LOCUS       sequence-111443-        7133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for Bxb1-mediated recombination of the GFP-Dnmt3b1 cDNA into
            a MIN-tagged locus using Neomycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7133)
  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 7133)
  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-GFP-Dnmt3b1-Poly(A)-NeoR
FEATURES             Location/Qualifiers
     source          1..7133
                     /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             579..1373
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             579..1373
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1561..1580)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1605..1652
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1823..1860
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(1863..1865,1866..2576)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1863..1865,1866..2576)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1905..1926)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2166..2185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2513..2534
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2622..2631
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2711..2722
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    5248..5382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5272..5291)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5326..5345
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        5518..5565
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5584..6387
                     /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"
     rep_origin      complement(6516..7104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6596..6615)
                     /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 caagcttaat gattgaacaa gatggattgc
      601 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
      661 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
      721 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
      781 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
      841 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
      901 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
      961 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     1021 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     1081 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     1141 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     1201 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     1261 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     1321 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     1381 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     1441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     1561 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     1621 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     1681 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     1741 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     1801 ggcaagctta catcgagatc ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat
     1861 ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1921 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1981 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2041 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     2101 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2161 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2221 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2281 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2341 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2401 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2461 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2521 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagagcg
     2581 gcctgaggag cagagcccag gcgagcaaca gcgccgtgga cgccaccatg ggcgatcgca
     2641 tgaagggaga cagcagacat ctgaatgaag aagagggtgc cagcgggtat gaggagtgca
     2701 ttatcgttaa tgggaacttc agtgaccagt cctcagacac gaaggatgct ccctcacccc
     2761 cagtcttgga ggcaatctgc acagagccag tctgcacacc agagaccaga ggccgcaggt
     2821 caagctcccg gctgtctaag agggaggtct ccagccttct gaattacacg caggacatga
     2881 caggagatgg agacagagat gatgaagtag atgatgggaa tggctctgat attctaatgc
     2941 caaagctcac ccgtgagacc aaggacacca ggacgcgctc tgaaagcccg gctgtccgaa
     3001 cccgacatag caatgggacc tccagcttgg agaggcaaag agcctccccc agaatcaccc
     3061 gaggtcggca gggccgccac catgtgcagg agtaccctgt ggagtttccg gctaccaggt
     3121 ctcggagacg tcgagcatcg tcttcagcaa gcacgccatg gtcatcccct gccagcgtcg
     3181 acttcatgga agaagtgaca cctaagagcg tcagtacccc atcagttgac ttgagccagg
     3241 atggagatca ggagggtatg gataccacac aggtggatgc agagagcaga gatggagaca
     3301 gcacagagta tcaggatgat aaagagtttg gaataggtga cctcgtgtgg ggaaagatca
     3361 agggcttctc ctggtggcct gccatggtgg tgtcctggaa agccacctcc aagcgacagg
     3421 ccatgcccgg aatgcgctgg gtacagtggt ttggtgatgg caagttttct gagatctctg
     3481 ctgacaaact ggtggctctg gggctgttca gccagcactt taatctggct accttcaata
     3541 agctggtttc ttataggaag gccatgtacc acactctgga gaaagccagg gttcgagctg
     3601 gcaagacctt ctccagcagt cctggagagt cactggagga ccagctgaag cccatgctgg
     3661 agtgggccca cggtggcttc aagcctactg ggatcgaggg cctcaaaccc aacaagaagc
     3721 aaccagtggt taataagtcg aaggtgcgtc gttcagacag taggaactta gaacccagga
     3781 gacgcgagaa caaaagtcga agacgcacaa ccaatgactc tgctgcttct gagtcccccc
     3841 cacccaagcg cctcaagaca aatagctatg gcgggaagga ccgaggggag gatgaggaga
     3901 gccgagaacg gatggcttct gaagtcacca acaacaaggg caatctggaa gaccgctgtt
     3961 tgtcctgtgg aaagaagaac cctgtgtcct tccaccccct ctttgagggt gggctctgtc
     4021 agagttgccg ggatcgcttc ctagagctct tctacatgta tgatgaggac ggctatcagt
     4081 cctactgcac cgtgtgctgt gagggccgtg aactgctgct gtgcagtaac acaagctgct
     4141 gcagatgctt ctgtgtggag tgtctggagg tgctggtggg cgcaggcaca gctgaggatg
     4201 ccaagctgca ggaaccctgg agctgctata tgtgcctccc tcagcgctgc catggggtcc
     4261 tccgacgcag gaaagattgg aacatgcgcc tgcaagactt cttcactact gatcctgacc
     4321 tggaagaatt tgagccaccc aagttgtacc cagcaattcc tgcagccaaa aggaggccca
     4381 ttagagtcct gtctctgttt gatggaattg caacggggta cttggtgctc aaggagttgg
     4441 gtattaaagt ggaaaagtac attgcctccg aagtctgtgc agagtccatc gctgtgggaa
     4501 ctgttaagca tgaaggccag atcaaatatg tcaatgacgt ccggaaaatc accaagaaaa
     4561 atattgaaga gtggggcccg ttcgacttgg tgattggtgg aagcccatgc aatgatctct
     4621 ctaacgtcaa tcctgcccgc aaaggtttat atgagggcac aggaaggctc ttcttcgagt
     4681 tttaccactt gctgaattat acccgcccca aggagggcga caaccgtcca ttcttctgga
     4741 tgttcgagaa tgttgtggcc atgaaagtga atgacaagaa agacatctca agattcctgg
     4801 catgtaaccc agtgatgatc gatgccatca aggtgtctgc tgctcacagg gcccggtact
     4861 tctggggtaa cctacccgga atgaacaggc ccgtgatggc ttcaaagaat gataagctcg
     4921 agctgcagga ctgcctggag ttcagtagga cagcaaagtt aaagaaagtg cagacaataa
     4981 ccaccaagtc gaactccatc agacagggca aaaaccagct tttccctgta gtcatgaatg
     5041 gcaaggacga cgttttgtgg tgcactgagc tcgaaaggat cttcggcttc cctgctcact
     5101 acacggacgt gtccaacatg ggccgcggcg cccgtcagaa gctgctgggc aggtcctgga
     5161 gtgtaccggt catcagacac ctgtttgccc ccttgaagga ctactttgcc tgtgaatagg
     5221 cggccgcagt taacgaattc tctagaggat ccagacatga taagatacat tgatgagttt
     5281 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     5341 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     5401 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     5461 tacaaatgtg gtatggctga ttatgatcct gcaagcctcg tcgccgcggt ttattctgtt
     5521 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     5581 accatgggat cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     5641 gagaggctat tcggctatga ctgggcacaa cagacgatcg gctgctctga tgccgccgtg
     5701 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     5761 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     5821 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5881 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5941 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     6001 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     6061 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     6121 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     6181 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     6241 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     6301 gctgaccgct tcctcgtgct ttacggtatc gccgcccccg attcgcagcg catcgccttc
     6361 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaataa agaccgacca
     6421 agcgacgtct gagagctccc tggcgaattc ggtaccaata aaagagcttt attttcatga
     6481 tctgtgtgtt ggtttttggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6541 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6601 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6661 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6721 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6781 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6841 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6901 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6961 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7021 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7081 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctagtgt gcg
//
