LOCUS       sequence-111438-        7796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for Bxb1-mediated recombination of the mCh-Dnmt3b1 cDNA into
            a MIN-tagged locus using Puromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7796)
  AUTHORS   Mulholland CB, Smets M, Schmidtmann E, Leidescher S, Markaki Y, 
            Hofweber M, Qin W, Manzo M, Kremmer E, Thanisch K, Bauer C, Rombaut 
            P, Herzog F, Leonhardt H, Bultmann S
  TITLE     A modular open platform for systematic functional studies under 
            physiological conditions.
  JOURNAL   Nucleic Acids Res. 2015 May 24. pii: gkv550.
  PUBMED    26007658
REFERENCE   2  (bases 1 to 7796)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2015 May 24. pii: gkv550."
COMMENT     Sequence Label: attB-mCh-Dnmt3b1-Poly(A)-PuroR
FEATURES             Location/Qualifiers
     source          1..7796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..561
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(97..120)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     494..513
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             585..1181
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(585..604)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1081..1101
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1188..2183
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(2236..2253)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2242..2466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2495..2532
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2535..3239
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2677..2695)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2945..2964
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      3285..3294
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3374..3385
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    5911..6045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5935..5954)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5989..6008
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        6181..6228
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6247..7050
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6310..6329)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6920..6939
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7179..7767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7259..7278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatgccagct cattcctccc actcatgatc tatagatccc ccgggctgca ggaattctac
       61 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
      121 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
      181 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
      241 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
      301 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
      361 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
      421 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
      481 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
      541 tcctcatctc cgggcctttc gaccgatcat caagcttgat cctcatgacc gagtacaagc
      601 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
      661 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
      721 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
      781 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
      841 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
      901 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
      961 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     1021 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     1081 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     1141 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggatccatg cccacgctac
     1201 tgcgggttta tatagacggt ccccacggga tggggaaaac caccaccacg caactgctgg
     1261 tggccctggg ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcggg
     1321 tgctgggggc ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg
     1381 gtgagatatc ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca
     1441 tgccttatgc cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct
     1501 cacatgcccc gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc
     1561 tgtgctaccc ggccgcgcgg taccttatgg gcagcatgac cccccaggcc gtgctggcgt
     1621 tcgtggccct catcccgccg accttgcccg gcaccaacat cgtgcttggg gcccttccgg
     1681 aggacagaca catcgaccgc ctggccaaac gccagcgccc cggcgagcgg ctggacctgg
     1741 ctatgctggc tgcgattcgc cgcgtttacg ggctacttgc caatacggtg cggtatctgc
     1801 agtgcggcgg gtcgtggcgg gaggactggg gacagctttc ggggacggcc gtgccgcccc
     1861 agggtgccga gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta
     1921 ccctgtttcg ggcccccgag ttgctggccc ccaacggcga cctgtataac gtgtttgcct
     1981 gggccttgga cgtcttggcc aaacgcctcc gttccatgca cgtctttatc ctggattacg
     2041 accaatcgcc cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga
     2101 cccacgtcac cacccccggc tccataccga cgatatgcga cctggcgcgc acgtttgccc
     2161 gggagatggg ggaggctaac tgagctctag agcggccagt gtcgcggtat cgatgatcta
     2221 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2281 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2341 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2401 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2461 ctatggcttc tgaggcggaa agaaccagca tcccggcttg tcgacgacgg cggtctccgt
     2521 cgtcaggatc atccgtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     2581 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     2641 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     2701 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     2761 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     2821 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     2881 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2941 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     3001 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     3061 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     3121 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     3181 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaaga
     3241 gcggcctgag gagcagagcc caggcgagca acagcgccgt ggacgccacc atgggcgatc
     3301 gcatgaaggg agacagcaga catctgaatg aagaagaggg tgccagcggg tatgaggagt
     3361 gcattatcgt taatgggaac ttcagtgacc agtcctcaga cacgaaggat gctccctcac
     3421 ccccagtctt ggaggcaatc tgcacagagc cagtctgcac accagagacc agaggccgca
     3481 ggtcaagctc ccggctgtct aagagggagg tctccagcct tctgaattac acgcaggaca
     3541 tgacaggaga tggagacaga gatgatgaag tagatgatgg gaatggctct gatattctaa
     3601 tgccaaagct cacccgtgag accaaggaca ccaggacgcg ctctgaaagc ccggctgtcc
     3661 gaacccgaca tagcaatggg acctccagct tggagaggca aagagcctcc cccagaatca
     3721 cccgaggtcg gcagggccgc caccatgtgc aggagtaccc tgtggagttt ccggctacca
     3781 ggtctcggag acgtcgagca tcgtcttcag caagcacgcc atggtcatcc cctgccagcg
     3841 tcgacttcat ggaagaagtg acacctaaga gcgtcagtac cccatcagtt gacttgagcc
     3901 aggatggaga tcaggagggt atggatacca cacaggtgga tgcagagagc agagatggag
     3961 acagcacaga gtatcaggat gataaagagt ttggaatagg tgacctcgtg tggggaaaga
     4021 tcaagggctt ctcctggtgg cctgccatgg tggtgtcctg gaaagccacc tccaagcgac
     4081 aggccatgcc cggaatgcgc tgggtacagt ggtttggtga tggcaagttt tctgagatct
     4141 ctgctgacaa actggtggct ctggggctgt tcagccagca ctttaatctg gctaccttca
     4201 ataagctggt ttcttatagg aaggccatgt accacactct ggagaaagcc agggttcgag
     4261 ctggcaagac cttctccagc agtcctggag agtcactgga ggaccagctg aagcccatgc
     4321 tggagtgggc ccacggtggc ttcaagccta ctgggatcga gggcctcaaa cccaacaaga
     4381 agcaaccagt ggttaataag tcgaaggtgc gtcgttcaga cagtaggaac ttagaaccca
     4441 ggagacgcga gaacaaaagt cgaagacgca caaccaatga ctctgctgct tctgagtccc
     4501 ccccacccaa gcgcctcaag acaaatagct atggcgggaa ggaccgaggg gaggatgagg
     4561 agagccgaga acggatggct tctgaagtca ccaacaacaa gggcaatctg gaagaccgct
     4621 gtttgtcctg tggaaagaag aaccctgtgt ccttccaccc cctctttgag ggtgggctct
     4681 gtcagagttg ccgggatcgc ttcctagagc tcttctacat gtatgatgag gacggctatc
     4741 agtcctactg caccgtgtgc tgtgagggcc gtgaactgct gctgtgcagt aacacaagct
     4801 gctgcagatg cttctgtgtg gagtgtctgg aggtgctggt gggcgcaggc acagctgagg
     4861 atgccaagct gcaggaaccc tggagctgct atatgtgcct ccctcagcgc tgccatgggg
     4921 tcctccgacg caggaaagat tggaacatgc gcctgcaaga cttcttcact actgatcctg
     4981 acctggaaga atttgagcca cccaagttgt acccagcaat tcctgcagcc aaaaggaggc
     5041 ccattagagt cctgtctctg tttgatggaa ttgcaacggg gtacttggtg ctcaaggagt
     5101 tgggtattaa agtggaaaag tacattgcct ccgaagtctg tgcagagtcc atcgctgtgg
     5161 gaactgttaa gcatgaaggc cagatcaaat atgtcaatga cgtccggaaa atcaccaaga
     5221 aaaatattga agagtggggc ccgttcgact tggtgattgg tggaagccca tgcaatgatc
     5281 tctctaacgt caatcctgcc cgcaaaggtt tatatgaggg cacaggaagg ctcttcttcg
     5341 agttttacca cttgctgaat tatacccgcc ccaaggaggg cgacaaccgt ccattcttct
     5401 ggatgttcga gaatgttgtg gccatgaaag tgaatgacaa gaaagacatc tcaagattcc
     5461 tggcatgtaa cccagtgatg atcgatgcca tcaaggtgtc tgctgctcac agggcccggt
     5521 acttctgggg taacctaccc ggaatgaaca ggcccgtgat ggcttcaaag aatgataagc
     5581 tcgagctgca ggactgcctg gagttcagta ggacagcaaa gttaaagaaa gtgcagacaa
     5641 taaccaccaa gtcgaactcc atcagacagg gcaaaaacca gcttttccct gtagtcatga
     5701 atggcaagga cgacgttttg tggtgcactg agctcgaaag gatcttcggc ttccctgctc
     5761 actacacgga cgtgtccaac atgggccgcg gcgcccgtca gaagctgctg ggcaggtcct
     5821 ggagtgtacc ggtcatcaga cacctgtttg cccccttgaa ggactacttt gcctgtgaat
     5881 aggcggccgc agttaacgaa ttctctagag gatccagaca tgataagata cattgatgag
     5941 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     6001 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     6061 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     6121 ctctacaaat gtggtatggc tgattatgat cctgcaagcc tcgtcgccgc ggtttattct
     6181 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     6241 taaaccatgg gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     6301 gtggagaggc tattcggcta tgactgggca caacagacga tcggctgctc tgatgccgcc
     6361 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     6421 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     6481 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     6541 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     6601 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     6661 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     6721 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     6781 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     6841 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     6901 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     6961 tgggctgacc gcttcctcgt gctttacggt atcgccgccc ccgattcgca gcgcatcgcc
     7021 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa taaagaccga
     7081 ccaagcgacg tctgagagct ccctggcgaa ttcggtacca ataaaagagc tttattttca
     7141 tgatctgtgt gttggttttt ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     7201 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     7261 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     7321 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7381 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7441 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7501 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7561 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     7621 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7681 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7741 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctag tgtgcg
//
