LOCUS       sequence-429732-        5538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses binding-deficient K299E/D333A mutant of 2xDNMT3A PWWP 
            domain fused to C-terminal part of IFP2.0 as negative control for 
            H3K36me2/3 detection in BiAD sensors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5538)
  AUTHORS   Kohler AR, Hausser J, Harsch A, Bernhardt S, Haussermann L, Brenner 
            LM, Lungu C, Olayioye MA, Bashtrykov P, Jeltsch A
  TITLE     Modular dual-color BiAD sensors for locus-specific readout of 
            epigenome modifications in single cells.
  JOURNAL   Cell Rep Methods. 2024 Mar 26:100739. doi: 
            10.1016/j.crmeth.2024.100739.
  PUBMED    38554702
REFERENCE   2  (bases 1 to 5538)
  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: "Cell Rep 
            Methods. 2024 Mar 26:100739. doi: 10.1016/j.crmeth.2024.100739."
COMMENT     Sequence Label: 2x3APWWP_K299E_D333A-IFP2.0C
FEATURES             Location/Qualifiers
     source          1..5538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        73..376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        377..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     531..551
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      619..628
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1066..1086
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1087..1096
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    2149..2214
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2338..2459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2375..2394)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2429..2448
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2466..2921)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2603..2624)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2815..2834
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2948..3052
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3054..3411
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3054..3245
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3077..3097)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3262..3397
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3446..4240
                     /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             3446..4240
                     /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(3500..3519)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4110..4129
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4428..4447)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4472..4519
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4848..5436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5337..5356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
       61 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      121 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      181 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      241 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      301 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      361 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      541 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      601 ccgctagcgc taccggtcgc caccatggag tacgaggacg gccggggctt tggcattggg
      661 gagctggtgt ggggggaact gcggggcttc tcctggtggc caggccgcat tgtgtcttgg
      721 tggatgacgg gccggagccg agcagctgaa ggcacccgct gggtcatgtg gttcggagcc
      781 ggcaaattct cagtggtgtg tgttgagaag ctgatgccgc tgagctcgtt ttgcagtgcg
      841 ttccaccagg ccacgtacaa caagcagccc atgtaccgca aagccatcta cgaggtcctg
      901 caggtggcca gcagccgcgc ggggaagctg ttcccggtgt gccacgacag cgatgagagt
      961 gacactgcca aggccgtgga ggtgcagaac aagcccatga ttgaatgggc cctggggggc
     1021 ttccagcctt ctggccctaa gggcctggag ccaccaactg gatcgcccaa gaaaaaacgc
     1081 aaggtggcca ccatggagta tgaggacggc cggggctttg gcattgggga gctggtgtgg
     1141 ggggaactgc ggggcttctc ctggtggcca ggccgcattg tgtcttggtg gatgacgggc
     1201 cggagccgag cagctgaagg cacccgctgg gtcatgtggt tcggagccgg caaattctca
     1261 gtggtgtgtg ttgagaagct gatgccgctg agctcgtttt gcagtgcgtt ccaccaggcc
     1321 acgtacaaca agcagcccat gtaccgcaaa gccatctacg aggtcctgca ggtggccagc
     1381 agccgcgcgg ggaagctgtt cccggtgtgc cacgacagcg atgagagtga cactgccaag
     1441 gccgtggagg tgcagaacaa gcccatgatt gaatgggccc tggggggctt ccagccttct
     1501 ggccctaagg gcctggagcc accaactgga tcgtcacaaa cctcgggttc atccacgtcg
     1561 gaaggccagg agggcacgga ctctattgga ccccacgctc tgagaaatgc aatgttcgct
     1621 ctggagagtg ctcctaatct ccgggcactg gctgaagtcg caacccaaac agtccgggaa
     1681 ctgtcaggtt tcgaccgggt gatgctgtac aagtttgcac cagacgcaac aggagaggtt
     1741 attgccgaag caaggcgcga gggcatgcag gcttacctcg ggcataggtt tcccgcatcc
     1801 accacccctg cacaagctag ggccctctac acaagacacc tgctccggct gaccgcagac
     1861 accagggctg cagcagtgcc cctcgacccc gtgctgaatc cccagacaaa tgctcctaca
     1921 cctctgggcg gagctgtcct cagagctaca tccccaatgc acatgcagta cctgaggaat
     1981 atgggagtgg gctcctccct gagcgtcagc gtcgtggtcg gcggccagct gtggggactg
     2041 attgtctgcc accatcagac accctacgtg ctgccacccg atctgcggac caccctggag
     2101 tatctgggga ggctcctgtc cctgcaggtg cagaggaaag aagcctaatc cggactcaga
     2161 tctcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccaccgga
     2221 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2281 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2341 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2401 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     2461 cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2521 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2581 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2641 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2701 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2761 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2821 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2881 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     2941 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3001 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     3061 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3121 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3241 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3301 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3361 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3421 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3481 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3541 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3601 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3661 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3721 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3781 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3841 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3901 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3961 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4021 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4081 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4141 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4201 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4261 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4321 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4381 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4441 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4501 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4561 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4621 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4681 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4741 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4801 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4861 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4921 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4981 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5041 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5101 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5161 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5221 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5281 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5341 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5401 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5461 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5521 tctgtggata accgtatt
//
