LOCUS       sequence-370524-        7186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell line generation via recombination-mediated cassette exchange 
            (RMCE) and stable expression of MBD1_MBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7186)
  AUTHORS   Villasenor R, Pfaendler R, Ambrosi C, Butz S, Giuliani S, Bryan E, 
            Sheahan TW, Gable AL, Schmolka N, Manzo M, Wirz J, Feller C, von 
            Mering C, Aebersold R, Voigt P, Baubec T
  TITLE     ChromID identifies the protein interactome at chromatin marks.
  JOURNAL   Nat Biotechnol. 2020 Mar 2. pii: 10.1038/s41587-020-0434-2. doi: 
            10.1038/s41587-020-0434-2.
  PUBMED    32123383
REFERENCE   2  (bases 1 to 7186)
  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: "Nat 
            Biotechnol. 2020 Mar 2. pii: 10.1038/s41587-020-0434-2. doi: 
            10.1038/s41587-020-0434-2."
COMMENT     Sequence Label: pv7_MBD1_MBD_noEGFP
FEATURES             Location/Qualifiers
     source          1..7186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    409..442
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     enhancer        447..826
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        828..1104
                     /label=chicken beta-actin promoter
     intron          1105..2121
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2045..2067
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2129..2148
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2189..2233
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2231..2251
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2540..2560
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2591..2647
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(2660..2662,2663..3376)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2660..2662,2663..3373)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2702..2723)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2963..2982)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3310..3331
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3499..3961
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3555..3572)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3782..3801
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3975..4574
                     /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(3975..3994)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4471..4491
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4743..4864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4780..4799)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4834..4853
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4898..4931)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     primer_bind     complement(4965..4981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4965..4981)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4978..5000)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4989..5005
                     /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(5013..5019,5020..5037,5038..5043))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5058..5079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5196..5213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5367..5955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5447..5466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6126..6917,6918..6986))
                     /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     6749..6768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6987..7091)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7159..7177
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctcgagccat aacttcgtat
      421 aatgtatact atacgaagtt atatcggaca ttgattattg actagttatt aatagtaatc
      481 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      541 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      601 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      661 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      721 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      781 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      841 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      901 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggnsg cgcgccrggs
      961 ggggsggggs ggggsgrggg gsggggsggg gcgaggcgga gaggtgcggc ggcagccaat
     1021 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
     1081 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     1141 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     1201 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
     1261 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
     1321 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
     1381 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
     1441 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     1501 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     1561 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     1621 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     1681 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
     1741 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     1801 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     1861 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     1921 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1981 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     2041 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     2101 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     2161 tttggcaaag aattctagca tggctggtgg cctgaatgac atctttgagg cccagaagat
     2221 cgagtggcat gagaacctgt acttccaggg agtcgacgaa gagtctggtt ctgtttctgg
     2281 tggtgatgct gagtcctggc aggactgccc agccctgggc cctggctgga aacgccgaga
     2341 gtcctttcga aagtcagggg cctcgtttgg acgctcagac atctattacc agagccccac
     2401 aggagaaaag atccgaagca aagttgagct gactcggtac ttgggccctg catgtgatct
     2461 taccctgttt gacttcagac aaggcacctt atgccatccc atcggtggcg gaggctctgg
     2521 aggtggaggc tccgcacccc ccaaaaagaa acgcaaggtt ggataccggg atccatcgcg
     2581 aggaagcgga gctactaact tcagcctgct gaagcaggct ggagatgtgg aggagaaccc
     2641 tggacctccg gtcgccaccg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2701 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2761 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2821 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2881 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2941 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3001 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3061 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3121 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3181 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3241 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     3301 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3361 cgagctgtac aagtaaagcg agctcgacag atctaggagc ggttaacgag gtagcggccg
     3421 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     3481 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
     3541 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     3601 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     3661 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     3721 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     3781 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     3841 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa
     3901 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata
     3961 atatggccac aaccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     4021 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     4081 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     4141 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     4201 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     4261 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     4321 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     4381 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     4441 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     4501 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     4561 agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcagtgc
     4621 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4681 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4741 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4801 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4861 cttatcatgt ctgtataccg tggatccggc cggccccata acttcgtata gtatacatta
     4921 tacgaagtta tcccctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     4981 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5041 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5101 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5161 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5221 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5281 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5341 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5401 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5461 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5521 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5581 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5641 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5701 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5761 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5821 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5881 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5941 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6001 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6061 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6121 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6181 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6241 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6301 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6361 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6421 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6481 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6541 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6601 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6661 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6721 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6781 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6841 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6901 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6961 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7021 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7081 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7141 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
