LOCUS       sequence-385263-        7152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-RUNX1 Mutant (G363Afs*229) Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7152)
  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 7152)
  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-RUNX1-MUT
FEATURES             Location/Qualifiers
     source          1..7152
                     /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      3452..3461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3465..3599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3502..3521)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3556..3575
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3668..4025
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3668..3859
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3691..3711)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3876..4011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4045..4062)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4045..4061)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4054..4076)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4274..4729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4361..4380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4571..4592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4746..4765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4865..4887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4925..4943)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5011..5115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5116..5184,5185..5976)
                     /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(5334..5353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6147..6735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6636..6655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6889..6906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7023..7044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7059..7064,7065..7082,7083..7089)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7097..7113
                     /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     7102..7124
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7121..7137
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7121..7137
                     /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 ggcatctgat tctatcttcg
     1681 aatctttccc cagctatcct caatgtttta tgcgcgagtg tattctcggc atgaacccaa
     1741 gccgggatgt acatgacgca tctacttccc ggcgctttac cccaccaagc acagccctct
     1801 ctcctggtaa aatgtcagaa gcccttcccc tcggggctcc tgatgcagga gcagctctcg
     1861 cagggaaact tcgttctgga gataggtcca tggttgaagt ccttgcggat catccagggg
     1921 aacttgtccg gacagattct cctaattttc tttgttcagt tcttccgact cattggagat
     1981 gtaataaaac tctccctatt gcatttaaag tcgttgcgtt aggcgacgtg cctgacggga
     2041 ccttagttac agtcatggcc ggaaacgacg agaattatag cgcagaactt cgcaacgcca
     2101 cagccgcaat gaagaatcaa gtcgcgcggt tcaacgatct tcgcttcgtt ggcaggtccg
     2161 gtcgaggcaa gagttttacg ttgactatta ccgtatttac gaatccccct caggtggcaa
     2221 catatcatag ggctattaag attactgttg acggcccacg cgagccaaga cgccacagac
     2281 aaaagctgga cgaccaaaca aaaccgggct ccctctcatt ctctgaaagg cttagcgagc
     2341 tcgaacaact taggcggact gctatgcggg ttagtccgca tcatccggca ccaaccccaa
     2401 atccacgcgc atctcttaat cattcaaccg cattcaatcc ccaaccccaa tcacaaatgc
     2461 aagacacccg ccaaatacag ccttcaccgc cctggagcta tgaccaaagc tatcagtatt
     2521 taggctctat agcaagcccc agcgtccatc ctgcgacccc aatcagccca ggtagggcat
     2581 ccggaatgac tactctgtca gctgagctgt catccagact gtccaccgcc cctgatctta
     2641 ccgcattttc agatccccgt caatttcctg cccttccaag catttctgat ccgcgaatgc
     2701 attaccccgg agcttttacg tattctccca ctcccgttac ttccgccagt gcctgccgcc
     2761 cctgggccag acctagggcc acaactccca catgtcggcg tccaactcct gcccgcagga
     2821 aacggagaga agcacgctct aaacccgcac ggccgcccac aacatgcacc acagctccac
     2881 gccccgcacc aacatccagt ccctggtggg ccgcttccgc ccgaagacga gcaagttgtc
     2941 gtcccgcgcc tactccccca cccgcaccta gatgtagcac acctgcttca aggacacgtg
     3001 ccacttggtg gagacctcgg gcggcaaccg ccaccccgcc cccaacctgg cgaccaccaa
     3061 gagcatggcg gcgcccttgt ggtgggccca ccgaagctcc gggacttgcg cgtctcggac
     3121 cggccggaag aagattgcgg cttagggccc gaggaccacc agtaagagat aaaccagcag
     3181 gaatacccgg tccaggacca gggcatcgcc ccggagcaga aggagctaga aggcccggca
     3241 gtagatgcag atccgggccc aggtcattgt tgcgaccgga agctcatgcg gcagctgtgt
     3301 gttggcggcc gggtccacgg ggcggagtgc gtggggatgc ccccaggggt tgtcccccag
     3361 cacccgctcc gcggggcagg gccggtaaac agactggtag gtttcctgaa ggcaattgcg
     3421 agtgtttcta aagtcgacct gcagaagctt ggccgccatg gcccaacttg tttattgcag
     3481 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3541 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     3601 ggaattaatt cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     3661 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3901 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3961 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4021 gcaaaaagct gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4081 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4141 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4201 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4261 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4321 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4381 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4441 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4501 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4561 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     4621 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4681 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4741 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4801 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4861 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4921 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4981 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5041 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5101 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5161 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5221 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5281 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5341 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5401 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5461 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5521 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5581 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5641 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5701 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5761 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5821 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5881 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5941 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6001 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6061 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6121 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6181 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6241 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6301 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6361 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6421 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6481 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6541 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6601 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6661 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6721 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6781 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6841 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6901 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     6961 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7021 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7081 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg
     7141 attacgaatt aa
//
