LOCUS       pPBpuro-EGFP-eDHFR(69K6 8162 bp ds-DNA     circular     10-NOV-2021
DEFINITION  .
KEYWORDS    "Creator:Shinya Tsukiji"                                           
FEATURES             Location/Qualifiers
     rep_origin      complement(135..441)
                     /label="F1 ori"
     CDS             complement(460..528)
                     /label="LacZ alpha"
     primer_bind     599..616
                     /label="M13-fwd"
     primer_bind     625..645
                     /label="T7"
     misc_feature    670..983
                     /label="piggyBac 5' terminal repeat"
     misc_feature    1009..2726
                     /label="CAG promoter"
     promoter        1025..1337
                     /label="CMV immediate early enhancer"
     promoter        1091..1378
                     /label="CAG enhancer"
     gene            2754..3467
                     /label="EGFP"
     primer          complement(2796..2817)
                     /label="EGFP_N_primer"
     feature         2934..2963
                     /label="Y66 (EGFP)"
     primer          3404..3425
                     /label="EGFP_C_primer"
     misc_feature    3498..3992
                     /label="DHFR(69K6)"
     promoter        4067..4613
                     /label="IRES"
     CDS             4623..5216
                     /label="puromycin N-acetyltransferase"
     primer_bind     complement(5519..5539)
                     /label="M13-rev"
     misc_binding    complement(5545..5567)
                     /label="LacO"
     promoter        complement(5571..5601)
                     /label="lac promoter"
     misc_feature    complement(5696..5937)
                     /label="piggyBac 3' terminal repeat"
     primer_bind     complement(5972..5991)
                     /label="T3"
     promoter        complement(5974..5992)
                     /label="T3 promoter"
     primer_bind     complement(6009..6029)
                     /label="M13-rev"
     misc_binding    complement(6035..6057)
                     /label="LacO"
     promoter        complement(6061..6091)
                     /label="lac promoter"
     rep_origin      complement(6397..7079)
                     /label="ColE1 origin"
     CDS             complement(7177..7836)
                     /label="AmpR"
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     1441 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     1501 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     1561 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     1621 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     1681 ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg gctctgactg
     1741 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1801 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc
     1861 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
     1921 ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
     1981 gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
     2041 gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     2101 agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg
     2161 ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg gggctcgccg
     2221 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     2281 gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc gaggcgcggc
     2341 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     2401 ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg
     2461 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     2521 gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg gctgccttcg
     2581 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc
     2641 tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttgt
     2701 tgtgctgtct catcattttg gcaaagaatt GGgatccacc ggtcgccacc atggtgagca
     2761 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2821 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2881 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2941 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     3001 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     3061 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     3121 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3181 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3241 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3301 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     3361 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3421 tcgtgaccgc cgccgggatc actctcggca tggacgagct GTACAAGTCT GGGTCCGCAG
     3481 CCAGTAGCGC CGGAAGTATG ATCAGTCTGA TTGCGGCGTT AGCGGTAGAT CGCGTTATCG
     3541 GCATGGAAAA CGCCATGCCG TGGAACCTGC CTGCCGATCT CGCCTGGTTT AAACGCAACA
     3601 CCTTAAATAA ACCCGTGATT ATGGGCCGCC ATACCTGGGA ATCAATCGGT CGTCCGTTGC
     3661 CAGGACGCAA AAATATTATC CTCAGCAGTC AACCGGGTAC GGACAAAAAA AAGAAAAAGA
     3721 AAGATCGCGT AACGTGGGTG AAGTCGGTGG ATGAAGCCAT CGCGGCGTGT GGTGACGTAC
     3781 CAGAAATCAT GGTGATTGGC GGCGGTCGCG TTTATGAACA GTTCTTGCCA AAAGCGCAAA
     3841 AACTGTATCT GACGCATATC GACGCAGAAG TGGAAGGCGA CACCCATTTC CCGGATTACG
     3901 AGCCGGATGA CTGGGAATCG GTATTCAGCG AATTCCACGA TGCTGATGCG CAGAACTCTC
     3961 ACAGCTATTG CTTTGAGATT CTGGAGCGGC GGTAACCCGG GATAAgtcAA CTAACTTAAG
     4021 CTAGCAACGG TTTCCCTCTA GCGGGATCAA TTCCGCCCCC CCCCCCTAAC GTTACTGGCC
     4081 GAAGCCGCTT GGAATAAGGC CGGTGTGCGT TTGTCTATAT GTTATTTTCC ACCATATTGC
     4141 CGTCTTTTGG CAATGTGAGG GCCCGGAAAC CTGGCCCTGT CTTCTTGACG AGCATTCCTA
     4201 GGGGTCTTTC CCCTCTCGCC AAAGGAATGC AAGGTCTGTT GAATGTCGTG AAGGAAGCAG
     4261 TTCCTCTGGA AGCTTCTTGA AGACAAACAA CGTCTGTAGC GACCCTTTGC AGGCAGCGGA
     4321 ACCCCCCACC TGGCGACAGG TGCCTCTGCG GCCAAAAGCC ACGTGTATAA GATACACCTG
     4381 CAAAGGCGGC ACAACCCCAG TGCCACGTTG TGAGTTGGAT AGTTGTGGAA AGAGTCAAAT
     4441 GGCTCTCCTC AAGCGTATTC AACAAGGGGC TGAAGGATGC CCAGAAGGTA CCCCATTGTA
     4501 TGGGATCTGA TCTGGGGCCT CGGTGCACAT GCTTTACATG TGTTTAGTCG AGGTTAAAAA
     4561 ACGTCTAGGC CCCCCGAACC ACGGGGACGT GGTTTTCCTT TGAAAAACAC GATAATACCA
     4621 TGACCGAGTA CAAGCCCACG GTGCGCCTCG CCACCCGCGA CGACGTCCCC aGGGCCGTAC
     4681 GCACCCTCGC CGCCGCGTTC GCCGACTACC CCGCCACGCG CCACACCGTC GAtCCGGACC
     4741 GCCACATCGA GCGGGTCACC GAGCTGCAAG AACTCTTCCT CACGCGCGTC GGGCTCGACA
     4801 TCGGCAAGGT GTGGGTCGCG GACGACGGCG CCGCGGTGGC GGTCTGGACC ACGCCGGAGA
     4861 GCGTCGAAGC GGGGGCGGTG TTCGCCGAGA TCGGCCCGCG CATGGCCGAG TTGAGCGGTT
     4921 CCCGGCTGGC CGCGCAGCAA CAGATGGAAG GCCTCCTGGC GCCGCACCGG CCCAAGGAGC
     4981 CCGCGTGGTT CCTGGCCACC GTCGGCGTCT CGCCCGACCA CCAGGGCAAG GGTCTGGGCA
     5041 GCGCCGTCGT GCTCCCCGGA GTGGAGGCGG CCGAGCGCGC CGGGGTGCCC GCCTTCCTGG
     5101 AGACCTCCGC GCCCCGCAAC CTCCCCTTCT ACGAGCGGCT CGGCTTCACC GTCACCGCCG
     5161 ACGTCGAGTG CCCGAAGGAC CGCGCGACCT GGTGCATGAC CCGCAAGCCC GGTGCCTGAT
     5221 cgatgatcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5281 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5341 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5401 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5461 gcggtgggct ctatggcttc tgaggcggaa agaacctgca gcccaagctt ggcgtaatca
     5521 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5581 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5641 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatctTAAAA
     5701 GTTTTGTTAC TTTATAGAAG AAATTTTGAG TTTTTGTTTT TTTTTAATAA ATAAATAAAC
     5761 ATAAATAAAT TGTTTGTTGA ATTTATTATT AGTATGTAAG TGTAAATATA ATAAAACTTA
     5821 ATATCTATTC AAATTAATAA ATAAACCTCG ATATACAGAC CGATAAAACA CATGCGTCAA
     5881 TTTTACGCAT GATTATCTTT AACGTACGTC ACAATATGAT TATCTTTCTA GGGTTAATCT
     5941 AGTATACGCG Tttaattaat ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     6001 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     6061 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     6121 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6181 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6241 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6301 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6361 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6421 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6481 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6541 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6601 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6661 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6721 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6781 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6841 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6901 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6961 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7021 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7081 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7141 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7201 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7261 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7321 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7381 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7441 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7501 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7561 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7621 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7681 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7741 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7801 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7861 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7921 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7981 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8041 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8101 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8161 ac
//