LOCUS       sequence-370520-        7173 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell line generation via recombination-mediated cassette exchange 
            (RMCE) and stable expression of MECP2_MBD(T158M).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7173)
  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 7173)
  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_MECP2_MBD-T158M
FEATURES             Location/Qualifiers
     source          1..7173
                     /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"
     intron          1104..2120
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2044..2066
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2128..2147
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2188..2232
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2230..2250
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2599..2619
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2647..2649,2650..3363)
                     /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(2647..2649,2650..3360)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2689..2710)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2950..2969)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3297..3318
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3486..3948
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3542..3559)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3769..3788
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3962..4561
                     /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(3962..3981)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4458..4478
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4730..4851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4767..4786)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4821..4840
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4885..4918)
                     /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(4952..4968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4952..4968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4965..4987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4976..4992
                     /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(5000..5006,5007..5024,5025..5030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5045..5066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5183..5200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5354..5942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5434..5453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6113..6904,6905..6973))
                     /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     6736..6755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6974..7078)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7146..7164
                     /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 gggggggsgc gcgccrggsg
      961 gggsggggsg gggsgrgggg sggggsgggg sgaggcggag aggtgcggcg gcagccaatc
     1021 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     1081 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1141 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1201 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     1261 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     1321 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     1381 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     1441 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     1501 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     1561 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     1621 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     1681 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     1741 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     1801 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     1861 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     1921 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     1981 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     2041 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     2101 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     2161 ttggcaaaga attctagcat ggctggtggc ctgaatgaca tctttgaggc ccagaagatc
     2221 gagtggcatg agaacctgta cttccaggga gtcgacgaag agtctggttc tgtttctggt
     2281 ggtgataaac agcggcgctc cattatccgt gaccggggac ctatgtatga tgaccccacc
     2341 ttgcctgaag gttggacacg aaagcttaaa caaaggaagt ctggccgatc tgctggaaag
     2401 tatgatgtat atttgatcaa tccccaggga aaagcttttc gctctaaagt agaattgatt
     2461 gcatactttg aaaaggtggg agacacctcc ttggacccta atgattttga cttcatggta
     2521 actgggagag ggagcccctc caggagagag cagaaaccaa tcggtggcgg aggctctgga
     2581 ggtggaggct ccgcaccccc caaaaagaaa cgcaaggttg gataccggga tccaccggtc
     2641 gccaccgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2701 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2761 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2821 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2881 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2941 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3001 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3061 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3121 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3181 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3241 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3301 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3361 taaagcgagc tcgacagatc taggagcggt taacgaggta gcggccgccg aagccgcttg
     3421 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
     3481 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
     3541 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa
     3601 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
     3661 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
     3721 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
     3781 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
     3841 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
     3901 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac
     3961 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     4021 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     4081 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     4141 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     4201 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     4261 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     4321 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     4381 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     4441 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     4501 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     4561 acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcagtgctac gagatttcga
     4621 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4681 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4741 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4801 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4861 tataccgtgg atccggccgg ccccataact tcgtatagta tacattatac gaagttatcc
     4921 cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4981 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5041 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5101 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5161 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5221 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5281 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5341 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5401 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5461 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5521 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5581 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5641 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5701 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5761 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5821 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5881 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5941 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6001 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6061 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6121 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6181 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6241 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6301 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6361 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6421 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6481 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6541 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6601 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6661 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6721 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6781 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6841 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6901 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6961 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7021 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7081 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     7141 ataaaaatag gcgtatcacg aggccctttc gtc
//
