LOCUS       sequence-385262-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-HMGB1 Mutant (K184Rfs*44) Fusion 
            Protein, ""R del"" variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  AUTHORS   Mensah MA, Niskanen H, Magalhaes AP, Basu S, Kircher M, Sczakiel HL,
            Reiter AMV, Elsner J, Meinecke P, Biskup S, Chung BHY, Dombrowsky G,
            Eckmann-Scholz C, Hitz MP, Hoischen A, Holterhus PM, Hulsemann W, 
            Kahrizi K, Kalscheuer VM, Kan A, Krumbiegel M, Kurth I, Leubner J, 
            Longardt AC, Moritz JD, Najmabadi H, Skipalova K, Snijders Blok L, 
            Tzschach A, Wiedersberg E, Zenker M, Garcia-Cabau C, Buschow R, 
            Salvatella X, Kraushar ML, Mundlos S, Caliebe A, Spielmann M, Horn 
            D, Hnisz D
  TITLE     Aberrant phase separation and nucleolar dysfunction in rare genetic 
            diseases.
  JOURNAL   Nature. 2023 Feb;614(7948):564-571. doi: 10.1038/s41586-022-05682-1.
            Epub 2023 Feb 8.
  PUBMED    36755093
REFERENCE   2  (bases 1 to 6036)
  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"; doi: 
            "10.1038/s41586-022-05682-1"; journalName: "Nature"; date: 
            "2023-02"; volume: "614"; issue: "7948"; pages: "564-571"
COMMENT     Sequence Label: pRK5-mEGFP-HMGB1-MUT-Rdel
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(939..941,942..944,945..1652)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(984..1005)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1592..1613
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2336..2345
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2349..2483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2386..2405)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2440..2459
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2552..2909
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2552..2743
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2575..2595)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2760..2895
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2929..2946)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2929..2945)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2938..2960)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3158..3613
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3245..3264)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3455..3476
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3630..3649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3749..3771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3809..3827)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3895..3999
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4000..4068,4069..4860)
                     /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(4218..4237)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5031..5619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5520..5539
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5773..5790
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5907..5928
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5943..5948,5949..5966,5967..5973)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5981..5997
                     /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)."
     primer_bind     5986..6008
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6005..6021
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6005..6021
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgtaccg gtgccaccat ggtgagcaag ggcgaggagc
      961 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1021 tcagcgtgcg cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     1081 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1141 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1201 ccatgcccga aggctacgtc caggagcgca ccatctcctt caaggacgac ggcacctaca
     1261 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1321 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca
     1381 gccacaacgt ctatatcacg gccgacaagc agaagaacgg catcaaggcg aacttcaaga
     1441 tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1501 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     1561 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1621 ccgggatcac tctcggcatg gacgagctgt acacggcgat gggcaaagga gatcctaaga
     1681 agccgagagg caaaatgtca tcatatgcat tttttgtgca aacttgtcgg gaggagcata
     1741 agaagaagca cccagatgct tcagtcaact tctcagagtt ttctaagaag tgctcagaga
     1801 ggtggaagac catgtctgct aaagagaaag gaaaatttga agatatggca aaagcggaca
     1861 aggcccgtta tgaaagagaa atgaaaacct atatccctcc caaaggggag acaaaaaaga
     1921 agttcaagga tcccaatgca cccaagaggc ctccttcggc cttcttcctc ttctgctctg
     1981 agtatcgccc aaaaatcaaa ggagaacatc ctggcctgtc cattggtgat gttgcgaaga
     2041 aactgggaga gatgtggaat aacactgctg cagatgacaa gcagccttat gaaaagaagg
     2101 ctgcgaagct gaaggaaaaa tatgaaaagg atattgctgc atatcgagct aaaggaaagc
     2161 ctgatgcagc aaaaaaggga gttgtcaagg ctgaaaaaag caagaaaagg aaaaagatga
     2221 agatgaagat gaaaatgaag aagatgaaga tgaagaagaa gatgatgatg atgaataagt
     2281 tggttctagc gcagttcttc ttctcctgtc tataaagtcg acctgcagaa gcttggccgc
     2341 catggcccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2401 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2461 atgtatctta tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa
     2521 cctctgaaag aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg
     2581 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2641 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2701 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     2761 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     2821 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     2881 gcttttttgg aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt
     2941 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     3001 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3061 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     3121 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     3181 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3241 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3301 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3361 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3421 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3481 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     3541 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3601 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3661 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3721 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3781 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3841 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3901 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3961 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4021 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4081 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4141 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4201 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4261 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4321 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4381 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4441 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4501 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4561 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4621 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4681 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4741 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4801 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4861 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4921 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4981 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5041 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5101 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5161 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5221 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5281 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5341 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5401 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5461 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5521 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5581 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5641 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5701 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5761 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     5821 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     5881 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     5941 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     6001 cacacaggaa acagctatga catgattacg aattaa
//
