME Editing 
     The impact of prime editing and integrase parameters on the integration efficiency of PRIME editing was assessed. 
     Relevant pegRNA parameters for PASTE include the primer binding site (PBS), reverse transcription template (RT), and attB site lengths, as well as the relative locations and efficacy of the pegRNA spacer and nicking guide ( FIG.  31 A ). A range of PBS and RT lengths were tested at two loci, ACTB and lamin B1 (LMNB1), and rules governing efficiency were found to vary between loci, with shorter PBS lengths and longer RT designs having higher editing at the ACTB locus ( FIG.  31 B ) and longer PBS and shorter RT designs performing better at LMNB1 ( FIG.  31 C ). 
     The length of the attB landing site must balance two conflicting factors: the higher efficiency of prime editing for smaller inserts and reduced efficiency of Bxb1 integration at shorter attB lengths. AttB lengths were evaluated at ACTB, LMNB1, and nucleolar phosphoprotein p130 (NOLC1), and the optimal attB length was found to be locus dependent. At the ACTB locus, long attB lengths could be inserted by prime editing ( FIG.  29 B ) and overall PASTE efficiencies for the insertion of GFP were highest for long attB lengths ( FIG.  31   d   ). In contrast, intermediate attB lengths had higher overall integration efficiencies (&gt;20%) at LMNB1 ( FIG.  31 E ) and NOLC1 ( FIG.  31 F ), indicating that the increased efficiency of installing shorter attB sequences overcame the reduction of Bxb1 integration at these sites. 
     The PE3 version of prime editing combines PE2 and an additional nicking guide to bias resolution of the flap intermediate towards insertion. To test the importance of nicking guide selection on PASTE editing, editing at ACTB and LMNB1 loci was tested with two nicking guide positions. Suboptimal nicking guide positions were found to reduce the PASTE efficiency up to 30% ( FIG.  32 A ) in agreement with the 75% reduction of PASTE efficiency in the absence of nicking guide ( FIG.  29 G ). The pegRNA spacer sequence was found to be necessary for PASTE editing, and substitution of the spacer sequence with a non-targeting guide was found to eliminate editing ( FIG.  32 B ). 
     Rational mutations were also introduced in both the Bxb1 integrase and reverse transcriptase domain of the PE2 construct to optimize PASTE further. While some of these mutations were well tolerated by PASTE ( FIGS.  33 A-B ), none of them improved PASTE editing efficiency. 
     Short RT and PBS lengths can offer additional improvements for editing. A panel of shorter RT and PBS guides were tested at ACTB and LMNB1 loci and while shorter RT and PBS sequences did not increase editing at ACTB ( FIG.  31 G ), it was found that they had improved editing at LMNB1 ( FIG.  31 H ) with best performing guides reaching GFP insertion rates of ˜40% ( FIG.  31 I ). 
     Example 20 
     PASTE Tagging at Multiple Endogenous Genes 
     GFP insertion efficiency was measured at seven different gene loci—ACTB, SUP T16H, SRM2, NOLC1, DEPDC4, NES, and LMNB1—to test the versatility of the PASTE programming. A range of integration rates up to 22% was found ( FIG.  34 A ). Because PASTE does not require homology or sequence similarity on cargo plasmids, integration of diverse cargo sequences is modular and easily scaled across different loci. Six different gene cargos, varying in size from 969 bp to 4906 bp, were tested for insertion at ACTB and LMNB1 loci with PASTE. Integration frequencies between 5% and 22% depending on the gene and insertion locus were found ( FIGS.  34 B and  35   ). Additionally, a panel of seven common therapeutic genes, CEP290, OTC, HBB, PAH, GBA, BTK, and ADA was evaluated for insertion at the ACTB locus, and the efficient integration of these cargos were found between 5%-20% ( FIG.  34 C ). 
     The precise insertions of PASTE for in-frame protein tagging or expressing cargo without disruption of endogenous gene expression was assessed. As Bxb1 leaves residual sequences in the genome (termed attL and attR) after cargo integration, these genomic scars can serve as protein linkers. The frame of the attR sequence was positioned through strategic placement of the attP on the minicircle cargo, achieving a suitable protein linker, GGLSGQPPRSPSSGSSG (SEQ ID NO: 427). Using this linker, four genes (ACTB, SRRM2, NOLC1, and LMNB1) were tagged with GFP using PASTE. To assess correct gene tagging, the subcellular location of GFP was compared with the tagged gene product by immunofluorescence. For all four targeted loci, GFP co-localized with the tagged gene product, indicating successful tagging ( FIGS.  34 D-E ). 
     Example 21 
     Orthogonal Sequence Preferences for Bxb1 Integration 
     The central dinucleotide of Bxb1 is involved in the association of attB and attP sites for integration, and changing the matched central dinucleotide sequences can modify integrase activity and provide orthogonality for insertion of two genes. Expanding the set of attB/attP dinucleotides can enable multiplexed gene insertion with PASTE. The efficiency of GFP integration at the ACTB locus with PASTE across all 16 dinucleotide attB/attP sequence pairs was profiled to find optimal attB/attP dinucleotides for PASTE insertion. Several dinucleotides with integration efficiencies greater than the wild-type GT sequence were found ( FIG.  36 A ). A majority of dinucleotides had 75% editing efficiency or greater compared to wild-type attB/attP efficiency, implying that these dinucleotides can be orthogonal channels for multiplexed gene insertion with PASTE. 
     The specificity of matched and unmatched attB/attP dinucleotide interactions was then assessed. The interactions between all dinucleotide combinations in a scalable fashion using a pooled assay to compare attB/attP integration were profiled ( FIG.  36 B ). By barcoding 16 attP dinucleotide plasmids with unique identifiers, co-transfecting this attP pool with the Bxb1 integrase expression vector and a single attB dinucleotide acceptor plasmid, and sequencing the resulting integration products, the relative integration efficiencies of all possible attB/attP pairs were measured ( FIG.  36 C ). Dinucleotide specificity was found to vary, with some dinucleotides (GG) exhibiting strong self-interaction with negligible crosstalk, and others (AA) showing minimal self-preference. Sequence logos of attP preferences ( FIG.  37   ) revealed that dinucleotides with C or G in the first position have stronger preferences for attB dinucleotide sequences with shared first bases, while other attP dinucleotides, especially those with an A in the first position, have reduced specificity for the first attB base. 
     GA, AG, AC, and CT dinucleotide pegRNAs were then tested for GFP integration at ACTB, either paired with their corresponding attP cargo or mispaired with the other three dinucleotide attP sequences. All four of the tested dinucleotides efficiently were found to integrate cargo only when paired with the corresponding attB/attP pair, with no detectable integration across mispaired combinations ( FIG.  36 D ). 
     Example 22 
     Multiplex Gene Integration with PASTE 
     Multiplexing in cells by using orthogonal pegRNAs that direct a matched attP cargo to a specific site in the genome was assessed ( FIG.  38 A ). Selecting the three top dinucleotide attachment site pairs (CT, AG, and GA), pegRNAs that target ACTB (CT), LMNB1 (AG), and NOLC1 (GA) and corresponding minicircle cargo containing GFP (CT), mCherry (AG), and YFP (GA) were designed. Upon co-delivering these reagents to cells, single-plex, dual-plex, and trip-plex editing of all possible combinations of these pegRNAs and cargo in the range of 5%-25% integration was found to be achieved ( FIG.  38 B ). 
     An application for multiplexed gene integration is for labeling different proteins to visualize intracellular localization and interactions within the same cell. PASTE was used to simultaneously tag ACTB (GFP) and NOLC1 (mCherry) or ACTB (GFP) and LMNB1 (mCherry) in the same cell. No overlap of GFP and mCherry fluorescence was observed and tagged genes were confirmed to be visible in their appropriate cellular compartments, based on the known subcellular localizations of the ACTB, NOLC1 and LMNB1 protein products ( FIGS.  15 A-B ). 
     Example 23 
     PASTE Efficiencies Compared With DSB-based Insertion Methods 
     PASTE efficiencies were found to exceed comparable DSB-based insertion methods. 
     PASTE editing was assessed alongside DSB-dependent gene integration using either NHEJ (i.e., homology-independent targeted integration, HITI) or HDR pathways. PASTE had equivalent or better gene insertion efficiencies than either HITI ( FIGS.  39 A-B ) or HDR ( FIGS.  39 C-D ). On a panel of 7 different endogenous targets, PASTE exceeded HITI editing at 6 out of 7 genes, with similar efficiency for the 7th gene ( FIG.  39 A ). As DSB generation can lead to insertions or deletions (indels) as an alternative and undesired editing outcome, the indel frequency of all three methods was assessed by next-generation sequencing, finding significantly fewer indels generated with PASTE than either HDR or HITI in both HEK293FT and HepG2 cells ( FIGS.  39 B,  39 D and  40 A ), showcasing the high purity of gene integration outcomes with PASTE. 
     Example 24 
     Off-target Characterization of PASTE and HITI Gene Integration 
     Off-target editing can be used in genome editing technologies. The specificity of PASTE at specific sites was assessed based on off-targets generated by Bxb1 integration into pseudo-attB sites in the human genome and off-targets generated via guide- and Cas9-dependent editing in the human genome ( FIG.  39 E ). While Bxb1 lacks documented integration into the human genome at pseudo-attachment sites, potential sites with partial similarity to the natural Bxb1 attB core sequence were computationally identified. Bxb1 integration by ddPCR across these sites was tested and no off-target activity was found ( FIGS.  39 F and  40 B -D). To assay Cas9 off-targets for the ACTB pegRNA, two potential off-target sites were identified via computational prediction and no off-target integration for PASTE was found ( FIGS.  39 G and  40 A -D), but substantial off-target activity by HITI at one of the sites was found ( FIGS.  39 H and  40 A -D). 
     Genome-wide off-targets due to either Cas9 or Bxb1 through tagging and PCR amplification of insert-genomic junctions were additionally assessed ( FIG.  39 I ). Single cell clones were isolated for conditions with PASTE editing and negative controls missing PE2, and deep sequencing of insert genomic junctions from these clones showed all reads aligning to the on-target ACTB site, confirming no off-target genomic insertions ( FIGS.  39 J-L ). 
     Expression of reverse transcriptases and integrases involved in PASTE can have detrimental effects on cellular health. The complete PASTE system, the corresponding guides and cargo with only PE2, and the corresponding guides and cargo with only Bxb1 were transfected and compared to both GFP control transfections and guides without protein expression via transcriptome-wide RNA sequencing to determine the extent of these effects. While Bxb1 expression in the absence of Prime editing was found to have several significant off targets, the complete PASTE system had only one differentially regulated gene with more than a 1.5-fold change ( FIGS.  41 A-B ). Genes upregulated by Bxb1 overexpression included stress response genes, such as TENT5C and DDIT3, but these changes were not seen in the expression of the PASTE system ( FIG.  41 C ), potentially due to the decreased expression of Bxb1 from the P2A linker on the PASTE construct. 
     Example 25 
     PASTE Efficiency in Non-Dividing Cell 
     PASTE activity in non-dividing cells was assessed. Cas9 and HDR templates or PASTE were transfected into HEK293FT cells and cell division was arrested via aphidicolin treatment ( FIG.  42 A ). In this model of blocked cell division, PASTE was found to maintain a GFP gene integration activity greater than 20% at the ACTB locus whereas HDR-mediated integration was abolished ( FIGS.  42 B and  43 A ). 
     Example 26 
     Production and Secretion of Therapeutic Transgene 
     PASTE with larger transgenes and in additional cell lines were assessed. 
     To evaluate the size limits for therapeutic transgenes, insertion of cargos up to 13.3 kb in length in both dividing and aphidicolin treated cells was assessed. Insertion efficiency greater than 10% was found ( FIG.  42 C ), enabling insertion of ˜99.7% of all full-length human cDNA transgenes. To overcome reduction of large insert delivery to cells because of delivery inefficiencies, delivering larger DNA amounts of insert was found to significantly improve gene integration efficiency ( FIG.  43 B ). PASTE editing to additional cell types such as PASTE in the K562 lymphoblast line and in primary human T cells were also assessed. Both PE2-P2A-Bxb1 (PASTE) and separate delivery of PE2 and Bxb1 were found to result in efficient editing in both cell types ( FIGS.  42 D-E ). Lastly, as therapeutic delivery of PASTE in vivo might require viral delivery of the DNA cargo, whether AAV could deliver an attP containing payload that could be integrated into the genome via Bxb1 was evaluated. Targeting the ACTB locus, AAV was found to be capable of delivering the appropriate template for integrase mediated insertion with rates up to 4% in a dose dependent fashion ( FIGS.  42 F and  43 C ). 
     To improve the efficiency of PASTE, PE2* NLS was incorporated for prime editing and improved PASTE integration at multiple loci was found ( FIG.  44 A ). Furthermore, PE2* resulted in more robust integration at lower titrations of cargo plasmid, demonstrating integration at amounts as low as 8 ng of plasmid ( FIG.  44 B ). To combat reductions in PASTE efficiency due to incomplete plasmid delivery, a puromycin resistance gene was co-delivered and found to increase the PASTE efficiency in the presence of drug selection ( FIG.  45   ). 
     Programmable gene integration provides a modality for expression of therapeutic protein products, and protein production was assessed for therapeutically relevant proteins Alpha-1 antitrypsin (encoded by SERPINA1) and Carbamoyl phosphate synthetase I (encoded by CPS1), involved in the diseases Alpha-1 antitrypsin deficiency and CPS1 deficiency, respectively. By tagging gene products with the luminescent protein subunit HiBiT, the transgene production and secretion were assessed independently in response to PASTE treatment ( FIG.  42 G ). PASTE was transfected with SERPINA1 or CPS1 cargo in HEK293FT cells and a human hepatocellular carcinoma cell line (HepG2) and efficient integration at the ACTB locus was found ( FIG.  42 H-I ). This integration resulted in robust protein expression, intracellular accumulation of transgene products ( FIGS.  42 J and  46 A -B), and secretion of proteins into the media ( FIG.  42 K ). 
     Example 27 
     Optimized PASTE Constructs 
     To optimize complex activity, a panel of protein modifications were screened, including alternative reverse transcriptase fusions and mutations, various linkers between the reverse transcriptase domain and integrase and between the Cas9 and reverse transcriptase domain, and reverse transcriptase and BxbINT domain mutants ( FIG.  47 A  and  FIG.  49 C - FIG.  49 F ). A number of protein modifications, including a 48 residue XTEN linker between the Cas9 and reverse transcriptase and the fusion of MMuLV to the Sto7d DNA binding domain (Oscorbin et al. FEBS Lett. 594. 4338-4356. 2020) improved editing efficiency ( FIG.  47 A  and  FIG.  49 C - FIG.  49 D ). When these top modifications were combined with a GGGGS linker (SEQ ID NO: 420) between the reverse transcriptase-Sto7d domain and the BxbINT, they produced—55% gene integration, highlighting the importance of directly recruiting the integrase to the target site ( FIG.  47 A ). This optimized construct was referred to as SpCas9-(XTEN-48)-RT-Sto7d-(GGGGS)-BxbINT. The optimized contruct achieved precise integration of templates as large as ˜36,000 bp with ˜20% integration efficiency ( FIG.  47 A ), with complete integration of the full-length cargo confirmed by Sanger sequencing. 
     Additionally, pegRNAs containing different AttB length truncations were tested and found that prime editing was capable of inserting sequences up to 56 bp at the beta-actin (ACTB) gene locus, with higher efficiency at lengths below 31 bp ( FIG.  48 A - FIG.  48 B ). A panel of multiple enzymes was evaluated, including Bxb1 (i.e., BxbINT), TP901 (i.e., Tp9INT), and phiBT1 (i.e., Bt1INT) phage serine integrases. Prime editing successfully inserted all landing sites tested, with efficiencies between 10-30% ( FIG.  48 C - FIG.  48 D ) 
     Example 28 
     Viral Delivery &amp; In Vivo Editing 
     In order to package the complete PASTE system in viral vectors, an AdV vector was utilized ( FIG.  50 B ). Adenovirus was evaluated for if it could deliver a suitable template for BxbINT-mediated insertion along with plasmids for SpCas9-RT-BxbINT and guide expression, or AdV delivery of guides and BxbINT with plasmid delivery of SpCas9-RT, finding that 10-20% integration of the ˜36 kb adenovirus genome carrying EGFP in HEK293FT and HepG2 cells was achieved ( FIG.  50 C ). Upon packaging and delivering the cargo and PASTE system components across 3 AdV vectors, the complete PASTE system (Cas9-reverse transcriptase, integrase and guide RNAs, or cargo) could be substituted by adenoviral delivery, with integration of up to ˜50-60% with viral-only delivery in HEK293FT and HepG2 cells ( FIG.  50 D ). 
     To further demonstrate PASTE would be amenable for in vivo delivery, an mRNA version of the PASTE protein components was developed as well as chemically-modified synthetic atgRNA and nicking guide against the LMNB1 target ( FIG.  50 E ). Electroporation of the mRNA and guides along with delivery of the template via adenovirus or plasmid yielded high efficiency integration up to ˜23% ( FIG.  50 E - FIG.  50 F ). More sustained BxbINT expression could allow for integration into newly placed AttB sites in the genome, so circular mRNA expression was tested and found to boost the efficiency of integration to ˜30% ( FIG.  50 G - FIG.  50 I ). 
     Example 29 
     Simultaneous Deletion &amp; Insertion With PASTE 
     The PASTE system was used to simultaneously delete one sequence and insert another. 130 bp and 385 bp deletions of first exon of LMNB1 with combined insertion of AttB nucleic acid sequence was performed ( FIG.  51 A ). This data shows that it is possible to replace DNA sequence using the PASTE system. 
     A130 bp deletion of the first exon of LMNB1 with combined insertion of a 967 bp cargo using the PASTE system was also performed. 
     One of two attP sequences were inserted using the mini circle template that has mutated AttP, as described above. This AttP mutants shows better integration kinetics and efficiency, especially for the shorter AttBs (38-44 bp). The LMNB1 AttB used in this experiment is 38 bp ( FIG.  51 B ).