LOCUS       sequence-370471-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell line generation via recombination-mediated cassette exchange 
            (RMCE) and stable expression of MBD1_MBD(R22A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  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 7113)
  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: pv6_MBD1_MBD-R22A
FEATURES             Location/Qualifiers
     source          1..7113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     890..912
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     974..993
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1034..1078
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1076..1096
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1385..1405
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1433..1435,1436..2149)
                     /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(1433..1435,1436..2146)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1475..1496)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1736..1755)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2083..2104
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2272..2734
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2328..2345)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2555..2574
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2748..3347
                     /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(2748..2767)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3244..3264
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3516..3637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3553..3572)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3607..3626
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3671..3704)
                     /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(3738..3754)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3738..3754)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3751..3773)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3762..3778
                     /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(3786..3792,3793..3810,3811..3816))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3831..3852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3969..3986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4140..4728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4220..4239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4899..5690,5691..5759))
                     /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     5522..5541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5760..5864)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5932..5950
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5988..6010)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6110..6129
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6323..6345
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6337..6354
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6338..6354
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6368..6401
                     /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        6406..6785
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          join(7063..7113,1..966)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
       61 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
      121 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
      181 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
      241 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
      301 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
      361 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
      421 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
      481 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
      541 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
      601 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
      661 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
      721 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
      781 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
      841 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
      901 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
      961 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1021 tagcatggct ggtggcctga atgacatctt tgaggcccag aagatcgagt ggcatgagaa
     1081 cctgtacttc cagggagtcg acgaagagtc tggttctgtt tctggtggtg atgctgagtc
     1141 ctggcaggac tgcccagccc tgggccctgg ctggaaacgc cgagagtcct ttgcaaagtc
     1201 aggggcctcg tttggacgct cagacatcta ttaccagagc cccacaggag aaaagatccg
     1261 aagcaaagtt gagctgactc ggtacttggg ccctgcatgt gatcttaccc tgtttgactt
     1321 cagacaaggc accttatgcc atcccatcgg tggcggaggc tctggaggtg gaggctccgc
     1381 accccccaaa aagaaacgca aggttggata ccgggatcca ccggtcgcca ccgtgagcaa
     1441 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1501 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1561 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1621 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1681 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1741 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1801 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1861 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     1921 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     1981 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2041 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2101 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcgagctcga
     2161 cagatctagg agcggttaac gaggtagcgg ccgccgaagc cgcttggaat aaggccggtg
     2221 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     2281 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     2341 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     2401 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     2461 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     2521 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     2581 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     2641 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     2701 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg accgagtaca
     2761 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     2821 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     2881 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     2941 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     3001 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3061 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3121 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3181 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3241 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3301 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     3361 acccgcagcg cccgaccgaa aggagcgcag tgctacgaga tttcgattcc accgccgcct
     3421 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3481 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3541 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3601 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtggatcc
     3661 ggccggcccc ataacttcgt atagtataca ttatacgaag ttatcccctg caggcatgca
     3721 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3781 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3841 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3901 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3961 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4021 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4081 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4141 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4201 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4261 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4321 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4381 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4441 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4501 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4561 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4621 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4681 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4741 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4801 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4861 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4921 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4981 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5041 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5101 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5161 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5221 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5281 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5341 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5401 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5461 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5521 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5581 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5641 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5701 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5761 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5821 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5881 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5941 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     6001 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     6061 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     6121 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     6181 aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     6241 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     6301 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc
     6361 tcgagccata acttcgtata atgtatacta tacgaagtta tatcggacat tgattattga
     6421 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     6481 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     6541 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     6601 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     6661 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     6721 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     6781 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
     6841 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
     6901 ggggggsgcg cgccrggsgg ggsggggsgg ggsgrggggs ggggsggggs grggcggrga
     6961 ggkgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg
     7021 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc
     7081 cttcgccccg tgccccgctc cgccgccgcc tcg
//
