LOCUS       sequence-385254-        6231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-RAX Mutant (S222Rfs*63) Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6231)
  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 6231)
  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-RAX-MUT
FEATURES             Location/Qualifiers
     source          1..6231
                     /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      2531..2540
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2544..2678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2581..2600)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2635..2654
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2747..3104
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2747..2938
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2770..2790)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2955..3090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3124..3141)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3124..3140)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3133..3155)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3353..3808
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3440..3459)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3650..3671
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3825..3844)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3944..3966
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4004..4022)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4090..4194
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4195..4263,4264..5055)
                     /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(4413..4432)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5226..5814
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5715..5734
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5968..5985
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6102..6123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6138..6143,6144..6161,6162..6168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6176..6192
                     /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     6181..6203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6200..6216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6200..6216
                     /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 gcatctccct ggttgtgctc
     1681 ccgcaatggc cgacggctcc ttttcactgg caggacattt gcttcggtct ccaggtggct
     1741 ctacatcccg actccattcc attgaagcta ttctcggctt cactaaagat gatggaatac
     1801 tgggaacatt tcccgcagaa agaggggcac gcggggctaa agaaagagac cggcgtttag
     1861 gggctaggcc tgcttgtcct aaagcccctg aagagggatc tgaaccgagc ccaccacccg
     1921 ctcctgcccc agctcctgaa tatgaggcac ccagaccata ttgtccaaaa gaacctggcg
     1981 aagcgaggcc atctcctgga ctcccggtag gccctgcaac aggtgaggct aagctctctg
     2041 aagaagagca acccaagaag aaacacaggc gcaataggac aacatttact acctatcaac
     2101 tccacgaact tgaacgggcc tttgaaaagt ctcattatcc agatgtctat agtagggaag
     2161 aattagctgg aaaagttaat ctgcccgaag tgagggtaca agtttggttt caaaatcggc
     2221 gagcaaaatg gcgcagacaa gaaaagctcg aggtatcatc tatgaaactc caagatagcc
     2281 ctctgctgtc attttccaga tctccaccga gtgccactct ccgaccatct ggaagggcaa
     2341 gggctgctgt cgctggtaga ctcggagcca ggtgtaggtg gtcacccgga tctggtcggc
     2401 gttgtagggc cggtgcacca aggagatgta gggcatgtcg agcaagcgga aggagaagaa
     2461 gggcttgtct ccccgcgact aggcatcgac gacgattgcg tccatcatag agtcgacctg
     2521 cagaagcttg gccgccatgg cccaacttgt ttattgcagc ttataatggt tacaaataaa
     2581 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2641 tgtccaaact catcaatgta tcttatcatg tctggatcgg gaattaattc ggcgcagcac
     2701 catggcctga aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga
     2761 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2821 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2881 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2941 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3001 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     3061 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctg ttaacagctt
     3121 ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3181 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3241 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     3301 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3361 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3421 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3481 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3541 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3601 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3661 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     3721 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3781 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     3841 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3901 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3961 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4021 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4081 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4141 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4201 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4261 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4321 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4381 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4441 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4501 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4561 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4621 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4681 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4741 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4801 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4861 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4921 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4981 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5041 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5101 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5161 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5221 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5281 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5341 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5401 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5461 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5521 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5581 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5641 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5701 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5761 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5821 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5881 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5941 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     6001 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     6061 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     6121 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     6181 agcggataac aatttcacac aggaaacagc tatgacatga ttacgaatta a
//
