LOCUS       sequence-323732-        8019 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Double-Floxed Inverted Open reading frame for mouseMeCP2(alpha) 
            Under EF1a promoter.
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 Apr 14, 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_mMeCP2-HaloTag
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        172..1350
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          403..1341
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1298..1318
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1381..1414)
                     /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(1465..1498)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1527..2411)
                     /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(2772..2792)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     protein_bind    3945..3978
                     /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    4029..4062
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4087..4675
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4140..4160)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4558..4569)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4707..5183
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4906..4925)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5223..5363
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5223..5352
                     /label=AAV2 ITR
     rep_origin      5438..5893
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5525..5544)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5735..5756
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5910..5929)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6029..6051
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6089..6107)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6175..6279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6280..6348,6349..7140)
                     /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(6498..6517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7311..7899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7800..7819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7961..8019,1..71)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
       61 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
      121 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      181 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      241 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      301 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      361 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      421 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      481 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      541 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      601 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      661 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      721 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      781 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
      841 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
      901 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
      961 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1021 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1081 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1141 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1201 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1261 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1321 agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt cgactccgga
     1381 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
     1441 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
     1501 cgcgccttac ttgtacacat gtcctaaccg gaaatctcga gggtagacag ccagcgcgcg
     1561 atctcgctgc cgatcaggtc cgggttgtct tcttgcagca gattcagacc cgggccgatg
     1621 tccacagcct tgcagttagg caggcttttg gccaggcgag cggcttcggc cggtgggatc
     1681 agaacgcctg gggtgcccca gaacagcagc ttcgggacag gggactggtg cagccagtcc
     1741 atgtattctt cgaccagcgc gacgatgttc gctggctcac cggcgattgg cagctcgttt
     1801 gggaagcgcc acagtggctc gcggtcaaca ggattcagga acggctcgcg gtaatggtcc
     1861 atctcgactt cagtcagcgg gcggacgaca cccatcggca gcgtaccctc gataaaaacg
     1921 ttctgatcga tgatcagctt gcggccgacg tcggtggtgc ggaaggcctg gaaggtctcg
     1981 cgggcaaatt ctggccattc gtcccaggtc gggatagggc ggatgaactc cataaatgca
     2041 atacctttga cgcgctctgg attgcgcttg gcccagtgga aacccagagc ggagccccag
     2101 tcgtgaatga ccaggacgac ctcttccaga cccagggctt cgatgaaggc atccatgaag
     2161 cggacgtggt cgtcgaagaa ataacccagg tctggtttgt cggatttgcc cataccgatc
     2221 aggtctggag caatgcagcg atgggtcggt gcaacatgcg ggatgatgtt gcgccacacg
     2281 taggaggagg tcgggttacc gtgcaggaac agcacagggg tgccatcgcg cggaccaaca
     2341 tcgacgtagt gcatgcgctc gcccaggact tccacataat gggggtcgaa tggaaagcca
     2401 gtaccgattt cggtggcgac cggtggatcc ttacagctaa ctctctcggt cacgggcgtc
     2461 cggctgtcca caggctcctc tctgtttggc cttggcatgg aagatgaaac aatgtctttg
     2521 cgctctccct cccctcggtg tttgtacttt tctgcaactg tggtagtggt ggcgaccata
     2581 ggctgagtct tagctggctc cttggggcag ccatcgcttt ccagtgagcc tcctcggggc
     2641 atcttctctt ctttgcagat gctgctgctc aagtcctgag gctcaggggg gctgatgggg
     2701 tcctcagagc tctcaggctc aggtgggggt ggggatggga gcagtggcat gggggccttt
     2761 gtggactctg agtgatggtg gtgatgatgg tgctccttct taggtgggga ggaggcactg
     2821 ctgctgcgcc ccttggggct gctctccttg cttttacgcc cagggctctt gcaggtcttc
     2881 agtcccttcc cgcttttctc accaagggtg gacaccagca ggggcttcac cacttccttg
     2941 acctcgatgc tgaccgtctc ccgggtcttg cgcttcttga tggggagcac agtctcatgc
     3001 acagaccgta tggaagactc cttcacggct ttctttttgg cctcagctgc agcagctgcc
     3061 accacactcc caggctttct accccgtttc ttaggaatgg cctgggggtc agcttcagct
     3121 tttcgctttc tgccagggcg tttgatcacc atgacctggg cagatgtggt agccccacct
     3181 ccctcaccct tacccccagg cgatgcttgg aaaggcatct tgacaacaag tttcccaggg
     3241 ctcttctcca ggaccctttt cacctgaaca ccttctgatg ctgctgcctt tggtctccca
     3301 gtgccgctcc ctttggggcg tccccgaccc ctgccagttc ctggagcttt gggagatttg
     3361 ggcttcttag gtggtttctg ctctctcctg gaggggctcc ctctcccagt taccgtgaag
     3421 tcaaaatcat tagggtccaa ggaggtgtct cccacctttt caaagtatgc aatcaattct
     3481 actttagagc gaaaagcttt tccctgggga ttgatcaaat atacatcata ctttccagca
     3541 gatcggccag acttcctttg tttaagcttt cgtgtccaac cttcaggcaa ggtggggtca
     3601 tcatacatag gtccccggtc acggataatg gagcgccgct gtttggggga agccgaggct
     3661 tctggcactg ctggggcaga gcctgagctt tctgatgttt ctgctttgcc tgcctctgct
     3721 ggctctgcag aatggtgggc tgaaggttgt agtggctcat gcttgccttc tttgtcctcc
     3781 ttcttgtctt tcttcgcctt cttaaacttc agtggcttgt ctctgaggcc ctggagatcc
     3841 tggtcttctg acttttcctc cagtctctcc tcctcgcctc ctcctcctcc tccgctcggc
     3901 gcggcggcgg cggcggcggt ggcggcagcg gcggccatgc tagcataact tcgtataaag
     3961 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg
     4021 tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt
     4081 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4141 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4201 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4261 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4321 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4381 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4441 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     4501 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4561 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4621 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     4681 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     4741 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     4801 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     4861 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     4921 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     4981 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     5041 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     5101 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     5161 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     5221 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5281 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5341 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5401 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5461 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5521 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5581 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5641 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5701 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5761 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5821 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5881 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5941 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6001 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6061 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6121 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6181 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6241 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6301 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6361 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6421 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6481 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6541 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6601 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6661 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6721 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6781 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6841 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6901 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6961 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     7021 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7081 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7141 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7201 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7921 ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca gctgcgcgct
     7981 cgctcgctca ctgaggccgc ccgggcgtcg ggcgacctt
//
