Matrix attachment region and use for promoting gene expression

Abstract

The present invention relates to newly identified matrix attachment regions (MARs) and chimeric sequences. The present invention also discloses nucleotide constructs comprising the MARs at appropriate positions relative to protein coding sequences for optimal expression of the proteins.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 776,643, filed December 7, 2018, pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference. Background Technology

[0003] Transcription of gene sequences (i.e., the generation of mRNA) is controlled at many different levels. Transcription start sites, or promoters, have varying strengths, and the transcription initiation frequency of a given gene can also be increased by enhancer sequences. Pauses during transcription can affect the transcription rate, thus affecting the amount of transcript produced within a given time period. The rates of precursor mRNA splicing, polyadenylation, and cleavage can also affect the level of mRNA produced by transcription units. Furthermore, the sequence within the mRNA molecule can regulate its transport from the nucleus to the cytoplasm, as well as its turnover rate (i.e., its cytoplasmic stability).

[0004] The expression of peptides (such as therapeutic antibodies and growth factors) in vitro is important for the pharmaceutical industry, and there is a need for methods to maximize protein expression.

[0005] Overview

[0006] This invention describes a novel technique for evaluating the strength of matrix attachment regions (MARs) and demonstrates its potential for identifying previously unknown novel MAR sequences. The invention also describes chimeric MAR sequences.

[0007] The present invention also describes nucleotide constructs comprising cis-MAR placements and other associated sequences for optimal expression of a gene of interest (GOI).

[0008] Therefore, according to one embodiment of the present invention, a recombinant polynucleotide is provided comprising: a coding sequence; a promoter configured to initiate transcription of the coding sequence; and a matrix attachment region (MAR) core selected from SEQ ID NO: 1, 5, 9 and 13 and nucleic acid sequences having at least 75% sequence identity with any one of SEQ ID NO: 1, 5, 9 and 13, wherein the MAR core is capable of attaching to the nuclear matrix of mammalian cells.

[0009] Another embodiment provides a method for transfecting a coding sequence into a cell, comprising: contacting the cell with a first polynucleotide comprising a coding sequence and a promoter for initiating transcription of the coding sequence and a second polynucleotide comprising a matrix attachment region (MAR) core comprising a nucleic acid sequence selected from SEQ ID NO: 1, 5, 9 and 13 and having at least 75% sequence identity with any one of SEQ ID NO: 1, 5, 9 and 13, wherein the MAR core is capable of attaching to the mammalian cell nuclear matrix, under conditions for transfecting first and second polynucleotides into the cell.

[0010] The present invention also provides a chimeric matrix attachment region (MAR) comprising: (a) a MAR core selected from SEQ ID NO: 1, 5, 9 and 13 and a nucleic acid sequence having at least 75% sequence identity with any one of SEQ ID NO: 1, 5, 9 and 13, wherein the MAR core is capable of attaching to the nuclear matrix of mammalian cells; (b) a 5' flanking region selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30 and 34 and a nucleic acid sequence having at least 75% sequence identity with any one of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30 and 34; and (c) a 3' flanking region selected from SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31 and 35 and a nucleic acid sequence having at least 75% sequence identity with any one of SEQ ID NO: 1, 5, 9 and 13, wherein the MAR core is capable of attaching to the nuclear matrix of mammalian cells; (b) a 5' flanking region selected from SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31 and 35 and a nucleic acid sequence having at least 75% sequence identity with any one of SEQ ID NO: 1, 5, 9 and 13; and (c) a 3' flanking region selected from SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31 and 35; and (d ... Any one of the nucleic acid sequences 3, 7, 11, 15, 19, 23, 27, 31, and 35 that has at least 75% sequence identity, wherein the MAR core, 5' flanking region, and 3' flanking region do not originate from the same natural MAR. sequence list <110> Sersien Corporation <120> Matrix attachment zone and its use in promoting gene expression <130> 21F-1869-CNP <150> 62 / 776643 <151> 2018-12-07 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 186 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 1 aaataattaa ttagcaaata atttatatat ttatatattt atatatatat atatatatat 60 atatatatat atatatatat atatatatat atatatatat atatatatat atatatatat 120 atatatatat atatatatat atatatatat aaatatataa atatatatat atatatatat 180 attggt 186 <210> 2 <211> 185 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 2 aaatatttca aaaattaagc aaagaaactt tcacagtatg actaataata ttttttcttc 60 tggagaaagt cttatttgtc ttatttcggc tagaataaaa acagttttta attttttaaa 120 caccatttta cggacaaaat tattagcccc tttaagctat atttattttc aatagtctac 180 agaac 185 <210> 3 <211> 98 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 3 gggagaagca gtccaatgtg gctgtatata tatattatta tttttttttt tttaaataca 60 aacaattaag ctgtcgaact gttgtataaa cacaatat 98 <210> 4 <211> 1879 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 4 atgcaaagga ctctttctgt aaaatatctg ctgtaaaaat ctaaaggatt tgagggttgc 60 actcccaaag gctcctgaga gcttgtgtaa aataagcctt cctaactgct gctttctcac 120 tttagtcaat tttggccttt tttatattca tatacagtaa atgttgctct aatcaatgtg 180 gatgatcatt atcagtatat gactcgaaaa atattttaaa cttctctttg cttctagacc 240 aggatttgaa ggaatattct atttttaatg acactgcttt gcaatattta tgattcttaa 300 agtcaaacag agtgaaacag aagtaaacat gtcatgatgt acactcacat gaaacaatct 360 ctgaaatgaa gaatgaatga agaaagttgt gttgctaaca aaatggagga agactcacga 420 atagacaaaa gcagggcaga aacgagacac gcacccgtgt acatatatac agttgaagtc 480 agacttatta gccccccttt gatttttttt tcttttttta aatatttcaa aaattaagca 540 aagaaacttt cacagtatga ctaataatat tttttcttct ggagaaagtc ttatttgtct 600 tatttcggct agataaaaa cagtttta ttttaaac accattttac ggacaaaatt 660 attagcccct ttaagctata tttattttca atagtctaca gaacataa ttattagca 720 Attattattattattattattattattattattattattattattattattattattattattattattattatat 780 grandfather father father father father father father father daddy daddy daddy daddy daddyttggt gggagagca 900 gtccaatgtg gctgtatata tatatta tttttttttt ttataaca aacattaag 960 ctgtcgaact gttgtataaa cacaatca ctcgagttgc agtccgatgt gactatatat 1020 tggcactggt gggagatttg cattcattgt gatac agccacattg cattgctacg 1080 agtgtgatat tatgtttata caccagttca acagcataat cgtgtgtaat aaaagataa 1140 tcaacacgg agaggaact actttctcc gccattcatt cacatctgca gcttacgtca 1200 gaacagcaga agctgttgct cattcatata tatatat aataatttgt atacttacag 1260 acgacacagt ggctcagtgg ttagcactgt ctcctcacag acgaaggtc actgttca 1320 gtccaggctg ggtccgccgg catttgtgtg tggagtttgc ttgtttttcc cgtgttaatg 1380 tgtatggatg tttcccagta ctgggttgca gctggaaggg catccgctgt gtaaaactta 1440 tgctggataa gttggcagtt cattccgctg tgtcaacccc tgatgaataa agggattaag 1500 ccaaatgaaa atgaatgaat gatttaatac tcttgtttaa gtcttagtgc cgattatata 1560 tagatatgtg gtgttagatc aaaccagtgt tcattttgac agcaaaattt gatttagttt 1620 tagtcatttt ttagtcttcc atattcgtta tagcatcagt ctatatacag tcaactaaat 1680 taaacatgat tttagtcaac ttcatgcatt ttcacatgga caaggtcatt aaaattctac 1740 aggatcaggt tgtaccaact ctatctcatg gaaatttgta actttttgat tgtggctaat 1800 tcgtatgaat ttgtatgatc tcatttgtac aatttagtat tatttgctca tcccccagtg 1860 atggttggat ttagggttg 1879 <210> 5 <211> 225 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 tatccaaata tatatatata tatatatata tatatactat atatatatat atatatatat 60 atatatatat atatatatat atatatatat atatatatat atatatatat atatatatat 120 atatatatat atatatatat atatatatat atatatatat atatatatat atatatatat 180 atatatatat atatatatat tttttttttt ttttttaaga aaaca 225 <210> 6 <211> 106 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 6 caaatttgct atacaaaaac ctaaatgtaa taaagttaca tccataaaag tgacttttta 60 ttacttttga aacacaacag atttatgccc tgtctatatc cagttc 106 <210> 7 <211> 109 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 gttataataa ttttataatc tctcacaata ttgctgtttt actgattttt gaataaatgc 60 agccttggtg agcataagag actttttcta aaaactttga aaagtctta 109 <210> 8 <211> 2679 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 8 tgttacatta ggctgaaaac acactgtgta tgtgcatatt ttcccttaaa tacataaaga 60 agtaatgggt ggcgtacctg tattggttat agactccagg gcagtataac atggctgtga 120 atagactctg ttgtggacat acactccata acaccaaact gatcccgtag aacgatgtca 180 ccaggaaaag aaggagcgtc aaaatgaact gcctgtgatt ggcttgacct acacagctgt 240 ttatcctgtc atggaaagtc acatacatac aagacacaaa tataagcaa cacatttaca 300 gatattttca ttcaaacaat ttagaacaat tatactattt tctctcttgt caatgtagct 360 ttaatgagaa tttgtggct catggcatgt tcatatttat atttgtgcct gtgtgtgtgt 420 tgaagtagca tggagacaga ttggcctggt gagagctccc tttactgtgc tgaaaactgt 480 gttcctgttg cctttgcaca tgtctcacca gcctgcctgc tccagtgacg tctttggga 540 atcagcttga acatatgtgt gtgtctgatg gggtagggaa cacatgcata caagtgtgaa 600 gagatcagaa aatgaatcaa atcattggca tgaccactaa tttaaacaat tcattttgga 660 attgttcagc tacttctgta gtaaggattt gcaataagcc agatatctta gaaggaaagg 720 cacaaatatc aacattaaaa ttaatattg aaatacaga aaatattc ggttgactga 780 ttatccaaaacacaagtg taaagtgata ttttaatga acatctaaaa ttacaatgca 840 acggtaagtc tgtgaagctg agatgattaa atgtgatct ttatgaatga aagcagatgg 900 ccacaatgct ctctctcctt ttattgttta taaatcataa atcatcatt catatagtaa 960 cttttttaga ttttgaatat ttatttgt aaagttttaa atattattga atagtaata 1020 atttaatatg ataatcattt aaaagtgt atatttgtaa atacattat ttttaaata 1080 tataaatgat gaaaaaaatt taatatag aaaacact ttataaata atacaaggca 1140 aatttgctat aaaaacct aaatgtaata aagttacacc cataaagtg actttttatt 1200 acttttgaaa cacacagat ttatgccctg tctatatcca gttctatcca atatata 1260 father father father father father father father father father grandfather father father father father father father father father grandfather father father father father father father father 1440 atattttt tttttttttt aagaaaacag ttataataat tttataatct ctcacaatat 1500 tgctgtttta ctgatttttg aataaatgca gccttggtga gcataagaga cttttctaa 1560 aaactttgaa aagtcttacc gacaacatat tattgaatgg cagtgtaaat atagatcgtt 1620 ttgtcattta gagaatattt aattaatgtt tatacactt cacactagga atgaagtgct 1680 cgtactcaat ttttaaagaa gaaggaatga gaaacccacc aaacacagtg gtggtccatt 1740 cgaaggacac atgccccaca gatcctgcag tgccctgccc ttggtgggcg cacctgtcgg 1800 cacacagggc ataacttctt ctttccttcg ttttccccat ttgactgact gattcctttg 1860 cagggggcgg agtctccagg tatgtttgtg ctctgattgg ttgtacagat actgttaatt 1920 ccctgagcta atgactggga tttgacaaag cctggtcctt gtttggtgcg cacaagggag 1980 ataagggtca acatcatgcc tgtcgtcaca gtaaccagct gcagattact gacgtctcct 2040 ctgggaacga tctcagtaag gaaaagatag tacatgtagg caagggagaa aagtgccaag 2100 ctaaggaaaa acaatgtgcg tcttttcctt ctgtgagtca catagtaata ccaaagcacc 2160 agtcctggta gggcagttag gacaacaact cccaacatac agttaattgc tgctactcgc 2220 aacagaccag gaattaatac catgggaggt aagacagaga tttccaatcg aaggggtcct 2280 acggcacaac atggcaaacc caaacgatca gccatacaag agacgaaacg agagaagatg 2340 tctggcttgt cttgctctcc tctgactaac ctattgagaa agagtgacaa attcaccatt 2400 catcatttac acactattat gttatcagtt ttatgtatgg aaatgacaag gtgcaaacgg 2460 acaaaattat ttaattcaac tacacttttt aaagtgtaaa aatgtaaagt gtcaaaagtt 2520 tatatcaata aatgtaatgt aaaaccatct ctgcatgcca tgatcttaac tggttgacct 2580 ctgacctttc acaggcatca tctagttctt cacagtcaca gcagcagtac tctctttgct 2640 ggtcgatgtc tccacagcag cacagagggt catctggtt 2679 <210> 9 <211> 350 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 9 atatatatat gttagtgata tatatataaa atatatatca ctcttattat atattatata 60 tattatataa tatataatat attatattat ataatatatt atatattata taatatatat 120 aatatattat atattatata atataatata ttatatatta tataataata taatatatta 180 tatattatat aataatatat aatatattat ataataatat ataataatat ataatatatt 240 attatataat atataataat atataataat ataataatgt gattatatta tatataatat 300 gtaatataat aatgtatata tattatatat gcattatata taatgatttt 350 <210> 10 <211> 186 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 10 aattgcataa gccaatccct tgcaataaat aaaatagatc gagatgtaga tagacagaga 60 tccagatcct actggttcca ttttcctggc agaaccatga tggatatgat atatgtccca 120 taaagagcct cccagtggtg tctttttgtt ttgtaaagat ggttttaatc cacagattta 180 agagtg 186 <210> 11 <211> 153 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 11 ctatcaaatg tatatttaaa tctacctact tgtgttatat atttatatat gtgtgtgtga 60 tatgtataat tttaaaatat atctgtatag ctacttaatt tgtatttcca gcccaattca 120 gtctagtgac agaaaatata actcatcaag tct 153 <210> 12 <211> 4061 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 12 tacccagtaa ttcttaaact acttactcca gcacttggcc catggcaaat ttcctggtag 60 ccatatgtac atatacacat gcttttaaat acataaacgt atgtacatat acttttaatt 120 tactactaag attttataca ttttccagta gtttgtatga tgaacatgta tatacctccc 180 tttaaagtta aacaataaaa aaatcttaat ttttctacct taaaatctcc tctcatcacg 240 tcatccaatg atagccgctg ttaacatcat gttcctattt acttctgctt gtttctctga 300 agatgtacgt gagtgggtaa acacgatgta atttgacatt gcggttttta atagcatatc 360 acaaatattt tcctgttata atttcattca caaacatggt tctgatagtt acataatatt 420 acaccatatg atcatttcct agtttactta gctatttcct atggttggat attcttggct 480 540. tgtttccaat atttttctat tctaattcat ctccaacaaa agtcatcatc tgtgaatctt tctctgcatc tctaagtatt tccttagaat aaactcctag agtgaatctg ttagcacatt tgtttctctc ttttatta caactggaa atgattattg cagacacatt ctcttcaagt gaatttct tctcggctga actttgcttt actgagtatt tttccctccc ttggctgcat tttcattctt cttttgctgt tttgcgtttc actcccacct acaaaatatc caagttccct 780 840. ctcccaaaat aaaacccctc ttggttcatg gcagagtcaa ctttgttcag ctgtctttac cctgcaggtc caaaggtcta ttgcccagag tattgctga ggggataaga ggtgccaggt cctcatcaga gtaatctttg catcttccat tttagatgat gaggctgagg ctaaataact ttttgtgact tgaacaaaat cacgcccgtc attttggagc agagattgga cccagaccat ctgactgcag tgcacttggt tactacacaa cagccctgga agctgcaacc tgaggacctc cagcctcaca tgaatcatct tcatccacca accttctccc atctgggttc cacactcct cccatcaccc agcatgggg gcctcatggt gtggttgac ccttcctta tctccacctg 1200 catatggggc acatctccc siaacctcac tccagtccaa tccacccc acccactgt 1260 ctcgctcata gggttgagga agacagaat aaaccctggc tgggaaagt cactttagtc 1320 aatgtacggt cagttttacg tgtcagcttg gccaggctag agtccccagt catccaaacg 1380 cccagttgac tgtaagcaaa gactatccta gataatttgg tcggcctgag acatcagtt 1440 aaaagaccat agagcagaa ctgaaaacag aaaattctgc ctgtggactg cagcgccagc 1500 tcctgcctga gagtttccag cctgaccttc ctaaacttgc ctactaatcc cccacaattg 1560 cataagccaa tcccttgca taaataaat agatcgagat gtagatagac agagatccag 1620 atcctactgg ttccatttc ctggcagaac catgatggat atgatatatg tcccataaag 1680 agcctcccag tggtgtcttt ttgttttgta aagatggttt taatccacag atttaagagt 1740 fatherfather tgttagtgat fatherfather fatherfather actcttatta fatherfather 1800 great-grandfather great-grandfather great-grandfather great-grandfather 1860 Our great-grandfather's great-grandfather's great-grandfather's great-grandfather's 1920 father father father father father father father father 1980 fatherfather fatherfather fatherfatherg tgattatt fatherfather 2040 tgtaatataa taatgtatatat atattatat tgcattatat ataatgatt tctatcaaat 2100 gtatattaa atctacctac ttgtgttata tattatata tgtgtgtgtg atatgtataa 2160 ttttaaaata tatctgtata gctacttaat ttgtatttcc agcccaattc agtcttagtga 2220 cagaaaatat aactcatcaa gtctgggtaa tctcaata ttcggctaaa ttcccaatt 2280 catcaatttc tccctcctcc agcctccgca taattgattc taacttcag ggagttgaag 2340 cagcactgcc ctggggggct ctctccagc tccagcccc tccctgcca gctctggtgc 2400 ccacggggat gcgcccacgg ggatgcaagg ccctgtatga ccatcagacc tcctgtccctc 2460 acttgagatg tttatttcta gtccccagtc ctgccccac cacaggatgt acccagacc 2520 ctggtctcta ttccagatt catccttgtc aagcggttct cagcagcatg cctctgccac 2580 gcccagtcac agaggacgat attggctccc ctctttatgg ggggaggata aaaaggatca 2640 tatcccagac ccctgggctt aaggcttcca tgttcccatc tctactcttt aatgccagga 2700 tgacataggg tactttctgc aggcatagac aaccaggcag gaagaggggc ccatgttcct 2760 tgtaatctca gatcacaagt accctctggg gatctatcac cccaagcttg gtgcatggac 2820 aaggggtacc ttattctctt cctttcttct aagactatgc actgcctctc ccccaggcaa 2880 atggaatctt gtcaattcca agtgagtttg gaaaaactca ttctttttta cttattgttt 2940 agatcggaac ctaaaatcga aagttccagg aagctcaagt tcttataact aaaaaggctt 3000 ttctctctca agagcattgg ctctgtcttt tccatgttct actttgaaac acaaaagctg 3060 agctcgacat tgatttaatt gctttttatt tgaatgattg ctataagtga caccttttgg 3120 ggcagtatgt cctagctggt tgaattagag ttgggaaaca cattgcaaga aagggtagaa 3180 agaggcatac tttattgaat cttttggagt aacttttgtg cctgttacca caagcttatt 3240 tttgataaga tgcttcctaa ctttcctcgc tcacccctac ctcatctcag ggccaagcac 3300 gaaagttgtt ttatacatgc taggtttggc tgatttcaat gtgacttccc cagggcagaa 3360 gagacagtgg ttgaaggatg aagaatcaac ctacccgggg gaatcaaaga ttgatcccag 3420 ggaatggagc agatataatt aaataagttt tttctttccc cagtgtatct aatattcatt 3480 tttgtattta cttggagcaa atgaatttaa atgaattttt agttagagtt aaatcaactc 3540 attaaaacac attgaaggaa ttagaaatgt aaaatcattt cctgctgcaa ttcattcatc 3600 tctaagtcta taactgactt gaatctaggt aaaggtcatt gcttaaaggg gacagtggcc 3660 cttatcttg caacctacaa acttttacag tttgcatgga gttttcacat ccatgaactc 3720 aagcaagact gaataggcct gtatggtgaa caaacaggag taaatgcctc cactattcag 3780 gggaggaaag tgagctcaaa aaagtttaat attgttgcct aggatactc aaccagcaaa 3840 tggcaatatc gagacaggag caggtcttca gtccaaaccc acagagcaga gcttgcccag 3900 gaagctgatc attcttgtac caccttctcc atcactggac ctacaccctg cccttcttta 3960 cccagctctg acctctcaga tgctgaacct ggggtgccct tgtcattgaa cttcgagttg 4020 cattattttc ccattgtttt acctacaatt aacttgccgt c 4061 <210> 13 <211> 1700 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 13 agactctgtc tcaaatatat atatatttat atatatatat tttatattta atatatataa 60 tatatattat atataaattt attatatata atatatatta tatataaatt tattatatat 120 aatatatatt atatatatta tatgttatat atatttatta catataatat ataatatata 180 tttattatat ataatatata tttattatat attatatatt atatatttta tattttatat 240 atattatata attttatatt ttatatatat tatataatta atatttaata tatattatat 300 atatatatat aatatatatt tattatatat aatatatatt tattatatat tatatattat 360 atatttatat tttatatata ttatatattt atattttata tatattatat atttatattt 420 tatatatatt atatataata tatatattat atattttata tataatatat atttattata 480 catattttat attatatata atatatattt attatatatt tttatatata ttatatataa 540 grandfather grandfather father father father father father father 600 attattattattattattattattattattattattattattattattattadad 660 fatherfather father father father father father father 720 daddy daddy daddy daddy daddy daddy daddy 780 daddy daddy daddy daddy daddy daddy daddy daddyc 840 daddy daddy daddy daddy daddy daddy daddy daddy 900 fatherfather fatherfather fatherfather fatherfather fatherfather 960 grandfather father father father father father cgdad father 1020 attattatta tattttgt attattattattattattattattattattattattattattattattattattattattattattattattattattatta attattatgta tattttatat acaatacata attatattt tatatacaat attatatatta 1140 tattttatat acaatattattattattattattattattattattattattattattattattattattattattattatt 1200 daddy daddy daddy daddy daddy daddy daddy daddy 1260 taatatatat tttatactat atataatata tattttatac attatataat atatatttta 1320 tactatatat aatatatatt ttatacatta tataatatat attttatata ttatataata 1380 tatattttat gttatataat atatatttta tgtattatat aacatatatt ttatgtatta 1440 tataacgtat attttatata ttatgtaacg tatattttat atattatgta acgtatattt 1500 tatatattat gtaacgtata ttttatatat tatgtaatat atattttata tattatatat 1560 tttgtatatt atatattttg tatatgatat attatatatt ttatatatta tatattatat 1620 attttgtata ttatatatta tatattttat atataatata tcatatatat tttatatata 1680 tataaagcat cagtaaacaa 1700 <210> 14 <211> 177 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 14 taacatggtg aaatatcatc tctactaaaa atacaaaaaa ttagccaggc gtggtggcgg 60 gcgcctgtgg tcccagctac ttgggaggct gagggaggag aatggcgtga acccaggagg 120 catagcttgc agtgagccga gattgcacca ctgcactcca gcctgggcaa cagagca 177 <210> 15 <211> 249 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 15 ctctagagcc aggcaccgtg actcactcct gtgatcccag cttttcgaaa ggctgaggca 60 ggaggatcgc ttgaggccag gagttcaaga ccagccttgg caacatagtg agactctccc 120 gtctctaaaa caacaacaac aaaaggttaa ttaaaaatta aaagaaaaga aaactccaca 180 gccaccttct ccaggaaaat aagtcccaaa gccacttgcc actgatgcag aggtgcgcag 240 agcccgagg 249 <210> 16 <211> 5274 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 16 gacagatggg attattcatt tttataaaat atttaaccca acaaatccaa aatatcattt 60 cagcatgtaa ataatacaga tggtccccaa tttaaccatg gttccattaa aggatttttt 120 ttgactttaa taatggattt atcagaatat aacctcatca taagttgagg aacatctgta 180 taaaaatata ttaatgagat accctacatt cattatcctt tgtataccaa gtcttaccat 240 gtggattttg cacttcccac acatctcaat atggaccagc catatcacgt gccccctgcc 300 agatgcatct agtggctttc acatcggaca gctccacact agaatgacag taaggcacag 360 aaccagacag tccccccgct tccagctcct gatcaaccac ttgtagccat gggagctttg 420 gagactgcct taatagttat gaatctaagc taggtgtcat ggctcacacc tgtaatccca 480 actctttggg acgccaagat ggatggatag cttgaggctt tttttttttt tttttttttt 540 tttagacaag agtctcgctc tgttgccagg ctggagtgca gtggcacgat cttggctcac 600 tgcaacctcc gcctcccagg ttcaagggat tctcctgcct cagcctcccc ctgagtagct 660 gagactacag gtgcgcacca ccacgcccag ctaatttttg tatttttagt agagacagag 720 tttcaccatg ttggccagaa tggtctcgat cacttgacct catgatccac ccacctcagc 780 ctcacaaagt gctgggatta caggcgtgag ccaccgcgcc cggccagctt gacgcttgag 840 tccaggaatt caagaccagc ctgggcaaca tggtcgaaac cctgtctcta ctgaaaaaaa 900 aaaaaaaaaa ttatccgggc atggtgacac acgcctacag tcccagctac cccagaggct 960 gaggtgggag gatcattgaa ccctaaag tcaggctga agtgatccaa gattgcatca 1020 ctgaactcca gtctgggtga tgggagtgag accctgctc aaaaaaaaa aaaaatagaa 1080 aagctctgaa tctatgggtt ctagccctgg gaaattctgg atacatgga gttaacagtt 1140 cacctgcctc aggtgagata atgtaaaact agaaaggtcc attackgt gatgtctaat 1200 tgaatctatt tatcaaaca gtacccaat gcagtagtt cagaaacctt atactgttag 1260 gtataataa ccataaacaa gataaaaaat atgggctggg tgtggtggct cccactgtta 1320 tcctagcatt ttgggaggcg aaggcaggag gatcacttga ggctggcatt tgagacaaca 1380 cctgggcaac atagcaagac cccatctcta taaaaaaaca tataatata atataagat 1440 atatatattt tatatatata catatattg tacctgttaa cgtggggtga ggggtgcagg 1500 gagggtggaa aggagaagt catatatta agcatcagta ggccggcgc agtggctcat 1560 gcctgtaatc ccagcactt gggaggctga ggcgggcaga tcacaggtc aggagattga 1620 gatcatcctg gctacatgg tgaatatca tcttacta aaatacaaa aattagccag 1680 gcgtggtggc gggcgcctgt ggtcccagct acttgggagg ctgagggagg agaatggcgt 1740 gaacccagga gcatagctt gcagtgagcc gagattgcac cactgcactc cagcctgggc 1800 aacagagcaa gactctgtct chattattattattattattattattattattattattatta 1860 Grandfather grandfather grandfather grandfather grandfather grandfather 1920 great-grandfather great-grandfather great-grandfather great-grandfather great-grandfather 1980 father father father father father father father father 2040 attattattattattattattattattattattattattattattattattattattattattattattattattatta 2100 fatherfather fatherfather father father father father father daddy daddy daddy daddy daddy daddy daddy 2220 daddy daddy daddy daddy daddy daddy daddy 2280 tattattac tattattattattattattattattattattattattattattattattattattattattatat 2340 father father father father father father father father 2400 daddy attattatta daddy daddy attattatta daddy 2460 fatherfather fatherfather fatherfather fatherfather fatherfather 2520 fatherfather fatherfather fatherfather fatherfather 2580 fatherfather fatherfather fatherfather fatherfather fatherfather 2640 fatherfather fatherfather fatherfather fatherfather fatherfather 2700 daddy daddy daddy daddy daddy daddy daddy daddy 2760 fatherfather fatherfather fatherfather fatherfatherfather 2820 gdaddadttattadad fatherttgta fatherfather fatherfather fatherfather 2880 attattatata tattatgtat attattatata caatacataa tatatatttt atatacaata 2940 daddy daddy daddy daddy daddy daddy daddy daddy 3000 fatherfather father father father father father father 3060 daddy daddy daddy daddy daddy daddy daddy 3120 grandfather fatherfather fatherfather fatherfather fatherfather 3180 fatherfather fatherttattg fatherfather fatherfather gdadttatta acatatatttt 3240 tatgtattat ataacgtata ttttatat tatgtaacgt atattttatta tattatgtaa 3300 cgtatatttt atatattatg taacgtatat attatatatt atgtatatata tattttatat 3360 attatatatt ttgtatatta tattattgt attadadttattattatattt tatatattat 3420 fatherfather tttgdad father father father father father catatatatt 3480 ttatatat ataaagcatc agtaaacaac tctagagcca ggcaccgtga ctcactcctg 3540 tgatcccagc tttcgaag gctgaggcag gaggatcgct tgaggccagg agttcagac 3600 cagccttggc aacatagtga gactctcccg tctctaaaac aaaaacaa aaggttaat 3660 taaaaattta aagaaaagaa aactccacag ccaccttc caggaaaata agtcccaag 3720 ccacttgcca ctgatgcaga gtgcgcaga gcccgaggaa cacggagtca tagcagctct 3780 gcaattgat tttattccag gctaaagat gctatttctc aaaaaagga gctgggagcg 3840 tctctgttca tgaattcatt ttcaggggt ggggtgatt cagagtcca ggctgttcc 3900 tgaccatgca cactgttccg gcctggaagc ctcagacccc agccaggctg accacgagcc 3960 agacccggag taagcttcgt cccatgctc ctgtcggtcc gggcagcctg agttctcctgg 4020 tgacccttcc ctgcacccag ccattcaa ggtctggcaa ggcctggtgc cagccagaa 4080 aatctgaggc agccaggttt gttattcaa atctctaac cttcagacct ctgtgcttgg 4140 cttacatatg tgaagttaa aaaagttag tgtgtcgcag tggtgatt aattcaagta 4200 agcaagatc cctgcatgac cagccttcga atgtcagccc atgctgaagt taacacattt 4260 aactcataga aaaatagt taaaatgagg agtggatgca aaatggtact gccattctag 4320 aaaacagttg gcagttctt aaagttaa acgtacgctt accacagac ccagcaatca 4380 cccctcccaga gaatgaaaa tttgttca tataaaatat tgtacacaa taattataac 4440 agctttattt gtaatagtca tgtatcaatc aggatctcc aaggaacag gaccaataga 4500 agagagatat atacatata aatcatatat aataatgata tatataga gttatata 4560 tatacacaaa cacatacata tattgtgata acctaccttg ttttaacctg agtgactctc 4620 tcctagcaga gagagccaga cagactccat tttagttct tcattcacag ccccctttat 4680 cccccttaag ggaataacta gtgcaagctg actctaagca catccagtaa tgcacctgct 4740 gataagatat tgaggcaggc tgtaccagca gctcctggga atgtgctcgg tggaaggtat 4800 ctaaaagccc ctgcatttat ccttagtga tagtttaagc ccctgcacct ggaactgttt 4860 atcttttaca actgcttcta taaccaatta atttttaac tttttgcctg ttctgcttct 4920 gtaaaactgc ttcagttaaa ctccccctcc cctatttaga ccatagtata aaagagaatc 4980 tagccccttc ttcggggctg agagaatttt gagtgctagc tgtctctcag tcgccggcta 5040 ataaaggact cataatttg tctcaaagtg tggcgtttct ctataactcg cttggttaca 5100 acactatc tcagggatct ccaaagaaat agaacctata ggatatatct atctataata 5160 aatcattgcc aggcatggtg gctcacccag tactttggga ggccaagtca ggaggatcac 5220 ttgagcccag gagtttgagg ccacctgagc aacatagtga gaccctgtct ctac 5274 <210> 17 <211> 965 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 17 ccagctactc gggaggctga ggcaggagaa cggcttgaac ccaggaggtg gaggttgcag 60 tgagctgaga tcgcgccact gcactccagc ctgggcgaca gagcgagact ctgtctcaaa 120 aaaaaaatat atatatatat atatatacac atatatatat aaaatatata tatatacaca 180 catatatata taaaatatat atatatacac acatatatat aaaatatata tatatacaca 240 catatatata aaatatatat atacacacat atatataaaa tatatatata cacacatata 300 tataaaatat atatatacac acatatatat aaaatatata tatacacaca tatatataaa 360 atatatatat acacacatat atataaaata tatatataca cacatatata taaaatatat 420 atatacacac atatatataa aatatatata tacacacata tatataaaat atatatatac 480 acacatatat ataaaatata tatatacaca catatataaa atatatatat acacacatat 540 ataaaatata tatatacaca tatatataaa atatatatat acacatatat ataaaatata 600 tatacacaca tatatataaa atatatatat acacacatat atataaaata tatatataca 660 catatatata aaatatatat atacacatat atataaaata tatatatata cacatatata 720 taaaatatat atacacacat atatataaag tatatatata cacacatata tataaaatat 780 atatatacac atatatataa aatatatata tacacatata tataaaatat atatatacac 840 atatatataa aaatatatat atatattttt taaaatattc caattgtctc actttgtgga 900 tgagaaaaag aagtagttag aggtcaagta acttggccta catcttttct caagattgta 960 aactc 965 <210> 18 <211> 256 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 18 ctgtgctccc ttaacatcct attttatctg tatatatata tattcttcca aatatccatg 60 ggaaaaaaaa tctgatcata aaaatatttt aggctgggag tggtggctca cgcctgtaat 120 cccagcactt tgggaggctg aggtgggcgg atcatgaggt caagagatcg agaccatcct 180 gaccaatatg gtgaaacccc atctctacta aagatacaaa actattagct ggacgtggtg 240 gcacgtgcct gtagtc 256 <210> 19 <211> 240 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 19 ctagtgagca ataaccacat cttcattttc tttgtataaa acaagaaagt ttagcatgaa 60 aaaggtactc aattacaaat gtgttggatt gaattgaaga cccttggaag gggattttgt 120 acctgaggat ctctttcttt tggccatatt gttcaatgga caaaatttag ccttcgaagg 180 caggccgatt tgaggttaat actaccttta ccacttgata gctatgtgac cttggccatg 240 <210> 20 <211> 3612 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 20 gactctagat tataccaacc tcataaaata agagcatata taaaagcaaa tgctcttatc 60 ttgcagatcc ctgaactgag gaggcaagat cagtttggca gttgaagcag ctggaatctg 120 caattcagag aatctaagaa aagacaaccc tgaagagaga gacccagaaa cctagcagga 180 gtttctccaa acattcaagg ctgagggata aatgttacat gcacagggtg agcctccaga 240 ggcttgtcca ttagcaactg ctacagtttc attatctcag ggatcacaga ttgtgctacc 300 tattgcctac catctgaaaa cagttgcttc ctatatttca tccagtttaa tatttattta 360 aaccaagaag gttaatctgg caccagctat tccgttgtga gtggatgtga aagtaccaat 420 tccattctgt tttactatta actatccttt gccttaatat gtatcagtag gtggcttgtt 480 gctaggaaat attaaatgaa tggcatgttt cataggttgt gtttaaagtt gttttttgag 540 ttaaatcttt ctttaataat actttctgat gtcaaaaaca cttagaagtc atggtgttga 600 acatctatat agggttggat ctaaaatagc ttcttaacct ttcctaacca ctgtttttgt 660 ttgtttgttt ttaactaagc atccagtttg ggaaattctg aattagggga atcataaaag 720 gtttcatttt agctgggcca cataaggaaa gtaagatatc aaattgtaaa aatcgttaag 780 aacttctatc ccatctgaag tgtgggttag gtgcctcttc tctgtgctcc cttaacatcc 840 tattttatct gtatatatat atattcttcc aaatatccat gggaaaaaaa atctgatcat 900 aaaaatattt taggctggga gtggtggctc acgcctgtaa tcccagcact ttgggaggct 960 gaggtgggcg gatcatgagg tcaagagatc gagaccatcc tgaccaatat ggtgaaaccc 1020 catctctact aaagatacaa aactattagc tggacgtggt ggcacgtgcc tgtagtccca 1080 gctactcgggg aggctgaggc aggagaacgg cttgaaccca ggaggtggag gttgcagtga 1140 gctgagatcg cgccactgca ctccagcctg ggcgacagag cgagactctg tctcaaaaaa 1200 aaaatatata father flat father father father father flat 1260 father father father father father father father father father father 1320 father father flaw father father father father acatatat 1380 aaaatadad blackfather fatherfather blackfather fatherfather 1440 father cacatata father father father father father father father father rainbow papataaat father father rainbow father father father 1560 father father father father catacata 1620 father father father father father father father father father 1680 flat father father flat father father father flat 1740 father father father father father father father father 1800 father cacatata father father father father acadadtat aaadadtata 1860 tatacataata tatacatac acatatatat aaatatata tatacata 1920 tataaaaa tatatatata tattttttaattattccaa ttgtctcact ttgtggatga 1980 gaaaagaag tagttagagg tcaagtaact tggcctacat cttttctca gattgtaac 2040 tcctagtgag caataaccac atcttcatttt tctttgtata aaacaagaaa gtttagcatg 2100 aaaaaggtac tcattacaa atgtgttgga tgaattga gacctgga agggatttt 2160 gtacctgagg atctctttct tttggccata ttgttcaatg gataaaattt agccttcgaa 2220 ggcaggccga tttgaggtta atactacctt taccacttga tagctgtg accttggcca 2280 tgtggtttca acagtctgaa cctcattttc tctgtgtatg tgtggtcccctc cttacaagtt 2340 tgtgaaaaat gtgaagtcct tagccatgat agcccaat aacaggctaa atgataatag 2400 gtttatgttc ttttccttta tattctcaga taagcactgt ccaagtttga ggtgttttga 2460 ggtctcgcct gatttggatt gtttgagttt atgctattct ttgaattctt tgagctgttc 2520 tgaagcagtg tatcatgaac aaaaacatcc ccagttcagt ccaaacccct ggttacatat 2580 cattcttatg ccatgttata accagtttga gagtgttccc tctgttattg catttaagtt 2640 tcagcctcac acagaaattc agcagccaat ttctaagccc taagcataaa atctggggtg 2700 gggggggggg atggcctgaa gagcagcatt atgaatagca ccattataat taatgatctc 2760 tcaggaagat ttacaatcac aggtagcaga taaaacaaat agtactgctt ctgcacttcc 2820 cctcctttta ttcgctatga aattttatgg gaaatcagtc cagtgaaaaa tgtaagctct 2880 taatctttcc cagaaatcct acctcatttg atgaatactt tgagggaatg aattagagca 2940 tttttttctt ttatagtcta cttcgcattt acgaagtgag gacggtagct taggctgcct 3000 ggccaactga tgagaaggtc agaggcattt ttagagacct ctgttgtctt tcattcatgt 3060 tcattttcca caaggcaagt aatttccaac aaatcagtgt cttcattagt aataagatta 3120 ttaacaacaa taatagtcat agtaactatt cagtgagagt ccattatata tcaggcattc 3180 tacaaggtac tttatataca tctgagtaaa cctcacacaa ttctacaggg aggtatttct 3240 atccccattt aacaaataag gaaacgaagt ccaagtaaat taacttgccc aaggtcacac 3300 agatagtacc tggcagaaca ggaatttaaa cctaaatttg tccaactcca aaagcagcct 3360 tctatttgtt ataaatgctg cctctcatta tcacatattt tattattaac aacaacaaac 3420 ataccaatta gcttaagata caatacaacc agataatcat gatgacaaca gtaattgtta 3480 tactattata ataaaataga tgttttgtat gttactataa tcttgaattt gaatagaaat 3540 ttgcatttct gaaagcatgt tcctgtcatc taatatgatt ctgtatctat taaaatagta 3600 ctacatctag ag 3612 <210> 21 <211> 599 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 21 atatgtatat atgtatatat gtatatatgt atatatgtat atatgtatat atgtatatat 60 gtatatatgt atatatgtat atatgtatat atgtatatat gtatatatgt atatatgtat 120 atatgtatat atgtatatat gtatatatgt atatatgtat atatgtatat atgtatatat 180 gtatatatgt atatatgtat atatgtatat atgtatatat gtatatatgt atatatgtat 240 atatgtatat atgtatatac gtatatatgc atatacgtat atatgtatat atgtatatat 300 gtatatatgt atatatgtat atatgtatat atgtatatat gtatatatgt atgtatgtat 360 gtatgtatgt atatatgtat atatgtatgt atgtatgtat gtatgtatgt atgtatatat 420 gtatatatat atgtatgtat gtatgtatgt atgtatgtat atgtgtatat gtgtatatgt 480 gtatatgtgt atatgtgtat atatgtatat atgtatatat gtatatatgt atatgtgtat 540 atgtgtatat gtgtatatat gtatatatgt atatatgtat atatgtatat atataacat 599 <210> 22 <211> 205 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 22 gctctttcct ttagttaagc ttatgaaata gtgtttctct catgtttcct ctatattctc 60 tcttttgcct tcctgtttct tcctgttgat tccatcccat tggagtgaaa tcttatgatc 120 ttttggcatc aacaaagtga tctgcatcca aataattcca catctcattc catgttgact 180 gtggatctat atatatatat atgta 205 <210> 23 <211> 352 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 23 agtattaaat tatatataca tatataagtg aaatgtcaca atcttctaga acttgctctg 60 tatgtccact taacatggta gagtgagcta tgtcagcatt ttctatttcc tgtgaatcat 120 tctgtgtgtt gccaagaaga aatatgatat attctgaggt tatgaaatga tattttggtc 180 atcatgtttc tcatcctatt ttcatattac ctaaatactt ttgcttttaa aattattatt 240 attaataata atataattat ttatacaata atatttaaat aatatattta tttaatataa 300 ttattatatt tcacataaaa gcaatagttc cagtgttaca aattgtaggc aa 352 <210> 24 <211> 5460 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 24 ctcgaggtct caagataaga atgactgctg taactcaaat ccaccaaagc tatttgtgtt 60 agaatgcttt cctttggtaa taacataata ccacagagtg agtgaatgta tcaagcaaag 120 tactcactca taatctctcc acccaaatga ctttgtcttc taaaattaaa cccttcccag 180 aggcctctcc ccttaatacc atattgggct cttcacactt cttccaacat cgccttccat 240 cctggccctt ccaacctccc ttctgtttgt gctaggaaca gctcaaggcc tcctatctac 300 cacagagtta catggcttgc cccttgccaa ccccccagta ccacacagtg agtgcaaaat 360 ctcaccacat tcagaaccca gtcactattc aaatcatatt ttaacctttg cagtactgac 420 tacttttgat tcatctaaac attactgaac tttattctag aaaacattta agaaatttgt 480 agttaggttc atcctttgag accttacatt taatttcttt ctatgtaaac ggaaagcatt 540 gttcagtccc acgctcatta tggcaaccca cttccaagta cttcgtttac tacgtgggct 600 ggaatcatac agttttctgt tgtgcttgtg ggagcagatc cccctaacct ctgctgattt 660 ttctcaccac ttatcataca tttattacat gcatgcactg ctgtgtgagt ttctaaatac 720 ttgggtagca attctctact attactttaa ttttcctact tgtctgcaaa tacgaaaagt 780 agcttgaaag aacttcagat ctttgttgtt atctgttgca aacactccat ttttctgttg tagcaaaaaaa aaaaaaaaag acatccatag ttgtcaatga gaatgcaaga tacatacatt ctgcacctgt gtgctaacat aagtggctgc cctgtgactc agagattgct tgtccttctc 960 ctaagcctat ccttttttgt tactttggat acttttgttc aatgaatcca gaaaaagtgt ttttcagatt caccatgtga ccctcattta aaacctgtaa tccccctatg gttaagttcc tgcttttgtt tctgttttct ttctttcagt aaaaggaatt gaacccagtc cttccactta ctatctgagc atatggctct tttagattat gtgttggtg gtgttcattg gtctcaccaa aatgctaaag aagccttcat cttctacttg tgggtagtct ttacattcat tactgcaagt ttagtttatg tggtagtacc agatcctttg cttcttttga cttcatgcct acctaacagc gctctttcct ttagttaagc ttatgaaata gtgtttctct catgtttcct ctatattctc tcttttgcct tcctgtttct tcctgttgat tccatcccat tggagtgaaa tcttatgatc 1440. ttttggcatc aacaaagtga tctgcatc aataattcca catctcattc catgttgact gtggatctat atatatatat atgtaatatg tatatatgta tatatgtata tatgtatata 1560 tgtatatattg tatatatgta tatatgtata tattgatata tgtatatat tatatatgta 1620 tatatgtata tatgtatata tgtatatatt tatatatg tatattgta tatgtatata tatgtatata 1680 tgtatatatt tatatatgta tatatgtata tattgatata tgtatatat tatatatgta 1740 tatatgtata tatgtatata tgtatatatg tatatatgta tatacgtata tatgcatata 1800 cgtatatatt tatatatgta tatatgtata tattgatata tgtatatat tatatatgta 1860 1920 1980 tgtatatgtg tatatgtgta tatgtgtata tgtgtatat ttatatat tatatatgta 2040 2100 tttatatatg tatatatata acatagtatt aaattatata tacatatata agtgaaatgt 2160 cacaatcttc tagacttgc tctgtatgtc cacttaacat ggtagagtga gctatgtcag 2220 cattttctat ttcctgtgaa tcattctgtg tgttgccaag aagaaatatg atatattctg 2280 aggttatgaa atgatatttt ggtcatcatg tttctcatcc tattttcata ttacctaaat 2340 acttttgctt tattattat tattattattattattac daddyttt 2400 aaatatata tttatttaat attattatta tattcacat aaagcaata gttccagtgt 2460 tacaaattgt aggcaatggg ctgttctgat tatctaagtt gggcccagga tatgtgctga 2520 atagttaaag cacatgccca gcatgtatga gggtaaaagg atgggtggat gtagtgaccc 2580 atttgtaatt taagccttag caggcagagg tgtgacccat attgcaagt actagtcat 2640 tataaggtca tctatatcac aatctctgga ttagatgat tgaacctgct cagtgaccaa 2700 tgtgttagca atacagga ggatgataac atcaacgtca gagacacat tgaagggctt 2760 acaaatagtg cccatttact ttatacaga aaaattcaat gtaccctcta ggcaatttca 2820 acttttagtc tcttggtagg atagtctaca tttagaatgg ctaattcata attagaaag 2880 cttcttcacc ccctactttt ctggttattt ctctatgaat gtggtaggca tgagttagta 2940 cacatgtttc catgtacatg tgtctctatg tgtctgcatg catatggtag aatgtactca 3000 tattctatgt acagttagaa caatatttat attgtcaaag aaatcaaaag gagtatta 3060 agcttcagaa ataaggataa gtttgaaata ttcattgttt tattttttac agtatttttt 3120 cctttgagaa ttctatgtaa agtactttga acatatttgc cttcaactcc tccctcactt 3180 tcaccctctc ttcattcctc cctttccttt ccactcaaag ttgagattcc tttatttatt 3240 tattatcct tcaaatatca ctggtactat ccacatgatc tcaggattga ggtctgctct 3300 gacgtgtcat cctgctttca tgcaatggcc ttataggtgg aacaacatta tgaactaacc 3360 agtaccccgg agctcttgac tctagctgca tatatatcaa aagatggcct agtcggccat 3420 cactggaaag agaggctcat tggacttgca aactttatat gccccagtac aggggaacac 3480 cagggccaaa aagggggagt gggtgggcag gggagtgggg gtgggtggat atgggggact 3540 tttggtatag cattggaaat gtaaatgagt taataccta ataaaaaatg gaaaaaaaaa 3600 gttctaatg tgtgtttcta gaaacttcct ctcttaaagc aacaacatgt ccatgagcaa 3660 tatagaattg aagatcacca tcaatcctc tttattcctc attgttcca tcatgtacta 3720 ccagacctct ttaaagtgta gtacagtgtg ttaggaatg agcagatt cctgggtatg 3780 tgctaaatta gctactgagt caaatacat tttgctga acatagtg ttggtcatt 3840 tctgggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg ro ro have 3900 ggaaggaagg aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaahaaaahes tgttctgac 3960 agcatctgtc tgagtcaggc agtgaatga agggaatc ctagagaatg cacaggaaag 4020 cagcccaagg agagtgtggg ctgaaaggca tcatgttaga aacatgcact cgatgacaga 4080 accttgagaa aaggaactc aaagcaaagc acttatta attgtaaa cgcactttat 4140 tcatagccat gggggatgtc atattccaa gcataagaat gatcagttttc caatcactgt 4200 gaacccccaa aacacaagt gaaaacccac tactttatt gatgagattt ggggttgctc 4260 tattaattta taaatcaga gtaagacacg atataatga aacgattgta gttctaaagc 4320 agcggcactt ccctgaacag tgtcatttg acagtaact gctacactt tcaggtcaca 4380 gcgactgaag aaaaagtagg gaaagaagc tggctgtgct gtttgacatt ttctttctctt 4440. atctggtgac atgaagagaa gctctgggtc cccctactct tgttcatata tctgttgctt ttatgctgca tcctgaggtt tgaagaaatg catttggcac tgagaaaga tgaggagaga atgccttgga catggtccta acatgctttg gtactgagaa aagagagcag aggagatgac 4680. tggcctattt tggccttcat ctgagtgata gattttactt aacaataga aacaaagttt tacttataa cagaaccaat gacctgtgtc atctctgata 4800. 4800. 4800. 4800. 4800. 4800. 4800. 4800. 4800. 4800. 4800 agggctattt gtatagtttc agtgatagtg tgacaaagtg taatctaaat ttctaaaaag 4920. 4920. 4920. 4920. 4920. 4920. 4920. 4920. 4920. 4920. 4920 4980. 4980. 4980. 4980. 4980. 4980. 4980. 4980. 4980. 4980. 4980 catgaatatt tttattattt agctaaaga ccaatgttca tgtactcaag aagtgtattg aggaacttag gaattagtc tgaacaggtg agagggtgcg ccagagaacc tgacagcttc tggaacaggc ggaagcacag aggcactgag gcagcaccct gtgtgggccg gggacagccg 5160 gccaccttcc ggaccggagg acaggtgccc gcccggctgg ggaggcgacc taagccacag 5220 cagcagcggt cgccatcttg gtccgggacc cgccgaactt aggaaattag tctgaacagg 5280 tgagagggtg cgccagagaa cctgacagct tctggaacag gcagaagcac agaggcgctg 5340 aggcagcacc ctgtgtgggc cggggacagc cggccacctt ccggaccgga ggacaggtgc 5400 ccacccggct ggggaggcgg cctaagccac agcagcagcg gtcgccatct tggtcccggg 5460 <210> 25 <211> 220 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 25 taggcaacag agtgagatca tgtgtcatat atatatatat atatatatat atatatatat 60 atatatacac acacacacac atatatatat acacatatat atacgtatat atatatatgt 120 atatatatac atatatatac atatatatat atacgtatat atatacgtat atatatatca 180 atgtaaatta tttgggaaat ttggtatgaa tagtcttccc 220 <210> 2; <211> 134 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 26 aaatacaaaa attagccagg tgtggtggca tgtgcctgta gtcctaccta ctcgggaggc 60 tgaggcacaa gaatcgcttg aatgtgggag gtggaggttg cagtgacctg agatcgtgcc 120 actgcactcc agcc 134 <210> 27 <211> 246 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 27 tgtgaacaca gatcataaaa tcatatatca agcagacaaa taagtagtag tcacttatat 60 gctttatactt gtaacttaaa gtaaaagaat tacaaaagca tatgacaaag actaatttta 120 agatatccta atttaaattg ttttctaaaa gtgtgtatac cattttacct atcatatgaa 180 taatttagaa acatgtttat aaaattaatg tccaaatcca ttcaaaagtt ttgtaatgca 240 gatcac 246 <210> 28 <211> 3361 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 28 agcgccgaat tcgatccctt tataaaacca caataatg gagtgctata atttcaaca 60 gtgttttggtc tgctggcaga gtggtcattc taacagcagt cacagtagag tagaatag 120 actgcagtat atctaggca aaagctgag gtttcaggag ctgaaggta aagaggaga 180 aagaaatggg atgggaatt ggaagacaa atatcgttaa gagaaatttg cttttaggag 240 agggaaaga atctatgtgt acttaagact atggaatca tcccatttaa gctgggaac 300 tagtttcata tataactaat aaattttatt tacagaatat ctattacct gatctaggct 360 tcaagccaaa gggactgtgt gaaaaaccat cagttctgtc atattcctaa aaaaaaatta 420 aaagttaa aaaaaaaaaaact tctttttcttt caaataatc aaggtgctta 480 ttcacatcca ttccaatttg gggaaatact tattttccta tgattagcga agagaaaagt 540 aacttgcatt tcaattcaag ttgatacatg tcacttta gaggtcact atatttgct 600 agttgagcta accatatagg ctttaaatac ttcatagta gaaagaaaat gaaaatcatt 660 agtgaactgt aataaataga tcatactttt tgaaagaatc agactgaagt ttccgaaaaa 720 aagagtaag cttcaatgaa aaggtaagtg aatttagcat ttactcagca tctactatgg 780 acttaacacc taacagtaga taatctgaag gcaacatat tgtatagg actgcagaat 840 gatagatgat aaatatcatc tcttctattt gatgaat tttttcaaat ctttcacaca 900 cagtggtttg ctatggaaag atttgtagta cattaaaca atctgaagat ggagttagaa 960 agcttaggct atgttttgag cacacatat aatttctctg tgattgttc ttcatctttc 1020 aaatgaggtt actgtgaga ttaatgaga taactaatg atgataaat aatgtaatct 1080 tagcagcacc tattaatc tgtgcaacaa ctctgtgag tgagtaggc tcagcttcag 1140 tcacttctct gccatttatt aactaagata gtttggaag tcaccatct cttcagctgt 1200 aaaatgatga ggatcatacc tatttgg ggctgctttt aggtacaaat atacaggcaa 1260 gcactttgtt atactaaag cattacacca attagtttta ctctttcca ttcacatg 1320 aaattaatgt atcagaatt ctgtagatta cctaaatctt ctgttaacac gtgatatgca 1380 gttcaggtta aatgtcagtt gagttaccaa agcacataca tactcaccac cctatccaaa 1440 tctacaagcc tcccagttg tctcagttg tctacagcc aaaatttcac tgatccaat gaaaaaa atattaca aaactcacac ctgtaatctc 1560 aacattttgg gaggccagg caggtagatc acttgaggcc gaggttca gaccaggcctg 1620 atcacatgg tgaaccctg tcttactaa atacaaaaa ttagccaggt gtggtggcat 1680 gtgcctgtag tcctacctac tcgggaggct gaggcacaag aatcgcttga atgtgggagg 1740 tggaggttgc agtgacctga gatcgtgcca ctgcactcca gcctaggcaa cagagtgaga 1800 tcatgtgtca grandfather grandfather grandfather grandfather 1860 cacatatata tatacacata tatatacgta tatatatata tgtatatata tacatatata 1920 tacatatata tatatacgta tatatatatacg tatatatata tcaatgtaaa ttatttggga 1980 aatttggtat gatagtctt ccctgtgaac acgatcata aaatcatata tcaagcagac 2040 aaataagtag tagtcactta tatgcttata cttgtaactt aaagtaaag attacaaa 2100 gcatatgaca aagactatt ttaagatatc ctaatttaaa ttgttttcta aaagtgtgta 2160 taccatttta cctatcatat gaataattta gaaacatgtt tataaatta atgtccaaat 2220 ccattcaaaa gttttgtaat gcagatcacc cacaacaaca aagaatccta gcctattaaa 2280 aaagcaacac cacctacata taatgaaata ttagcagcat ctatgtaacc aaagttacac 2340 agtgaatttg ggccatccaa cactttgagc aaagtgttga attcatcaaa tgaatgtgta 2400 atcatttact tactaatgcc aatacacttt aaggtaatct taagtagaag agatagagtt 2460 tagaattttt taaattattc tcttgttgta aagcaataga cttgaataaa taaattagaa 2520 gaatcagtca ttcaagccac cagagtattt gatcgagatt tcacaaactc taactttctg 2580 atacccattc tcccaaaac gtgtaacctc ctgtcgatag gaacaaccca ctgcagggat 2640 gtttctcgtg gaaaaaggaa atttcttttg cattggtttc agacctaact ggttacaaga 2700 aaaaccaaag gccattgcac aatgctgaag tacttttttc aaatttaaaa tttgaaagtt 2760 gttcttaaaa tctatcattt attttaaaat acggatgaat gagaaagcat agatttgata 2820 aagtgaattc ttttctgcaa tctacagaca cttccaaaaa tcactacaga cactacagac 2880 actacagaaa atcataaata aacaagtgct agtatcaata tttttaccaa aaaatggcat 2940 tcttagaatt ttttataggc tagaaggttt gtacaaacta atctgccacg gattttaaaa 3000 tatgagtgaa taaattatat tgcaaaaaaa atcaggttac agagaactgg caaggaagac 3060 tcttatgtaa aacacagaaa acatacaaaa cgtattttta agacaaataa aaacagaact 3120 tgtacctcag atgatactgg agattgtgtt gacatattag cattatcact gtcttgctaa 3180 aacataaaaa taaaaagatg gaagatgaaa ttacaataca aatgatgatt taaacatata 3240 aaaggaaaat aaaaattgtt ctgaccaact actaaaggaa gacctactaa agatatgcca 3300 tccagcacat tgccactcta catgtggtct gtaaaccagc agcataggcg gccgcattag 3360 c 3361 <210> 29 <211> 143 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 29 attcaacagc tgaatcctaa attgcaaact cagtggctaa taacaacttt gaacaatgag 60 caccttatac acgctactgt attttctttt ctttcttttt tttttttttt ttttttttta 120 aaccgggtag cagtgagaga ggt 143 <210> 30 <211> 64 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 30 tgtagtcatt ttgcaatcct taaagctgaa ttgtgcaatg agctcgatga aggaagatac 60 tatc 64 <210> 31 <211> 73 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 31 ttctttaagt gccttggggc gaggagtccg gaataagaag acttctttgg gttttaaagt 60 gtaggataag caa 73 <210> 32 <211> 1697 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 32 agccctgccc ccatccgacc tccgccctcg ttggcttcgc aacgctgtgg tctctgtggc 60 cagtagaggg cacacttact ttactttcac aaatccgaga gccacaaccc gggtggtggg 120 gggtgagggg gcggggaaag agtctctgca gcaaaacgca gactagggat tggtggctct 180 tggtgtttga ggcaaaatcc tagaggctgt agtcattttg caatccttaa agctgaattg 240 tgcaatgagc tcgatgaagg aagatactat cattcaacag ctgaatccta aattgcaaac 300 tcagtggcta ataacaactt tgaacaatga gcaccttata cacgctactg tattttcttt 360 tctttctttt ttttttttttttttttttt aaaccgggta gcagtgagag aggtttcttt 420 aagtgccttg gggcgaggag tccggaataa gaagacttct ttgggtttta aaggtgtagga 480 taagcaaatc ccgagggaat atgcattata taataaatct agaaccaatg cacagagcaa 540 aagactcatg tttctggttg gttaataagc tagattatcg tgtatatata aagtgtgtat 600 gtatacgttt ggggattgta cagaatgcac agcgtagtat tcaggaaaaa ggaaactggg 660 aaattaatgt ataaattaaa atcagctttt aattagctta acacacacat acgaaggcaa 720 aaatgtaacg ttactttgat ctgatcaggg ccgacttttt tttttaagtg cataattacg 780 attccagtaa taaaagggga aagcttgggt ttgtcctggg aggaaggggt taacggtttt 840 ctttattcta gggtctctgc aggctcccca gatctgggtt ggcaattcac tcctccccct 900 ttctgggaag tccgggtttt ccccaacccc ccaattcatg gcatattctc gcgtctagcg 960 ccttgatttt ccccacccca gctcctaaac cagagtctgc tgcaaactgg ctccacaggg 1020 gcaaagagga tttgcctctt gtgaaaaccg actgtggccc tggaactgtg tggaggtgta 1080 tggggtgtag accggcagag actcctcccg gaggagccgg gtagagcgca cccgccgcca 1140 ctttactgga ctgcgcaggg agacctacag gggaaagagc cgcctccaca ccacccgccg 1200 ggtggaagtc cgaaccggag gtgctggagt gtgtgtgtgg gggggggggg gaatctgcct 1260 tttggcagca aattgggggg gggggtcgtt ctggaaagaa tgtgcccagt caacataact 1320 gtacgaccaa aggcaaaata cacaatgcct tccccgcgag atggagtggc tgtttatccc 1380 taagtggctc tccaagtata cgtggcagtg agttgctgag caattttaat aaaattccag 1440 acatcgtttt tcctgcatag acctcatctg cggttgatca ccctctatca ctccacacac 1500 tgagcggggg ctcctagata actcattcgt tcgtccttcc ccctttctaa attctgtttt 1560 ccccagcctt agagagacgc ctggccgccc gggacgtgcg tgacgcggtc cagggtacat 1620 ggcgtattgt gtggagcgag gcagctgttc cacctgcggt gactgatata cgcagggcaa 1680 gaacacagtt cagccga 1697 <210> 33 <211> 146 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 33 tcacagacac tacagaatgt tttggagatt atgtaatatt taaaatattc aaattatact 60 aaaaatgtat gtaaaatgta ttgaacatag gcaagtttca atacatagat tttgagtgaa 120 tgcttgcaac tttggttcca ttctct 146 <210> 34 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 34 tctgactctg cactctgcaa cctactctaa tattacatat gataacatga gtctatgcag 60 ctgttctcta tagatat 77 <210> 35 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 35 ctacttcctt aaggtggtgt ccaagagtac atttttataa ataaaaagtt atagtacact 60 cctaagggca gcaagtagaa aacgtgctag ggagactcga tctcactttg 110 <210> 36 <211> 2195 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 36 aagcttcctt ggaattaatg gtcagataag gagctctagc aatatcactt taaatgctta 60 atatacaata tttagaaaac cttatgattg taaagagctt aaaaaagatg tgaaagaaca 120 atcactaaag cattgacaac atatgtgtgt tagtgaacaa tatgtaccaa aatggacaga 180 tgagaggtgt acattggggt gtgagttgat aatccagcag actgtgggac tagagggtct 240 acgaaaaaca aaggaagaac atacaaacaa attttaaaac actgtctttc aacactaaaa 300 ctgttggaat agagagcaag aatacatatt ggattcacat tcagtttatt tctcagattc 360 agactagtgc tctgactctg cactctgcaa cctactctaa tattacatat gataacatga 420 gtctatgcag ctgttctcta tagatattca cagacactac agaatgtttt ggagattatg 480 taatatttaa aatattcaaa ttatactaaa aatgtatgta aaatgtattg aacataggca 540 agtttcaata catagattt gagtgaatgc ttgcaactt ggttccattc tcttacttc 600 cttaaggtgg tgtccaagag tacatttta taaataaaa gttatagtac actcctaagg 660 gcagcaagta gaaaacgtgc taggact cgatctcact ttggaatcta tcctgggaga 720 caatgccctc tacaatgga ttagaga cagttttaa gaggagata atcaggtaaa 780 atctggggtt tattagagaga aagaaagagg tagagaaaaatttcaagc tcgaacatcg 840 gatcaggtgg cacaatgcgg tcaatgcctg caactcagg gtaagtatta ttctccctgt 900 ttacagttcc gtggaggaga agtgacttgc ctgtggtcat acacagagc aaagaaaagg 960 cttgagctag aactcaggcc ttgttaggtcccccttcc tcctagcaca ttggcaatt 1020 gcatgaggaa agtagaggta cagttgagtt catgtacaac ataaggcat tcaggtaag 1080 tgaatgagggg cagaagtttt atgatttagg gaagtgta gaggaaaa tattctttgtt 1140 cccaattaag aaagagatcc cttgaccatc agttagagat tcccaagt ccctctttgc 1200 cataagtcac tgaactgag atccaaggca tggctctgt gagtcaggag agcttaaccc 1260 agaggagaga ttcagaaca ggatatttcc tattttgagt atcctgctca tgccagtcat 1320 ggataaattt gcatctggct taagaatta ctggatcagc attgttttgg gtagttcac 1380 ttcctgctgg gtggggtagc aggctctata aagagatcct ctgctgcacg acctttaac 1440 ccctgtacc tgagcactga tctgccttgg agaacctggt gagtcggctt ccttgagttc 1500 ctctgttctt tgtgccctga aatgttgagt ttaatctgaa tatggcagt tggtggatc 1560 caatcctatg aaaattgact tgatgctact tagtgatga aaatttaaga ttagagcaca 1620 attatatgct attttagctt tctttgtta tacaggtagg tattccatatg gagagagaag 1680 ttaggggta acctttgata tgagagaa aaagaacaa agtattttc tttattctct 1740 tgctttctag tgtcctttac aaaggtttgt gtcttagcag gtgtgaaaga ctacaattct 1800 ccctgagcag cccttgctc tatgcccaag tcagccact tggacttat aacagataat 1860 gatgatagga atagcatatt agattgcca gggtgtctga acttgtgact gccttttctg 1920 aattggttat ttcaggga aagatgct tgatcttta taacagagat aatttattg 1980 gaaaaattgt atgagaaaac acaggatttc ctagggacaa tgaagcaatt tgttaaagtg 2040 gaagggagaa accagaaagt cttgaaaagg taattaagaa tttaaataat ttcttggaga 2100 ttggagaaat aatatgccat ggtattacac aagctttggc ttctctctct ggaggattcc 2160 cttcccacga acactgttgt atcatttctt tcaga 2195 Attached Figure Description

[0011] Figure 1 The intensity of two MAR sequences was compared: MAR X29 (a known MAR) as a control, and MAR zebrafish identified by the technique of this invention.

[0012] Figure 2 The strength of the MAR zebrafish core is shown using a construct that does not contain any MAR sequence as a control.

[0013] Figure 3 This is a carrier diagram of the construct containing the MAR core described in Example 2.

[0014] Figure 4 This is a carrier diagram of a control construct that does not contain the MAR core element, as described in Example 2.

[0015] Detailed description

[0016] I. Definition

[0017] All numerical designations (including ranges), such as pH, temperature, time, concentration, and molecular weight, are approximate values, varying by increments of 0.1 (+) or (-). It should be understood that, although not always explicitly stated, the term "approximately" precedes all numerical designations. It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary, and their equivalents are known in the art.

[0018] As used in the specification and claims, unless expressly stated in the context, the singular forms “an,” “a,” and “the” include the plural forms. For example, the term “an / a polynucleotide” includes a variety of polynucleotides, including mixtures thereof.

[0019] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure may be imparted before or after the assembly of the polynucleotide, if present. The sequence of the nucleotide may be broken down by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by coupling with a labeled component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the polynucleotides of the present invention includes double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0020] II. New matrix attachment region (MAR) sequence

[0021] This invention describes a method via computer ( in silico A method for quantifying the intensity of MAR elements, and a method for quantifying the intensity of new MAR sequences identified from a sequence database.

[0022] Through sequence comparison and structure-activity analysis, the inventors discovered that the MAR element can be composed of a central AT-rich core region and 5' and 3' flanking regions containing transcription factor binding motifs.

[0023] The core region can be enriched with (ATAT)n microsatellites. Binding motifs can target SATB1, NMP4, CEBP, Fast, and Hox transcription factors. Other nuclear matrix proteins that MAR can bind include ARBP protein (attachment site binding protein), which recognizes the consensual sequence ATTTCA in MAR. C / G TTGTAAAA; NMP-2 protein, located only in the nuclear matrix; Sp1, ATF, CCAAT, C / EBP, and AP-1 transcription factors; yeast ACBP protein (ARS co-binding protein), which interacts with ARS elements; tissue-specific human SATB1 protein, mainly expressed in the thymus, which binds to the minor groove of a special class of AT-rich MARs with A, T, or C on one chain but no G; Matrin 3 protein (an acidic protein in the internal nuclear matrix network of human and rat cells), matrin F / G; transcription factor RFP.

[0024] The analysis results helped the inventors find new MAR sequences, leading to the discovery of four new MAR sequences that were more efficient in promoting gene expression than the positive control MAR X29 (Arope et al. (2013) PLoS ONE 8(11):e79262).

[0025] This invention further demonstrates that the core of these newly identified MAR sequences can form a functional MAR with the 5' and 3' flanking regions from other MARs, providing additional useful chimeric MAR sequences.

[0026] Therefore, according to one embodiment of the invention, a recombinant polynucleotide is provided comprising: a coding sequence; a promoter configured to initiate transcription of the coding sequence; and a matrix attachment region (MAR) core and variants thereof as described herein. Examples of such novel MAR cores are listed in Table 1. See, for example, SEQ ID NO: 1, 5, 9 and 13.

[0027] Matrix attachment regions (MARs) are sequences in the DNA of eukaryotic chromosomes that attach to the nuclear matrix. As structural DNA components that organize the eukaryotic genome into functional units within the nucleus, MARs mediate the structural organization of chromatin within the nucleus. These elements form the DNA anchors of the chromatin scaffold and are used to organize chromatin into structural domains. The dynamic and complex organization of chromatin mediated by MAR elements plays a crucial role in the regulation of gene expression.

[0028] Variants of the MAR core are nucleic acid sequences that have a certain sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%) with reference MAR cores (e.g., SEQ ID NO: 1, 5, 9, and 13) and have the intended MAR function (e.g., the ability to attach to the mammalian nuclear matrix).

[0029] In some implementations, variants of the MAR core are enriched with AT, for example, having at least 75%, 80%, 85%, 90%, or 95% A or T in the sequence.

[0030] The MAR core can coexist with the 5' flanking region of the MAR (e.g., any one of SEQ ID NO: 2, 6, 10, 14, and 18 or a variant thereof (a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 2, 6, 10, 14, and 18)). In some embodiments, the 5' flanking region is located on the 5' side of the MAR core and within 100 nucleotides of the MAR core.

[0031] In some embodiments, the MAR core can coexist with the 3' flanking region of the MAR (e.g., any one of SEQ ID NO: 3, 7, 11, 15, and 19 or a variant thereof (a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 3, 7, 11, 15, and 19)). In some embodiments, the 3' flanking region is located on the 3' side of the MAR core and within 100 nucleotides of the MAR core.

[0032] Non-restrictive MAR sequences including a MAR core, a 5' flanking region, and a 3' flanking region include SEQ ID NO: 4, 8, 12, and 16 and nucleic acid sequences having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 4, 8, 12, and 16, wherein the MAR core is capable of attaching to the nuclear matrix of mammalian cells.

[0033] III. Chimeric MAR sequences

[0034] As provided, the core of these newly identified MAR sequences is capable of forming functional MARs with the 5' and 3' flanking regions from other MARs, providing additional useful chimeric MAR sequences.

[0035] In one embodiment, the chimeric MAR of the present invention includes a MAR core as disclosed herein, having a 5' wing region on the 5' side. In one embodiment, the chimeric MAR of the present invention includes a MAR core as disclosed herein, having a 3' wing region on the 3' side. In one embodiment, the chimeric MAR of the present invention includes a MAR core as disclosed herein, having a 5' wing region on the 5' side and a 3' wing region on the 3' side.

[0036] The 5' flanking region can be selected from any 5' flanking region or 3' flanking region known in the art or disclosed herein. In some embodiments, the 5' flanking region can be selected from any 5' flanking region known in the art or disclosed herein.

[0037] The 3' flanking region can be selected from any 5' flanking region or 3' flanking region known in the art or disclosed herein. In some embodiments, the 5' flanking region can be selected from any 3' flanking region known in the art or disclosed herein.

[0038] Each of the MAR core, MAR 5' flanking region, and MAR 3' flanking region can also be replaced by nucleic acid variants (e.g., those with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the reference sequence).

[0039] The MAR cores disclosed herein include SEQ ID NOs: 1, 5, 9, and 13. The 5' flanking regions disclosed herein include SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, and 34. The 3' flanking regions disclosed herein include SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, and 35.

[0040] In some implementations, the newly identified MAR flanking sequences can also be fused with other newly discovered or known core sequences to generate new chimeric MAR sequences. In some implementations, the chimeric MAR sequences include any of the MAR core regions in Table 1, any of the MAR 5' flanking sequences in Table 1, and any of the MAR 3' flanking sequences in Table 1.

[0041] IV. Gene expression

[0042] The MAR sequences and constructs disclosed herein can be used to promote gene expression in cells. When the gene of interest is contained in a construct containing a MAR (either alone or with a 5' and / or 3' flanking region), the construct can be introduced into a host cell to express the gene of interest.

[0043] The MAR sequences disclosed herein can also be used as trans-acting elements to promote gene expression trans-. For example, constructs containing any of the MAR sequences disclosed herein (wild-type or chimeric MARs alone or with 5' and / or 3' flanking regions) can be introduced into cells that have been further transfected (or already transfected) with a construct containing a gene of interest (GOI, or transgene), thereby enabling the MAR sequence to aid in GOI expression.

[0044] Therefore, according to one embodiment disclosed herein, a method for transfecting a coding sequence into cells is provided, comprising: contacting a cell with a first polynucleotide comprising the coding sequence and a promoter for initiating transcription of the coding sequence and a non-connected second polynucleotide comprising a matrix attachment region (MAR) core as disclosed herein, under conditions for transfecting first and second polynucleotides into cells. In some embodiments, the MAR core is selected from SEQ ID NO: 1, 5, 9 and 13 and nucleic acid sequences having at least 75% sequence identity with any one of SEQ ID NO: 1, 5, 9 and 13, wherein the MAR core is capable of attaching to the mammalian cell nuclear matrix.

[0045] In some embodiments, the second polynucleotide further comprises a 5' flanking region selected from SEQ ID NO: 2, 6, 10, 14, and 18 and nucleic acid sequences having at least 75% sequence identity with any one of SEQ ID NO: 2, 6, 10, 14, and 18. In some embodiments, the 5' flanking region is located on the 5' side of the MAR core and within 100 nucleotides of the MAR core.

[0046] In some embodiments, the second polynucleotide further comprises a 3' flanking region selected from SEQ ID NOs: 3, 7, 11, 15, and 19 and nucleic acid sequences having at least 75% sequence identity with any one of SEQ ID NOs: 3, 7, 11, 15, and 19. In some embodiments, the 3' flanking region is located on the 3' side of the MAR core and within 100 nucleotides of the MAR core.

[0047] In some embodiments, the complete MAR sequence is selected from SEQ ID NO: 4, 8, 12 and 16 and nucleic acid sequences having at least 75% sequence identity with any one of SEQ ID NO: 4, 8, 12 and 16, wherein the MAR is capable of attaching to the nuclear matrix of mammalian cells.

[0048] The present invention also provides transfected cells prepared by the method disclosed herein.

[0049] In any construct or vector disclosed in this invention, additional transcriptional regulatory sequences and / or post-transcriptional regulatory sequences may be included. Transcriptional regulatory sequences may include, for example, promoters, enhancers, and polyadenylation signals. Post-transcriptional regulatory sequences include, for example, introns and PREs.

[0050] In some implementations, a multiple cloning site (MCS) (also known as a "polylinker") is present in the vector to facilitate the insertion of a heterologous sequence. For example, the MCS can be positioned between the promoter and the polyadenylation signal to promote the insertion of the transgenic sequence. In a vector containing the transgenic sequence, the portion of the vector containing the promoter, the transgenic sequence, and the polyadenylation signal is called an "expression cassette."

[0051] Active promoters in eukaryotic cells are known in the art. Exemplary eukaryotic promoters include, for example, the SV40 early promoter, the SV40 late promoter, the cytomegalovirus (CMV) promoter, the CMV major immediate early (CMV-MIE) promoter, the EF1-α (translation elongation factor-1α subunit) promoter, the Ubc (ubiquitin C) promoter, the PGK (phosphoglycerate kinase) promoter, the actin promoter, etc. See also Boshart et al. , GenBankAccession No.K03104; Uetsuki et al. (1989) J. Biol. Chem. 264:5791-5798;Schorpp et al. (1996) Nucleic Acids Res. 24:1787-1788; Hamaguchi et al. (2000) J. Virology 74:10778-10784; Dreos et al. (2013) Nucleic Acids Res. 41 ( D1):D157-D164 and the eukaryotic promoter database at http: / / epd.vital-it.ch (accessed on July 16, 2014).

[0052] The vector may also contain enhancers. Non-limiting embodiments include enhancers in the CMV promoter and intron A sequences. It has been previously shown that five embryonic stem cell (ESC) transcription factors occupy superenhancers (Oct4, Sox2, Nanog, Klf4, and Esrrb), and many other transcription factors contribute to ESC control. Six additional transcription factors (Nr5a2, Prdm14, Tcfcp2l1, Smad3, Stat3, and Tcf3) occupy typical enhancers and superenhancers, and all of these are rich in superenhancers. Any of these, or further known in the art, may be used in this invention.

[0053] Active polyadenylation signals in eukaryotic cells are known in the art, including but not limited to SV40 polyadenylation, bovine growth hormone (BGH) polyadenylation, and herpes simplex virus thymidine kinase gene polyadenylation. Polyadenylation signals direct the 3' end cleavage of precursor mRNA, polyadenylation of precursor mRNA at the cleavage site, and transcription termination downstream of the polyadenylation signal. The core sequence AAUAAA is typically present in polyadenylation signals. See also Cole. et al. (1985) Mol. Cell. Biol. 5:2104-2113.

[0054] Exemplary introns that can be used in the vectors of the present invention include the β-globin intron and the first intron (also referred to as "intron A") of the major immediate early (MIE) gene of human / mouse / rat / other species cytomegalovirus.

[0055] Other post-transcriptional regulatory elements that may be included in the vector of the present invention include, but are not limited to, the 5'-untranslated region of the CMV MIE, the human Hsp70 gene, the SP163 sequence from the vascular endothelial growth factor (VEGF) gene, and a triplet leader sequence associated with late adenovirus mRNA. See, for example, Mariati et al. (2010) Protein Expression and Purification 69:9-15.

[0056] In some embodiments, the vectors disclosed herein contain a nucleotide sequence encoding a selection marker (i.e., a eukaryotic selection marker) that functions in eukaryotic cells, such that, when appropriate selection is applied, cells lacking the selection marker die or grow significantly slower than cells containing the selection marker. An exemplary selection marker functioning in eukaryotic cells is the glutamine synthase (GS) gene; selection is applied by culturing cells in a glutamine-deficient medium, or by selection with L-methionine sulfoxide, or both. Another exemplary selection marker functioning in eukaryotic cells is encoding a nucleotide sequence encoding a glutamine-containing phosphodiesterase (GS) gene. neo The selection process involves the administration of genes that confer resistance to neomycin, genimycin, or G418; selection is applied by culturing cells in a medium containing neomycin, genimycin, or G418. Other selection markers include dihydrofolate reductase (DHFR, which confers resistance to methotrexate), puromycin-N-acetyltransferase (which provides resistance to puromycin), and hygromycin kinase (which provides resistance to hygromycin B). Other selection markers that function in eukaryotic cells are known in the art.

[0057] The sequence encoding the aforementioned selection marker is operatively linked to the promoter and polyadenylation signal. As described above, promoters and polyadenylation signals that function in eukaryotic cells are known in the art.

[0058] In some embodiments, the vectors disclosed herein may contain two or more expression cassettes. For example, a vector containing two expression cassettes (one encoding the antibody heavy chain and the other encoding the antibody light chain) can be used to generate functional antibody molecules.

[0059] The vectors disclosed herein also contain an origin of replication that functions in prokaryotic cells (i.e., a prokaryotic origin of replication). Origins of replication that function in prokaryotic cells are known in the art and include, but are not limited to, those in *Escherichia coli*. oriC Origin point; plasmid origin, such as pSC101 origin, pBR322 origin ( rep The origin of prokaryotic replication (ROR) and pUC origin; and the origin of viral (i.e., bacteriophage) replication. Methods for identifying prokaryotic OCR origins have been provided, for example, in Sernova & Gelfand (2008). Brief. Bioinformatics 9(5):376-391.

[0060] The vector disclosed herein also contains a selection marker that functions in prokaryotic cells (i.e., a prokaryotic selection marker). Selection markers that function in prokaryotic cells are known in the art and include, for example, sequences encoding polypeptides that confer resistance to any one of ampicillin, kanamycin, chloramphenicol, or tetracycline. Examples of polypeptides conferring resistance to ampicillin (and other β-lactam antibiotics) include β-lactamases (…). bla Kanamycin resistance can be generated by the activity of the neomycin phosphotransferase gene; while chloramphenicol resistance is mediated by chloramphenicol acetyltransferase.

[0061] Exemplary transgenes include any recombinant protein or, for example, hormones (e.g., growth hormone), erythropoietin, antibodies, polyclonal or monoclonal antibodies (e.g., rituximab), antibody conjugates, fusion proteins (e.g., IgG-fusion proteins), interleukins, CD proteins, MHC proteins, enzymes, and coagulation factors. The antibody heavy chain and antibody light chain can be expressed by separate vectors or by the same vector containing both expression cassettes.

[0062] This invention provides a method for expressing recombinant peptides in cells. The method includes introducing a vector as described herein into cells and culturing the cells under conditions in which the vector is transiently or stably maintained in the cells. The cells can be prokaryotic or eukaryotic, such as stable cell lines generated through targeted integration with CRISP / Cas9. Cultured eukaryotic cells that can be used to express recombinant peptides are known in the art. Such cells include fungal cells (e.g., yeast), insect cells, plant cells, and mammalian cells. Accordingly, this invention provides cells comprising a vector as described herein.

[0063] Exemplary yeast cells include, but are not limited to, species of the genus Trichoderma ( Trichoderma sp.), Pichia pastoris (sp.), Pichia pastoris ( Pichiapastoris ), millet wine fission yeast ( Schizosaccharomyces pombae ) and brewer's yeast ( SaccharomycescerevisiaeExemplary insect cell lines include, but are not limited to, Sf9, Sf21, and fruit fly (Drosophila). Drosophila S2 cells. Exemplary plant cells include, but are not limited to, Arabidopsis thaliana (S2 cells). Arabidopsis ) cells and tobacco BY2 cells.

[0064] Cultured mammalian cell lines that can be used to express recombinant peptides include Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, virus-transformed HEK cells (e.g., HEK293 cells), NSO cells, SP20 cells, CV-1 cells, baby hamster kidney (BHK) cells, 3T3 cells, Jurkat cells, HeLa cells, COS cells, PERC.6 cells, and CAP cells. ® Cells and CAP-T ® Cells (the latter two cell lines are commercially available from Cevec Pharmaceuticals in Cologne, Germany). Many derivatives of CHO cells are also available, such as CHO-DXB11, CHO-DG-44, CHO-K1, CHO-S, or engineered CHO cells, such as CHO-M, CK1 SV CHO, and CHOZN. Primary mammalian cells may also be used.

[0065] In some embodiments, cells are cultured in a serum-free medium. For example, to produce therapeutic proteins for administration to patients, expression cells must be grown in a serum-free medium. In other embodiments, cells have been pre-acclimated to growth in a serum-free medium before use for peptide expression.

[0066] The vectors described herein can be introduced into any of the cells described herein using methods known in the art. Such methods include, but are not limited to, polyethylene glycol (PEG)-mediated methods, electroporation, bioprojectile delivery (i.e., particle bombardment), protoplast fusion, DEAE-dextran-mediated methods, and calcium phosphate coprecipitation. See also Sambrook. et al. “Molecular Cloning: A Laboratory Manual,” 3rd edition, Cold Spring Harbor Laboratory Press, 2001; and Ausubel et al. , “Current Protocols in Molecular Biology,” John Wiley & Sons, New York, 1987, and regularly updated.

[0067] Standard methods for cell culture are known in the art. See, for example, RI Freshney, “Culture of Animal Cells: A Manual of Basic Technique,” ​​5th ed., Wiley, New York, 2005. Example

[0068] The invention can be further understood by referring to the following embodiments, which are intended to be purely illustrative of the invention. The scope of the invention is not limited to the exemplary embodiments, which are merely intended to illustrate a single aspect of the invention. Any functionally equivalent methods are within the scope of the invention. Various modifications to the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and drawings. These modifications fall within the scope of the appended claims.

[0069] Example 1. Searching for new MARs

[0070] This embodiment develops a method for quantifying the intensity of matrix attachment region (MAR) elements via computer, and uses this method to quantify the intensity of some exemplary MAR sequences.

[0071] Motif-based MAR identification

[0072] A literature search was conducted to identify all motifs relevant to MAR identification, particularly looking for MAR-specific transcription factors, chromatin-binding domains, DNA bending, and DNA unwinding sequences. Potentially useful features were summarized and used for further MAR evaluation.

[0073] MAR elements can consist of a central AT-rich core region and 5' and 3' flanking regions containing transcription factor binding motifs. The core region may be rich in (ATAT)n microsatellites.

[0074] MARs can include "MAR identifying features" and are typically classified by the inclusion of these features. A bipartite element consists of two distinct sequences: AATAYAA and AWRTAANNWWGNNNC. Other MAR indicator sequences are composed of various transcription factors and DNA structural motifs. Exemplary sequences include DNA unwinding motifs, AP-1, A-box and T-box, NMP-2, SATB1, Hox4D, TEF, Pit1, and Fast.

[0075] Four new MAR elements were identified and named MAR zebrafish, MAR carp (Cyprinus Carpio), MAR 12-RP13, and MAR 17XX_fos. The complete sequences, cores, and 5' and 3' flanking regions of these elements, along with some known elements (MAR 1-68, MAR S4, MARX29, mouse c-myc 5MAR, and SPR2A-MAR), are listed in Table 1.

[0076] Table 1. MAR sequences

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] Note: In the full-length sequence, the core region is indicated by bold and underline, while the flanking regions are indicated by underline.

[0093] Example 2. Testing of MAR zebrafish

[0094] Experimental testing was conducted on one of the identified MAR elements (MAR zebrafish) and compared with the strongest known MAR from the literature, which showed twice the power.

[0095] In vitro comparison of MAR zebrafish (newly identified MAR element) with MAR X29 (control, see Arope et al. (2013) PLoS ONE 8(11): e79262).

[0096] Then, experimental tests were conducted on the MAR zebrafish core that did not include the flank regions of the full-length MAR zebrafish, and compared with the construct that did not contain any MAR sequence.

[0097] Materials and Methods

[0098] Neon transfection was performed using CT MCB cells. BalanCD + 4 mM Gln medium was used for transfection. Transfection was performed transiently via Bio-Rad (Gene Pulser II set to 900 uF, 300 v, 0 ohms). Cells divided 24 hours before transfection. Day 0 viability and VCD are expressed as million cells / ml. Transfection was performed using 5 μg of heavy and light chain DNA. 10 μg of GFP DNA was used as an external control to check transfection efficiency. Stable pools were generated by maintaining methionine sulfoxide (MSX) at levels that resulted in selective pressure favoring cell growth expressing the recombinant protein of interest (rituximab light chain) for 14 days. The resulting recombinant pools were compared in a 14-day fed-batch quantitative analysis. Titers were analyzed on harvest day and throughout the culture period by biolayer interferometry or ELISA. The above-described pool generation method was repeated using only one concentration of MSX (80 mM) to generate a pool for testing MAR zebrafish cores.

[0099] result

[0100] In vitro comparison results of MAR X29 and MAR zebrafish are as follows: Figure 1 As shown, at both selective marker concentrations (80 mM and 100 mM methionine sulfoxide (MSX)), MAR zebrafish significantly outperformed MAR X29. Therefore, this example validates the retrieval and computer-based method developed in Example 1.

[0101] In further experiments, the potency of the MAR core sequence alone was tested compared to a construct without any MAR components. Figure 2 As shown, the MAR zebrafish core sequence produced a significantly higher titer than the non-MAR control.

[0102] It should be understood that although the present invention has been described in conjunction with the above embodiments, the foregoing description and examples are intended to be illustrative and not to limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art.

Claims

1. A recombinant polynucleotide comprising: a coding sequence for a transgene; a promoter configured to initiate transcription of the coding sequence; and a matrix attachment region (MAR) nucleic acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4, wherein the MAR is capable of attaching to a mammalian cell nuclear matrix.

2. A cell comprising the polynucleotide of claim 1.

3. A method for transfecting a coding sequence for a transgene into a cell, comprising: contacting the cell with a first polynucleotide comprising the coding sequence and a promoter for initiating transcription of the coding sequence and an unlinked second polynucleotide comprising a matrix attachment region (MAR) nucleic acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4, under conditions for transfecting the first polynucleotide and the second polynucleotide into the cell, wherein the MAR is capable of attaching to the nuclear matrix of a mammalian cell.

4. A transfected cell prepared by the method of claim 3.

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