LOCUS       sequence-323737-        8019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double-Floxed Inverted Open reading frame for mouseMeCP2(alpha) 
            carrying R133C 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_R133C-HaloTag
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /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(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1586..2470)
                     /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(2831..2851)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     protein_bind    4004..4037
                     /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    4088..4121
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4146..4734
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4199..4219)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4617..4628)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4766..5242
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4965..4984)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5282..5422
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5282..5411
                     /label=AAV2 ITR
     rep_origin      5497..5952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5584..5603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5794..5815
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5969..5988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6088..6110
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6148..6166)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6234..6338
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6339..6407,6408..7199)
                     /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(6557..6576)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7370..7958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7859..7878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttact tgtacacatg tcctaaccgg aaatctcgag ggtagacagc cagcgcgcga
     1621 tctcgctgcc gatcaggtcc gggttgtctt cttgcagcag attcagaccc gggccgatgt
     1681 ccacagcctt gcagttaggc aggcttttgg ccaggcgagc ggcttcggcc ggtgggatca
     1741 gaacgcctgg ggtgccccag aacagcagct tcgggacagg ggactggtgc agccagtcca
     1801 tgtattcttc gaccagcgcg acgatgttcg ctggctcacc ggcgattggc agctcgtttg
     1861 ggaagcgcca cagtggctcg cggtcaacag gattcaggaa cggctcgcgg taatggtcca
     1921 tctcgacttc agtcagcggg cggacgacac ccatcggcag cgtaccctcg ataaaaacgt
     1981 tctgatcgat gatcagcttg cggccgacgt cggtggtgcg gaaggcctgg aaggtctcgc
     2041 gggcaaattc tggccattcg tcccaggtcg ggatagggcg gatgaactcc ataaatgcaa
     2101 tacctttgac gcgctctgga ttgcgcttgg cccagtggaa acccagagcg gagccccagt
     2161 cgtgaatgac caggacgacc tcttccagac ccagggcttc gatgaaggca tccatgaagc
     2221 ggacgtggtc gtcgaagaaa taacccaggt ctggtttgtc ggatttgccc ataccgatca
     2281 ggtctggagc aatgcagcga tgggtcggtg caacatgcgg gatgatgttg cgccacacgt
     2341 aggaggaggt cgggttaccg tgcaggaaca gcacaggggt gccatcgcgc ggaccaacat
     2401 cgacgtagtg catgcgctcg cccaggactt ccacataatg ggggtcgaat ggaaagccag
     2461 taccgatttc ggtggcgacc ggtggatcct tacagctaac tctctcggtc acgggcgtcc
     2521 ggctgtccac aggctcctct ctgtttggcc ttggcatgga agatgaaaca atgtctttgc
     2581 gctctccctc ccctcggtgt ttgtactttt ctgcaactgt ggtagtggtg gcgaccatag
     2641 gctgagtctt agctggctcc ttggggcagc catcgctttc cagtgagcct cctcggggca
     2701 tcttctcttc tttgcagatg ctgctgctca agtcctgagg ctcagggggg ctgatggggt
     2761 cctcagagct ctcaggctca ggtgggggtg gggatgggag cagtggcatg ggggcctttg
     2821 tggactctga gtgatggtgg tgatgatggt gctccttctt aggtggggag gaggcactgc
     2881 tgctgcgccc cttggggctg ctctccttgc ttttacgccc agggctcttg caggtcttca
     2941 gtcccttccc gcttttctca ccaagggtgg acaccagcag gggcttcacc acttccttga
     3001 cctcgatgct gaccgtctcc cgggtcttgc gcttcttgat ggggagcaca gtctcatgca
     3061 cagaccgtat ggaagactcc ttcacggctt tctttttggc ctcagctgca gcagctgcca
     3121 ccacactccc aggctttcta ccccgtttct taggaatggc ctgggggtca gcttcagctt
     3181 ttcgctttct gccagggcgt ttgatcacca tgacctgggc agatgtggta gccccacctc
     3241 cctcaccctt acccccaggc gatgcttgga aaggcatctt gacaacaagt ttcccagggc
     3301 tcttctccag gacccttttc acctgaacac cttctgatgc tgctgccttt ggtctcccag
     3361 tgccgctccc tttggggcgt ccccgacccc tgccagttcc tggagctttg ggagatttgg
     3421 gcttcttagg tggtttctgc tctctcctgg aggggctccc tctcccagtt accgtgaagt
     3481 caaaatcatt agggtccaag gaggtgtctc ccaccttttc aaagtatgca atcaattcta
     3541 ctttagagca aaaagctttt ccctggggat tgatcaaata tacatcatac tttccagcag
     3601 atcggccaga cttcctttgt ttaagctttc gtgtccaacc ttcaggcaag gtggggtcat
     3661 catacatagg tccccggtca cggataatgg agcgccgctg tttgggggaa gccgaggctt
     3721 ctggcactgc tggggcagag cctgagcttt ctgatgtttc tgctttgcct gcctctgctg
     3781 gctctgcaga atggtgggct gaaggttgta gtggctcatg cttgccttct ttgtcctcct
     3841 tcttgtcttt cttcgccttc ttaaacttca gtggcttgtc tctgaggccc tggagatcct
     3901 ggtcttctga cttttcctcc agtctctcct cctcgcctcc tcctcctcct ccgctcggcg
     3961 cggcggcggc ggcggcggtg gcggcagcgg cggccatgct agcataactt cgtataaagt
     4021 atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt
     4081 gcaaagaata acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta
     4141 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     4201 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     4261 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     4321 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     4381 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     4441 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     4501 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     4561 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     4621 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     4681 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     4741 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct
     4801 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg
     4861 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg
     4921 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag
     4981 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     5041 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     5101 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     5161 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     5221 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     5281 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5341 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5401 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5461 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5521 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5581 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5641 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5701 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5761 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5821 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5881 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5941 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6001 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6061 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6121 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6181 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6241 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6301 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6361 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6421 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6481 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6541 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6601 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6661 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6721 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6781 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6841 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6901 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6961 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7021 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     7081 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7141 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7201 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7261 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7321 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7381 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7441 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7501 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7561 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7621 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7681 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7741 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7801 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7861 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7921 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7981 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
