LOCUS       sequence-385207-        6024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-HMGB3 Mutant (K161Ifs*55) Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  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 6024)
  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-HMGB3-MUT
FEATURES             Location/Qualifiers
     source          1..6024
                     /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      2324..2333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2337..2471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2374..2393)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2428..2447
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2540..2897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2540..2731
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2563..2583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2748..2883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2917..2934)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2917..2933)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2926..2948)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3146..3601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3233..3252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3443..3464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3618..3637)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3737..3759
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3797..3815)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3883..3987
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3988..4056,4057..4848)
                     /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(4206..4225)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5019..5607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5508..5527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5761..5778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5895..5916
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5931..5936,5937..5954,5955..5961)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5969..5985
                     /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     5974..5996
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5993..6009
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5993..6009
                     /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 ggctaaaggt gaccccaaga
     1681 aaccaaaggg caagatgtcc gcttatgcct tctttgtgca gacatgcaga gaagaacata
     1741 agaagaaaaa cccagaggtc cctgtcaatt ttgcggaatt ttccaagaag tgctctgaga
     1801 ggtggaagac gatgtccggg aaagagaaat ctaaatttga tgaaatggca aaggcagata
     1861 aagtgcgcta tgatcgggaa atgaaggatt atggaccagc taagggaggc aagaagaaga
     1921 aggatcctaa tgctcccaaa aggccaccgt ctggattctt cctgttctgt tcagaattcc
     1981 gccccaagat caaatccaca aaccccggca tctctattgg agacgtggca aaaaagctgg
     2041 gtgagatgtg gaataattta aatgacagtg aaaagcagcc ttacatcact aaggcggcaa
     2101 agctgaagga gaagtatgag aaggatgttg ctgactatat aagtcgaaag gaaagtttga
     2161 tggtgcaaag ggtcctgcta aagttgcccg gaaaaaggtg gaagaggaag atgaagaaga
     2221 gaaggagaaa gaagaggaga agaagaagga gaaggatgaa taaagaaact gtttatctgt
     2281 ctccttgtga atacttagag tagagtcgac ctgcagaagc ttggccgcca tggcccaact
     2341 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2401 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     2461 atgtctggat cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag
     2521 gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2581 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2641 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2701 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2761 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2821 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2881 gcctaggctt ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg
     2941 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3001 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3061 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3121 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3361 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3481 atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3541 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     3601 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     3661 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3721 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3781 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3841 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3901 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3961 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4021 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4081 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4141 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4201 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4261 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4321 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4381 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4441 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4501 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4561 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4621 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4681 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4741 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4801 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4861 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4921 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4981 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5041 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5101 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5161 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5221 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5281 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5341 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5401 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5461 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5521 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5581 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5641 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5701 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5761 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     5821 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5881 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     5941 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     6001 agctatgaca tgattacgaa ttaa
//
