LOCUS       sequence-385206-        5574 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian Expression of mEGFP-HMGB1 Mutant (K184Rfs*44) IDR Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5574)
  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 5574)
  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"; 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-IDR2
FEATURES             Location/Qualifiers
     source          1..5574
                     /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      1874..1883
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1887..2021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1924..1943)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1978..1997
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2090..2447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2090..2281
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2113..2133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2298..2433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2467..2484)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2467..2483)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2476..2498)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2696..3151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2783..2802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2993..3014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3168..3187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3287..3309
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3347..3365)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3433..3537
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3538..3606,3607..4398)
                     /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(3756..3775)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4569..5157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5058..5077
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5311..5328
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5445..5466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5481..5486,5487..5504,5505..5511)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5519..5535
                     /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     5524..5546
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5543..5559
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5543..5559
                     /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 ggctaaagga aagcctgatg
     1681 cagcaaaaaa gggagttgtc aaggctgaaa aaagcaagaa aaggaagagg aggaagatga
     1741 ggaagatgaa gaggatgagg aggaggagga agatgaagaa gatgaagatg aagaagaaga
     1801 tgatgatgat gaataagttg gttctagcgc agttcttctt ctcctgtcta taaagtcgac
     1861 ctgcagaagc ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat
     1921 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1981 gtttgtccaa actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag
     2041 caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa
     2101 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2161 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2221 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2281 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2341 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2401 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag
     2461 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2521 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2581 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     2641 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     2701 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2761 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2821 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2881 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     2941 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3001 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     3061 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3121 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     3181 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3241 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3301 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3361 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3421 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3481 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3541 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3601 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3661 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3721 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3781 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3841 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3901 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3961 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4021 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4081 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4141 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4201 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4261 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4321 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4381 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4441 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4501 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4561 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4621 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4681 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4741 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4801 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4861 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4921 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4981 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5041 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5101 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5161 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5221 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5281 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5341 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5401 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     5461 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     5521 gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa ttaa
//
