<p>pNMDi3 / pNMDi-AdSA-Venus-EIFs7* (6651bp plamsid sequence)</p><p>CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA AAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGG CACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAA AATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTA AAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGCCTCGAGGCTAGAACTAGTGGAT CTGTAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTT CCAGTGGGGATCGACGGTATCGATAAGCTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGG CCCACCCTCGTGACCACCCTGGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGC TACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAAC GGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTG CTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCC GGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTGATAATAGTCTAGTCGGGCTGCAGATAACTTCGTATAAAGTATCCTATACGAAGTTATCCA TGGAATTCAACCACAACTTCTCTCAGCAGTGCCTGAGAATGGTGAGTTCACAGGAGCGGATGCGCTGGCCTGCTGGGTTAGGTCCCTGCTGCTGAGCCTCTG CTCTGCACTAGATGGTGGTGCGAGGTTGGCCAGGCTCGCCCACCTGGTAGCAGGGATCCGAGCCACAAAAAGGTCTAAAGGCATATTTCCTCTCTGTGGAGT TATTGATAACGCCTGTGTCACAGATGGGCTGTGGAATGCCTGTGGATAAAACCATTCGTTACCTTGAGGATACAGCACTGAACAATAGTCCCACCTTTTACT GTGATGGAGGAGGGAGAAAAGGCATTGCTTGCTGTCCTGTTAGCTACAGTCTGCCTGTAGTGAGGAAGGAAGCAGGGAGTGGCATGGTGGCTCTTACTCAGT GATTGTGTTGGGCTTGGTCCTTAATACACAATGAGCCTACATCCTTAAGGACAGAAGTACCAGCCAGGGCAGTCAAAAGCAATGCTGGGTCTGCCGACTTGA AGTCAACCCCAGCTCCTGAAAGCTTTGTCTCTTCTACAGGGAAGAGAAAGATACAATTTCCCCTAACTGCAGCTGAATGAAGAACCCAAACCCATTTGTGGA GGACGACATGGATAAGAATGAGATCGCCTCCGTCGCTTACCGGTAAGTCGGCTCTTGGGGACAGGTGACCCTGTCCTGTGCTGTCTGGGGTGGTCCATCCAC AGCACTGGATCTTGTAGTGAGTGCGTCTTGCTGTCCTTGTAGTTACCGCAGGTGGAAGCTTGGAGATGACATCGACCTTATCGTCCGCTGTGAACACGATGG TGTCATGACCGGGGCCAACGGGGAAGTGTCCTTCATCAACATCAAGACGCTCAATCTAGATAACTTCGTATAAAGTATCCTATACGAAGTTATACTAGAGCT CGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC AATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGACGGAAAGAACCAGCTGGGGCTCGATCCTCTAGCTACCGGGTAGGGGAGGCGCTTTT CCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCG CCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTG ACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTG TCCTTCGCTTTCTGGGTCAGAGGCTGGGAAGGGTGGGTCCGGGGGCGGGTCAGGGGCGGGTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGG CATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCAATATGGGATCGGCCATTGAACAA GATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGG CTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACG ACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCAC CTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGC ATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTC AAGGCGCGCATGCCCGACGGCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGAC TGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTG CTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCAATTCTCTAGCGCGCCTGAGTACTTC AGGGATTTGAATGTAAGTAATGCTTTTTTTTTTCTCGCTCTCGTTTTTCAAAAACCCACATAAAAATTGAGGAAAGGGAAGAATTGTTTTCTCCTTCCAGCA CCTCGTAATTTGACCCGACTGATGGTTCCCATTAGTCACATAAAGCTGTAGTCAAGTACAGACGTCCTTAGAACTGGAACTTGGCCAGGCTACGGTGACACT TCTTGCTGGCTGAAATAGTTGAACAGCTTTAATATAGGCGCGCTAGAGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTA ATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAA GCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA GCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCC TTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTT TTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA GACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGT GAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTA AAAGTGCTCATCATTGGAAAA Restriction Enzyme Map:</p><p>1 CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTG 80 1 GCAAGAAGCCCCGCTTTTGAGAGTTCCTAGAATGGCGACAACTCTAGGTCAAGCTACATTGGGTGAGCACGTGGGTTGAC 80 BssSI ApaLI </p><p>81 ATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA 160 81 TAGAAGTCGTAGAAAATGAAAGTGGTCGCAAAGACCCACTCGTTTTTGTCCTTCCGTTTTACGGCGTTTTTTCCCTTATT 160</p><p>161 GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG 240 161 CCCGCTGTGCCTTTACAACTTATGAGTATGAGAAGGAAAAAGTTATAATAACTTCGTAAATAGTCCCAATAACAGAGTAC 240 SspI BspHI </p><p>241 AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA 320 241 TCGCCTATGTATAAACTTACATAAATCTTTTTATTTGTTTATCCCCAAGGCGCGTGTAAAGGGGCTTTTCACGGTGGACT 320 BsrBI </p><p>321 CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAG 400 321 GCGCGGGACATCGCCGCGTAATTCGCGCCGCCCACACCACCAATGCGCGTCGCACTGGCGATGTGAACGGTCGCGGGATC 400</p><p>401 CGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTC 480 401 GCGGGCGAGGAAAGCGAAAGAAGGGAAGGAAAGAGCGGTGCAAGCGGCCGAAAGGGGCAGTTCGAGATTTAGCCCCCGAG 480 BsrBI NgoMIV NaeI </p><p>481 CCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCC 560 481 GGAAATCCCAAGGCTAAATCACGAAATGCCGTGGAGCTGGGGTTTTTTGAACTAATCCCACTACCAAGTGCATCACCCGG 560</p><p>561 ATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAA 640 561 TAGCGGGACTATCTGCCAAAAAGCGGGAAACTGCAACCTCAGGTGCAAGAAATTATCACCTGAGAACAAGGTTTGACCTT 640</p><p>641 CAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG 720 641 GTTGTGAGTTGGGATAGAGCCAGATAAGAAAACTAAATATTCCCTAAAACGGCTAAAGCCGGATAACCAATTTTTTACTC 720 PsiI </p><p>721 CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAAC 800 721 GACTAAATTGTTTTTAAATTGCGCTTAAAATTGTTTTATAATTGCGAATGTTAAAGGTAAGCGGTAAGTCCGACGCGTTG 800 SspI FspI </p><p>801 TGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAG 880 801 ACAACCCTTCCCGCTAGCCACGCCCGGAGAAGCGATAATGCGGTCGACCGCTTTCCCCCTACACGACGTTCCGCTAATTC 880 PvuI PvuII </p><p>881 TTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGG 960 881 AACCCATTGCGGTCCCAAAAGGGTCAGTGCTGCAACATTTTGCTGCCGGTCACTCGCGCGCATTATGCTGAGTGATATCC 960 BssHII </p><p>Human Adenovirus Type 3/5 SA 961 GCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGCCTCGAGGCTAGAACTAGTGGATCTGTAGGGCGCAGTAGTCCA 1040 961 CGCTTAACCTCGAGGTGGCGCCACCGCCGGCGAGATCGGAGCTCCGATCTTGATCACCTAGACATCCCGCGTCATCAGGT 1040 EcoICRI SacII BsrBI XhoI SpeI SacI EagI </p><p> splice site  L A V E D K L F A V 1041 GGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCT 1120 1041 CCCAAAGGAACTACTACAGTATGAATAGGACAGGGAAAAAAAAGGTGTCGAGCGCCAACTCCTGTTTGAGAAGCGCCAGA 1120</p><p>EYFP (Venus version) F P V G I D G I D K L M V S K G E E L F T G V V P I L 1121 TTCCAGTGGGGATCGACGGTATCGATAAGCTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG 1200 1121 AAGGTCACCCCTAGCTGCCATAGCTATTCGAGTACCACTCGTTCCCGCTCCTCGACAAGTGGCCCCACCACGGGTAGGAC 1200 ClaI </p><p>V E L D G D V N G H K F S V S G E G E G D A T Y G K L 1201 GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCT 1280 1201 CAGCTCGACCTGCCGCTGCATTTGCCGGTGTTCAAGTCGCACAGGCCGCTCCCGCTCCCGCTACGGTGGATGCCGTTCGA 1280</p><p>T L K L I C T T G K L P V P W P T L V T T L G Y G L 1281 GACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTACGGCCTGC 1360 1281 CTGGGACTTCGACTAGACGTGGTGGCCGTTCGACGGGCACGGGACCGGGTGGGAGCACTGGTGGGACCCGATGCCGGACG 1360 BssSI </p><p>Q C F A R Y P D H M K Q H D F F K S A M P E G Y V Q E 1361 AGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG 1440 1361 TCACGAAGCGGGCGATGGGGCTGGTGTACTTCGTCGTGCTGAAGAAGTTCAGGCGGTACGGGCTTCCGATGCAGGTCCTC 1440 PstI R T I F F K D D G N Y K T R A E V K F E G D T L V N R 1441 CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCG 1520 1441 GCGTGGTAGAAGAAGTTCCTGCTGCCGTTGATGTTCTGGGCGCGGCTCCACTTCAAGCTCCCGCTGTGGGACCACTTGGC 1520</p><p>I E L K G I D F K E D G N I L G H K L E Y N Y N S H 1521 CATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA 1600 1521 GTAGCTCGACTTCCCGTAGCTGAAGTTCCTCCTGCCGTTGTAGGACCCCGTGTTCGACCTCATGTTGATGTTGTCGGTGT 1600</p><p>N V Y I T A D K Q K N G I K A N F K I R H N I E D G G 1601 ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGC 1680 1601 TGCAGATATAGTGGCGGCTGTTCGTCTTCTTGCCGTAGTTCCGGTTGAAGTTCTAGGCGGTGTTGTAGCTCCTGCCGCCG 1680</p><p>V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S 1681 GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG 1760 1681 CACGTCGAGCGGCTGGTGATGGTCGTCTTGTGGGGGTAGCCGCTGCCGGGGCACGACGACGGGCTGTTGGTGATGGACTC 1760</p><p>NMDF Y Q S A L S K D P N E K R D H M V L L E F V T A A G 1761 CTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA 1840 1761 GATGGTCAGGCGGGACTCGTTTCTGGGGTTGCTCTTCGCGCTAGTGTACCAGGACGACCTCAAGCACTGGCGGCGGCCCT 1840 determine whether the single nucleotide insertion here is real I T L G M D E L Y K * E L I I loxP 1841 TCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTGATAATAGTCTAGTCGGGCTGCAGATAACTTCGTATAAAGTAT 1920 1841 AGTGAGAGCCGTACCTGCTCGACATGTTCATTCTTAACTATTATCAGATCAGCCCGACGTCTATTGAAGCATATTTCATA 1920 BsrGI PstI </p><p>EIFs7 exon 10 (splice donor) ↓ splice site 1921 CCTATACGAAGTTATCCATGGAATTCAACCACAACTTCTCTCAGCAGTGCCTGAGAATGGTGAGTTCACAGGAGCGGATG 2000 1921 GGATATGCTTCAATAGGTACCTTAAGTTGGTGTTGAAGAGAGTCGTCACGGACTCTTACCACTCAAGTGTCCTCGCCTAC 2000 NcoI BsrBI EcoRI </p><p>2001 CGCTGGCCTGCTGGGTTAGGTCCCTGCTGCTGAGCCTCTGCTCTGCACTAGATGGTGGTGCGAGGTTGGCCAGGCTCGCC 2080 2001 GCGACCGGACGACCCAATCCAGGGACGACGACTCGGAGACGAGACGTGATCTACCACCACGCTCCAACCGGTCCGAGCGG 2080 BseYI MscI </p><p>2081 CACCTGGTAGCAGGGATCCGAGCCACAAAAAGGTCTAAAGGCATATTTCCTCTCTGTGGAGTTATTGATAACGCCTGTGT 2160 2081 GTGGACCATCGTCCCTAGGCTCGGTGTTTTTCCAGATTTCCGTATAAAGGAGAGACACCTCAATAACTATTGCGGACACA 2160 BamHI NMDR </p><p>2161 CACAGATGGGCTGTGGAATGCCTGTGGATAAAACCATTCGTTACCTTGAGGATACAGCACTGAACAATAGTCCCACCTTT 2240 2161 GTGTCTACCCGACACCTTACGGACACCTATTTTGGTAAGCAATGGAACTCCTATGTCGTGACTTGTTATCAGGGTGGAAA 2240</p><p>2241 TACTGTGATGGAGGAGGGAGAAAAGGCATTGCTTGCTGTCCTGTTAGCTACAGTCTGCCTGTAGTGAGGAAGGAAGCAGG 2320 2241 ATGACACTACCTCCTCCCTCTTTTCCGTAACGAACGACAGGACAATCGATGTCAGACGGACATCACTCCTTCCTTCGTCC 2320</p><p>2321 GAGTGGCATGGTGGCTCTTACTCAGTGATTGTGTTGGGCTTGGTCCTTAATACACAATGAGCCTACATCCTTAAGGACAG 2400 2321 CTCACCGTACCACCGAGAATGAGTCACTAACACAACCCGAACCAGGAATTATGTGTTACTCGGATGTAGGAATTCCTGTC 2400 AflII </p><p>2401 AAGTACCAGCCAGGGCAGTCAAAAGCAATGCTGGGTCTGCCGACTTGAAGTCAACCCCAGCTCCTGAAAGCTTTGTCTCT 2480 2401 TTCATGGTCGGTCCCGTCAGTTTTCGTTACGACCCAGACGGCTGAACTTCAGTTGGGGTCGAGGACTTTCGAAACAGAGA 2480 BseYI BseYI HindIII </p><p> splice site EIFs7 exon 11 modified 2481 TCTACAGGGAAGAGAAAGATACAATTTCCCCTAACTGCAGCTGAATGAAGAACCCAAACCCATTTGTGGAGGACGACATG 2560 2481 AGATGTCCCTTCTCTTTCTATGTTAAAGGGGATTGACGTCGACTTACTTCTTGGGTTTGGGTAAACACCTCCTGCTGTAC 2560 PstI PvuII </p><p> splice site 2561 GATAAGAATGAGATCGCCTCCGTCGCTTACCGGTAAGTCGGCTCTTGGGGACAGGTGACCCTGTCCTGTGCTGTCTGGGG 2640 2561 CTATTCTTACTCTAGCGGAGGCAGCGAATGGCCATTCAGCCGAGAACCCCTGTCCACTGGGACAGGACACGACAGACCCC 2640 AgeI </p><p> splice site  EIFs7 exon 12 (splice acceptor) 2641 TGGTCCATCCACAGCACTGGATCTTGTAGTGAGTGCGTCTTGCTGTCCTTGTAGTTACCGCAGGTGGAAGCTTGGAGATG 2720 2641 ACCAGGTAGGTGTCGTGACCTAGAACATCACTCACGCAGAACGACAGGAACATCAATGGCGTCCACCTTCGAACCTCTAC 2720 HindIII </p><p>2721 ACATCGACCTTATCGTCCGCTGTGAACACGATGGTGTCATGACCGGGGCCAACGGGGAAGTGTCCTTCATCAACATCAAG 2800 2721 TGTAGCTGGAATAGCAGGCGACACTTGTGCTACCACAGTACTGGCCCCGGTTGCCCCTTCACAGGAAGTAGTTGTAGTTC 2800 BspHI </p><p> loxP BGHpA 2801 ACGCTCAATCTAGATAACTTCGTATAAAGTATCCTATACGAAGTTATACTAGAGCTCGCTGATCAGCCTCGACTGTGCCT 2880 2801 TGCGAGTTAGATCTATTGAAGCATATTTCATAGGATATGCTTCAATATGATCTCGAGCGACTAGTCGGAGCTGACACGGA 2880 XbaI EcoICRI SacI BclI 2881 TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTC 2960 2881 AGATCAACGGTCGGTAGACAACAAACGGGGAGGGGGCACGGAAGGAACTGGGACCTTCCACGGTGAGGGTGACAGGAAAG 2960 BGHpAR ATCAACGGTCGGTAGACAAC NMDiRex </p><p> polyadenylation 2961 CTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA 3040 2961 GATTATTTTACTCCTTTAACGTAGCGTAACAGACTCATCCACAGTAAGATAAGACCCCCCACCCCACCCCGTCCTGTCGT 3040</p><p>3041 AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGACGGAAAGAACCAGC 3120 3041 TCCCCCTCCTAACCCTTCTGTTATCGTCCGTACGACCCCTACGCCACCCGAGATACCGAAGACTCTGCCTTTCTTGGTCG 3120 SphI PvuII BseYI BseYI </p><p>PGK-1 promoter 3121 TGGGGCTCGATCCTCTAGCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGC 3200 3121 ACCCCGAGCTAGGAGATCGATGGCCCATCCCCTCCGCGAAAAGGGTTCCGTCAGACCTCGTACGCGAAATCGTCGGGGCG 3200 SphI BseYI </p><p>3201 TGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTC 3280 3201 ACCCGTGAACCGCGATGTGTTCACCGGAGACCGGAGCGTGTGTAAGGTGTAGGTGGCCATCCGCGGTTGGCCGAGGCAAG 3280 AgeI KasI NarI SfoI BbeI </p><p>3281 TTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAG 3360 3281 AAACCACCGGGGAAGCGCGGTGGAAGATGAGGAGGGGATCAGTCCTTCAAGGGGGGGCGGGGCGTCGAGCGCAGCACGTC 3360</p><p>3361 GACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCC 3440 3361 CTGCACTGTTTACCTTCATCGTGCAGAGTGATCAGAGCACGTCTACCTGTCGTGGCGACTCGTTACCTTCGCCCATCCGG 3440 BmgBI BmgBI BssSI StuI SpeI </p><p>3441 TTTGGGGCAGCGGCCAATAGCAGCTTTGTCCTTCGCTTTCTGGGTCAGAGGCTGGGAAGGGTGGGTCCGGGGGCGGGTCA 3520 3441 AAACCCCGTCGCCGGTTATCGTCGAAACAGGAAGCGAAAGACCCAGTCTCCGACCCTTCCCACCCAGGCCCCCGCCCAGT 3520 BseYI </p><p>3521 GGGGCGGGTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTG 3600 3521 CCCCGCCCAGTCCCCGCCCCGCCCGCGGGCTTCCAGGAGGCCTCCGGGCCGTAAGACGTGCGAAGTTTTCGCGTGCAGAC 3600 KasI BspEI BmgBI NarI SfoI BbeI </p><p> neoR M G S A I E Q D G L 3601 CCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATT 3680 3601 GGCGCGACAAGAGGAGAAGGAGTAGAGGCCCGGAAAGCTGGACGTCGGTTATACCCTAGCCGGTAACTTGTTCTACCTAA 3680 PstI </p><p>H A G S P A A W V E R L F G Y D W A Q Q T I G C S D 3681 GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATG 3760 3681 CGTGCGTCCAAGAGGCCGGCGAACCCACCTCTCCGATAAGCCGATACTGACCCGTGTTGTCTGTTAGCCGACGAGACTAC 3760 EagI </p><p>A A V F R L S A Q G R P V L F V K T D L S G A L N E L 3761 CCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTG 3840 3761 GGCGGCACAAGGCCGACAGTCGCGTCCCCGCGGGCCAAGAAAAACAGTTCTGGCTGGACAGGCCACGGGACTTACTTGAC 3840 KasI NarI SfoI BbeI </p><p>Q D E A A R L S W L A T T G V P C A A V L D V V T E A 3841 CAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC 3920 3841 GTCCTGCTCCGTCGCGCCGATAGCACCGACCGGTGCTGCCCGCAAGGAACGCGTCGACACGAGCTGCAACAGTGACTTCG 3920 PstI MscI FspI PvuII </p><p>G R D W L L L G E V P G Q D L L S S H L A P A E K V 3921 GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTAT 4000 3921 CCCTTCCCTGACCGACGATAACCCGCTTCACGGCCCCGTCCTAGAGGACAGTAGAGTGGAACGAGGACGGCTCTTTCATA 4000</p><p>S I M A D A M R R L H T L D P A T C P F D H Q A K H R 4001 CCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGC 4080 4001 GGTAGTACCGACTACGTTACGCCGCCGACGTATGCGAACTAGGCCGATGGACGGGTAAGCTGGTGGTTCGCTTTGTAGCG 4080</p><p>I E R A R T R M E A G L V D Q D D L D E E H Q G L A P 4081 ATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCC 4160 4081 TAGCTCGCTCGTGCATGAGCCTACCTTCGGCCAGAACAGCTAGTCCTACTAGACCTGCTTCTCGTAGTCCCCGAGCGCGG 4160 A E L F A R L K A R M P D G D D L V V T H G D A C L 4161 AGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGC 4240 4161 TCGGCTTGACAAGCGGTCCGAGTTCCGCGCGTACGGGCTGCCGCTACTAGAGCAGCACTGGGTACCGCTACGGACGAACG 4240 BssHII SphI NcoI </p><p>P N I M V E N G R F S G F I D C G R L G V A D R Y Q D 4241 CGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGAC 4320 4241 GCTTATAGTACCACCTTTTACCGGCGAAAAGACCTAAGTAGCTGACACCGGCCGACCCACACCGCCTGGCGATAGTCCTG 4320 NgoMIV NaeI BseYI </p><p>I A L A T R D I A E E L G G E W A D R F L V L Y G I A 4321 ATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGC 4400 4321 TATCGCAACCGATGGGCACTATAACGACTTCTCGAACCGCCGCTTACCCGACTGGCGAAGGAGCACGAAATGCCATAGCG 4400 BssSI </p><p>A P D S Q R I A F Y R L L D E F F * 4401 CGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCAATTCTCTAGCGCGCCTG 4480 4401 GCGAGGGCTAAGCGTCGCGTAGCGGAAGATAGCGGAAGAACTGCTCAAGAAGACTCCCCTAGTTAAGAGATCGCGCGGAC 4480 BsrBI BssHII </p><p>Mouse Hprt SD  splice site 4481 AGTACTTCAGGGATTTGAATGTAAGTAATGCTTTTTTTTTTCTCGCTCTCGTTTTTCAAAAACCCACATAAAAATTGAGG 4560 4481 TCATGAAGTCCCTAAACTTACATTCATTACGAAAAAAAAAAGAGCGAGAGCAAAAAGTTTTTGGGTGTATTTTTAACTCC 4560 ScaI </p><p>4561 AAAGGGAAGAATTGTTTTCTCCTTCCAGCACCTCGTAATTTGACCCGACTGATGGTTCCCATTAGTCACATAAAGCTGTA 4640 4561 TTTCCCTTCTTAACAAAAGAGGAAGGTCGTGGAGCATTAAACTGGGCTGACTACCAAGGGTAATCAGTGTATTTCGACAT 4640</p><p>4641 GTCAAGTACAGACGTCCTTAGAACTGGAACTTGGCCAGGCTACGGTGACACTTCTTGCTGGCTGAAATAGTTGAACAGCT 4720 4641 CAGTTCATGTCTGCAGGAATCTTGACCTTGAACCGGTCCGATGCCACTGTGAAGAACGACCGACTTTATCAACTTGTCGA 4720 ZraI MscI AatII </p><p>4721 TTAATATAGGCGCGCTAGAGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCG 4800 4721 AATTATATCCGCGCGATCTCAGCTGGAGCTCCCCCCCGGGCCATGGGTCGAAAACAAGGGAAATCACTCCCAATTAACGC 4800 BssHII SalI XhoI PspOMI KpnI BssHII TaqI ApaI BseYI Acc65I </p><p>4801 CGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGG 4880 4801 GCGAACCGCATTAGTACCAGTATCGACAAAGGACACACTTTAACAATAGGCGAGTGTTAAGGTGTGTTGTATGCTCGGCC 4880 iPCR3nestR iPCR3R BsrBI </p><p>4881 AAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC 4960 4881 TTCGTATTTCACATTTCGGACCCCACGGATTACTCACTCGATTGAGTGTAATTAACGCAACGCGAGTGACGGGCGAAAGG 4960 AseI </p><p>4961 AGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCT 5040 4961 TCAGCCCTTTGGACAGCACGGTCGACGTAATTACTTAGCCGGTTGCGCGCCCCTCTCCGCCAAACGCATAACCCGCGAGA 5040 PvuII AseI iPCR3F iPCR3nestF 5041 TCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAAT 5120 5041 AGGCGAAGGAGCGAGTGACTGAGCGACGCGAGCCAGCAAGCCGACGCCGCTCGCCATAGTCGAGTGAGTTTCCGCCATTA 5120 BsrBI </p><p>5121 ACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA 5200 5121 TGCCAATAGGTGTCTTAGTCCCCTATTGCGTCCTTTCTTGTACACTCGTTTTCCGGTCGTTTTCCGGTCCTTGGCATTTT 5200 PciI AflIII </p><p>5201 AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG 5280 5201 TCCGGCGCAACGACCGCAAAAAGGTATCCGAGGCGGGGGGACTGCTCGTAGTGTTTTTAGCTGCGAGTTCAGTCTCCACC 5280 TaqI </p><p>5281 CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC 5360 5281 GCTTTGGGCTGTCCTGATATTTCTATGGTCCGCAAAGGGGGACCTTCGAGGGAGCACGCGAGAGGACAAGGCTGGGACGG 5360 BssSI </p><p>5361 GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT 5440 5361 CGAATGGCCTATGGACAGGCGGAAAGAGGGAAGCCCTTCGCACCGCGAAAGAGTATCGAGTGCGACATCCATAGAGTCAA 5440</p><p>5441 CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC 5520 5441 GCCACATCCAGCAAGCGAGGTTCGACCCGACACACGTGCTTGGGGGGCAAGTCGGGCTGGCGACGCGGAATAGGCCATTG 5520 BseYI ApaLI </p><p>5521 TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG 5600 5521 ATAGCAGAACTCAGGTTGGGCCATTCTGTGCTGAATAGCGGTGACCGTCGTCGGTGACCATTGTCCTAATCGTCTCGCTC 5600</p><p>5601 GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG 5680 5601 CATACATCCGCCACGATGTCTCAAGAACTTCACCACCGGATTGATGCCGATGTGATCTTCCTGTCATAAACCATAGACGC 5680</p><p>5681 CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT 5760 5681 GAGACGACTTCGGTCAATGGAAGCCTTTTTCTCAACCATCGAGAACTAGGCCGTTTGTTTGGTGGCGACCATCGCCACCA 5760</p><p>5761 TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA 5840 5761 AAAAAACAAACGTTCGTCGTCTAATGCGCGTCTTTTTTTCCTAGAGTTCTTCTAGGAAACTAGAAAAGATGCCCCAGACT 5840</p><p>5841 CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA 5920 5841 GCGAGTCACCTTGCTTTTGAGTGCAATTCCCTAAAACCAGTACTCTAATAGTTTTTCCTAGAAGTGGATCTAGGAAAATT 5920 BspHI DraI </p><p>5921 ATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG 6000 5921 TAATTTTTACTTCAAAATTTAGTTAGATTTCATATATACTCATTTGAACCAGACTGTCAATGGTTACGAATTAGTCACTC 6000 DraI </p><p>6001 GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA 6080 6001 CGTGGATAGAGTCGCTAGACAGATAAAGCAAGTAGGTATCAACGGACTGAGGGGCAGCACATCTATTGATGCTATGCCCT 6080</p><p>6081 GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC 6160 6081 CCCGAATGGTAGACCGGGGTCACGACGTTACTATGGCGCTCTGGGTGCGAGTGGCCGAGGTCTAAATAGTCGTTATTTGG 6160</p><p>6161 AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA 6240 6161 TCGGTCGGCCTTCCCGGCTCGCGTCTTCACCAGGACGTTGAAATAGGCGGAGGTAGGTCAGATAATTAACAACGGCCCTT 6240 AseI </p><p>6241 GCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC 6320 6241 CGATCTCATTCATCAAGCGGTCAATTATCAAACGCGTTGCAACAACGGTAACGATGTCCGTAGCACCACAGTGCGAGCAG 6320 FspI AclI </p><p>6321 GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG 6400 6321 CAAACCATACCGAAGTAAGTCGAGGCCAAGGGTTGCTAGTTCCGCTCAATGTACTAGGGGGTACAACACGTTTTTTCGCC 6400</p><p>6401 TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT 6480 6401 AATCGAGGAAGCCAGGAGGCTAGCAACAGTCTTCATTCAACCGGCGTCACAATAGTGAGTACCAATACCGTCGTGACGTA 6480 PvuI </p><p>6481 AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG 6560 6481 TTAAGAGAATGACAGTACGGTAGGCATTCTACGAAAAGACACTGACCACTCATGAGTTGGTTCAGTAAGACTCTTATCAC 6560 ScaI </p><p>6561 TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA 6640 6561 ATACGCCGCTGGCTCAACGAGAACGGGCCGCAGTTATGCCCTATTATGGCGCGGTGTATCGTCTTGAAATTTTCACGAGT 6640 DraI </p><p>6641 TCATTGGAAAA 6651 6641 AGTAACCTTTT 6651 Restriction Enzyme Table:</p><p>Enzyme Recognition frequency Positions ______AatII G_ACGT'C 1 4656 Acc65I G'GTAC_C 1 4762 AclI AA'CG_TT 1 6279 AflII C'TTAA_G 1 2391 AgeI A'CCGG_T 2 2590, 3255 ApaI G_GGCC'C 1 4760 ApaLI G'TGCA_C 2 69, 5474 AseI AT'TA_AT 3 4931, 4990, 6225 BamHI G'GATC_C 1 2095 BbeI G_GCGC'C 3 3266, 3548, 3793 BclI T'GATC_A 1 2861 BmgBI CAC'GTC 3 3364, 3384, 3596 BseYI C'CCAG_C 10 2011, 2431, 2457, 3074, 3120 3200, 3492, 4294, 4766, 5464 BspEI T'CCGG_A 1 3559 BspHI T'CATG_A 3 237, 2758, 5880 BsrBI CCG'CTC 7 243, 407, 992, 1995, 4403, 4852 5093 BsrGI T'GTAC_A 1 1863 BssHII G'CGCG_C 5 936, 4187, 4473, 4731, 4799 BssSI C'ACGA_G 5 66, 1334, 3396, 4382, 5333 ClaI AT'CG_AT 1 1143 DraI TTT'AAA 3 5919, 5938, 6630 EagI C'GGCC_G 2 987, 3696 EcoICRI GAG'CTC 2 972, 2855 EcoRI G'AATT_C 1 1942 FspI TGC'GCA 3 796, 3892, 6275 HindIII A'AGCT_T 2 2469, 2709 KasI G'GCGC_C 3 3262, 3544, 3789 KpnI G_GTAC'C 1 4766 MscI TGG'CCA 3 2070, 3872, 4675 NaeI GCC'GGC 2 448, 4292 NarI GG'CG_CC 3 3263, 3545, 3790 NcoI C'CATG_G 2 1937, 4222 NgoMIV G'CCGG_C 2 446, 4290 PciI A'CATG_T 1 5160 PsiI TTA'TAA 1 679 PspOMI G'GGCC_C 1 4756 PstI C_TGCA'G 5 1362, 1901, 2520, 3646, 3843 PvuI CG_AT'CG 2 817, 6423 PvuII CAG'CTG 5 846, 2521, 3120, 3896, 4984 SacI G_AGCT'C 2 974, 2857 SacII CC_GC'GG 1 981 SalI G'TCGA_C 1 4741 ScaI AGT'ACT 2 4484, 6533 SfoI GGC'GCC 3 3264, 3546, 3791 SpeI A'CTAG_T 2 1012, 3390 SphI G_CATG'C 3 3074, 3184, 4195 SspI AAT'ATT 2 206, 759 StuI AGG'CCT 1 3439 XbaI T'CTAG_A 1 2810 XhoI C'TCGA_G 2 1000, 4747 ZraI GAC'GTC 1 4654</p><p>Enzymes that cut five or fewer times</p><p>Enzyme Recognition frequency Positions ______AatII G_ACGT'C 1 4656 Acc65I G'GTAC_C 1 4762 AclI AA'CG_TT 1 6279 AflII C'TTAA_G 1 2391 AgeI A'CCGG_T 2 2590, 3255 ApaI G_GGCC'C 1 4760 ApaLI G'TGCA_C 2 69, 5474 AseI AT'TA_AT 3 4931, 4990, 6225 BamHI G'GATC_C 1 2095 BbeI G_GCGC'C 3 3266, 3548, 3793 BclI T'GATC_A 1 2861 BmgBI CAC'GTC 3 3364, 3384, 3596 BspEI T'CCGG_A 1 3559 BspHI T'CATG_A 3 237, 2758, 5880 BsrGI T'GTAC_A 1 1863 BssHII G'CGCG_C 5 936, 4187, 4473, 4731, 4799 BssSI C'ACGA_G 5 66, 1334, 3396, 4382, 5333 ClaI AT'CG_AT 1 1143 DraI TTT'AAA 3 5919, 5938, 6630 EagI C'GGCC_G 2 987, 3696 EcoICRI GAG'CTC 2 972, 2855 EcoRI G'AATT_C 1 1942 FspI TGC'GCA 3 796, 3892, 6275 HindIII A'AGCT_T 2 2469, 2709 KasI G'GCGC_C 3 3262, 3544, 3789 KpnI G_GTAC'C 1 4766 MscI TGG'CCA 3 2070, 3872, 4675 NaeI GCC'GGC 2 448, 4292 NarI GG'CG_CC 3 3263, 3545, 3790 NcoI C'CATG_G 2 1937, 4222 NgoMIV G'CCGG_C 2 446, 4290 PciI A'CATG_T 1 5160 PsiI TTA'TAA 1 679 PspOMI G'GGCC_C 1 4756 PstI C_TGCA'G 5 1362, 1901, 2520, 3646, 3843 PvuI CG_AT'CG 2 817, 6423 PvuII CAG'CTG 5 846, 2521, 3120, 3896, 4984 SacI G_AGCT'C 2 974, 2857 SacII CC_GC'GG 1 981 SalI G'TCGA_C 1 4741 ScaI AGT'ACT 2 4484, 6533 SfoI GGC'GCC 3 3264, 3546, 3791 SpeI A'CTAG_T 2 1012, 3390 SphI G_CATG'C 3 3074, 3184, 4195 SspI AAT'ATT 2 206, 759 StuI AGG'CCT 1 3439 XbaI T'CTAG_A 1 2810 XhoI C'TCGA_G 2 1000, 4747 ZraI GAC'GTC 1 4654</p><p>Enzymes that do not cut: ______AfeI, AvrII, BfrBI, BglII, BmtI, BsiWI, BstBI, BstZ17I, EcoRV, HpaI, MfeI, MluI NdeI, NheI, NruI, NsiI, PmlI, SmaI, SnaBI, XmaI</p>