LOCUS       sequence-111442-        6944 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid for Bxb1-mediated recombination of the GFP-Dnmt3b6 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 6944)
  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 6944)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Dnmt3b6-Poly(A)-NeoR
FEATURES             Location/Qualifiers
     source          1..6944
                     /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    5059..5193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5083..5102)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5137..5156
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        5329..5376
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5395..6198
                     /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(6327..6915)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6407..6426)
                     /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 atgaacagga tcttcggctt ccctgctcac tacacggacg
     4921 tgtccaacat gggccgcggc gcccgtcaga agctgctggg caggtcctgg agtgtaccgg
     4981 tcatcagaca cctgtttgcc cccttgaagg actactttgc ctgtgaatag gcggccgcag
     5041 ttaacgaatt ctctagagga tccagacatg ataagataca ttgatgagtt tggacaaacc
     5101 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     5161 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     5221 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     5281 ggtatggctg attatgatcc tgcaagcctc gtcgccgcgg tttattctgt tgacaattaa
     5341 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggga
     5401 tcggccattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     5461 ttcggctatg actgggcaca acagacgatc ggctgctctg atgccgccgt gttccggctg
     5521 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     5581 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     5641 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     5701 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     5761 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     5821 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     5881 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     5941 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     6001 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     6061 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     6121 ttcctcgtgc tttacggtat cgccgccccc gattcgcagc gcatcgcctt ctatcgcctt
     6181 cttgacgagt tcttctgagc gggactctgg ggttcgaata aagaccgacc aagcgacgtc
     6241 tgagagctcc ctggcgaatt cggtaccaat aaaagagctt tattttcatg atctgtgtgt
     6301 tggtttttgg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6361 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6421 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6481 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6541 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6601 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6661 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6721 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6781 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6841 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6901 aaaaaaggat ctcaagaaga tcctttgatc ttttctagtg tgcg
//
