LOCUS       sequence-323735-        8019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double-Floxed Inverted Open reading frame for mouseMeCP2(alpha) 
            carrying R106W mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8019)
  AUTHORS   Piccolo FM, Liu Z, Dong P, Hsu CL, Stoyanova EI, Rao A, Tjian R, 
            Heintz N
  TITLE     MeCP2 nuclear dynamics in live neurons results from low and high 
            affinity chromatin interactions.
  JOURNAL   Elife. 2019 Dec 23;8. pii: 51449. doi: 10.7554/eLife.51449.
  PUBMED    31868585
REFERENCE   2  (bases 1 to 8019)
  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: "Elife."; 
            date: "2019-12-23"; volume: "8. pii: 51449. doi"; pages: " 
            10.7554/eLife.51449"
COMMENT     Sequence Label: EF1a_DIO_R106W-HaloTag
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        232..1410
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          463..1401
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1358..1378
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1441..1474)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1525..1558)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1587..2471)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(2832..2852)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     protein_bind    4005..4038
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4089..4122
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4147..4735
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4200..4220)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4618..4629)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4767..5243
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4966..4985)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5283..5423
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5283..5412
                     /label=AAV2 ITR
     rep_origin      5498..5953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5585..5604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5795..5816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5970..5989)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6089..6111
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6149..6167)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6235..6339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6340..6408,6409..7200)
                     /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     complement(6558..6577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7371..7959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7860..7879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
      181 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      241 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      301 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      361 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      421 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      481 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      541 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      601 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      661 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      721 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      781 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      841 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
      901 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
      961 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1021 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1081 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1141 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1201 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1261 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1321 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1381 agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt cgactccgga
     1441 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
     1501 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
     1561 cgcgccttac ttgtacacat gtcctaaccg gaaatctcga gggtagacag ccagcgcgcg
     1621 atctcgctgc cgatcaggtc cgggttgtct tcttgcagca gattcagacc cgggccgatg
     1681 tccacagcct tgcagttagg caggcttttg gccaggcgag cggcttcggc cggtgggatc
     1741 agaacgcctg gggtgcccca gaacagcagc ttcgggacag gggactggtg cagccagtcc
     1801 atgtattctt cgaccagcgc gacgatgttc gctggctcac cggcgattgg cagctcgttt
     1861 gggaagcgcc acagtggctc gcggtcaaca ggattcagga acggctcgcg gtaatggtcc
     1921 atctcgactt cagtcagcgg gcggacgaca cccatcggca gcgtaccctc gataaaaacg
     1981 ttctgatcga tgatcagctt gcggccgacg tcggtggtgc ggaaggcctg gaaggtctcg
     2041 cgggcaaatt ctggccattc gtcccaggtc gggatagggc ggatgaactc cataaatgca
     2101 atacctttga cgcgctctgg attgcgcttg gcccagtgga aacccagagc ggagccccag
     2161 tcgtgaatga ccaggacgac ctcttccaga cccagggctt cgatgaaggc atccatgaag
     2221 cggacgtggt cgtcgaagaa ataacccagg tctggtttgt cggatttgcc cataccgatc
     2281 aggtctggag caatgcagcg atgggtcggt gcaacatgcg ggatgatgtt gcgccacacg
     2341 taggaggagg tcgggttacc gtgcaggaac agcacagggg tgccatcgcg cggaccaaca
     2401 tcgacgtagt gcatgcgctc gcccaggact tccacataat gggggtcgaa tggaaagcca
     2461 gtaccgattt cggtggcgac cggtggatcc ttacagctaa ctctctcggt cacgggcgtc
     2521 cggctgtcca caggctcctc tctgtttggc cttggcatgg aagatgaaac aatgtctttg
     2581 cgctctccct cccctcggtg tttgtacttt tctgcaactg tggtagtggt ggcgaccata
     2641 ggctgagtct tagctggctc cttggggcag ccatcgcttt ccagtgagcc tcctcggggc
     2701 atcttctctt ctttgcagat gctgctgctc aagtcctgag gctcaggggg gctgatgggg
     2761 tcctcagagc tctcaggctc aggtgggggt ggggatggga gcagtggcat gggggccttt
     2821 gtggactctg agtgatggtg gtgatgatgg tgctccttct taggtgggga ggaggcactg
     2881 ctgctgcgcc ccttggggct gctctccttg cttttacgcc cagggctctt gcaggtcttc
     2941 agtcccttcc cgcttttctc accaagggtg gacaccagca ggggcttcac cacttccttg
     3001 acctcgatgc tgaccgtctc ccgggtcttg cgcttcttga tggggagcac agtctcatgc
     3061 acagaccgta tggaagactc cttcacggct ttctttttgg cctcagctgc agcagctgcc
     3121 accacactcc caggctttct accccgtttc ttaggaatgg cctgggggtc agcttcagct
     3181 tttcgctttc tgccagggcg tttgatcacc atgacctggg cagatgtggt agccccacct
     3241 ccctcaccct tacccccagg cgatgcttgg aaaggcatct tgacaacaag tttcccaggg
     3301 ctcttctcca ggaccctttt cacctgaaca ccttctgatg ctgctgcctt tggtctccca
     3361 gtgccgctcc ctttggggcg tccccgaccc ctgccagttc ctggagcttt gggagatttg
     3421 ggcttcttag gtggtttctg ctctctcctg gaggggctcc ctctcccagt taccgtgaag
     3481 tcaaaatcat tagggtccaa ggaggtgtct cccacctttt caaagtatgc aatcaattct
     3541 actttagagc gaaaagcttt tccctgggga ttgatcaaat atacatcata ctttccagca
     3601 gatcggccag acttcctttg tttaagcttc catgtccaac cttcaggcaa ggtggggtca
     3661 tcatacatag gtccccggtc acggataatg gagcgccgct gtttggggga agccgaggct
     3721 tctggcactg ctggggcaga gcctgagctt tctgatgttt ctgctttgcc tgcctctgct
     3781 ggctctgcag aatggtgggc tgaaggttgt agtggctcat gcttgccttc tttgtcctcc
     3841 ttcttgtctt tcttcgcctt cttaaacttc agtggcttgt ctctgaggcc ctggagatcc
     3901 tggtcttctg acttttcctc cagtctctcc tcctcgcctc ctcctcctcc tccgctcggc
     3961 gcggcggcgg cggcggcggt ggcggcagcg gcggccatgc tagcataact tcgtataaag
     4021 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg
     4081 tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt
     4141 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4201 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4261 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4321 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4381 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4441 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4501 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     4561 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4621 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4681 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     4741 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     4801 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     4861 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     4921 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     4981 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     5041 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     5101 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     5161 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     5221 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     5281 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5341 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5401 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5461 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5521 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5581 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5641 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5701 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5761 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5821 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5881 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5941 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6001 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6061 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6121 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6181 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6241 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6301 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6361 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6421 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6481 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6541 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6601 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6661 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6721 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6781 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6841 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6901 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6961 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7021 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     7081 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7141 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7201 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7501 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7621 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7681 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7741 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7801 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7861 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7921 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7981 ttacggttcc tggccttttg ctggcctttt gctcacatg
//
