LOCUS       sequence-429621-        6456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses binding-deficient R44Q mutant of MBD1 MBD domain fused to 
            C-terminal part of IFP2.0 as negative control for 5mC detection in 
            BiAD sensors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6456)
  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 6456)
  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: MBD_R44Q-IFP2.0C
FEATURES             Location/Qualifiers
     source          1..6456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     866..885
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        866..884
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             980..1045
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(2012..2030)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2013..2030)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2050..2067)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2056..2280
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2326..2754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2413..2432)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2623..2644
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2763..2783)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2768..3097
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2948..3083
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3010..3029
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3164..3958
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3218..3237)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3828..3847
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4132..4253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4169..4188)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4223..4242
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4302..4318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4302..4318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4315..4337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4326..4342
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4350..4356,4357..4374,4375..4380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4395..4416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4533..4550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4704..5292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4784..4803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5463..6254,6255..6323))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6086..6105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6324..6428)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac gataaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
      841 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagcttctc
      901 gggcccaata atcattttat tttgactgat agtgacctgt tcgttgcaac aaattgatga
      961 gcaatgcttt tttataatgg actataagga ccacgacgga gactacaagg atcatgatat
     1021 tgattacaaa gacgatgacg ataagatggc tgaggactgg ctggactgcc cggccctggg
     1081 ccctggctgg aagcgccgcg aagtctttcg caagtcaggg gccacctgtg gacgctcaga
     1141 cacctattac cagagcccca caggagacag gatacagagc aaagttgagc tgactcgata
     1201 cctgggccct gcgtgtgatc tcaccctctt cgacttcaaa caaggcatct tgtgctatcc
     1261 agcccccaag gcccatcccg tggcggttgc cagcaagaag cgaaagaagc cttcaaggcc
     1321 agccaagact cggaaacgtc aggttggacc ccagagtggt gaggtcagga aggaggcccc
     1381 gatcgatggt ggcggtggct ctggaggtgg tgggtcctcc ggagactcta ttggacccca
     1441 cgctctgaga aatgcaatgt tcgctctgga gagtgctcct aatctccggg cactggctga
     1501 agtcgcaacc caaacagtcc gggaactgtc aggtttcgac cgggtgatgc tgtacaagtt
     1561 tgcaccagac gcaacaggag aggttattgc cgaagcaagg cgcgagggca tgcaggctta
     1621 cctcgggcat aggtttcccg catccaccac ccctgcacaa gctagggccc tctacacaag
     1681 acacctgctc cggctgaccg cagacaccag ggctgcagca gtgcccctcg accccgtgct
     1741 gaatccccag acaaatgctc ctacacctct gggcggagct gtcctcagag ctacatcccc
     1801 aatgcacatg cagtacctga ggaatatggg agtgggctcc tccctgagcg tcagcgtcgt
     1861 ggtcggcggc cagctgtggg gactgattgt ctgccaccat cagacaccct acgtgctgcc
     1921 acccgatctg cggaccaccc tggagtatct ggggaggctc ctgtccctgc aggtgcagag
     1981 gaaagaagcc taataatcta gagggcccta ttctatagtg tcacctaaat gctagagctc
     2041 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2101 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2161 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2221 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2281 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2341 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2401 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2461 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2521 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2581 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2641 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2701 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     2761 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2821 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2941 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3001 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3061 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3121 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3181 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3241 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3301 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     3361 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3421 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3481 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3541 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3601 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3661 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     3721 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3781 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3841 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3901 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3961 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4021 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4081 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4141 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4201 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4261 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4321 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4381 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4441 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4501 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4561 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4621 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4681 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4741 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4801 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4861 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4921 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4981 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5041 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5101 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5161 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5221 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5281 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5341 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5401 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5461 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5521 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5581 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5641 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5701 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5761 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5821 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5881 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5941 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6001 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6061 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6121 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6181 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6241 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6301 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6361 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6421 gttccgcgca catttccccg aaaagtgcca cctgac
//
