LOCUS       sequence-385267-        6420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-RAX WT Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6420)
  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 6420)
  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-WT
FEATURES             Location/Qualifiers
     source          1..6420
                     /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      2720..2729
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2733..2867
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2770..2789)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2824..2843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2936..3293
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2936..3127
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2959..2979)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3144..3279
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3313..3330)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3313..3329)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3322..3344)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3542..3997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3629..3648)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3839..3860
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4014..4033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4133..4155
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4193..4211)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4279..4383
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4384..4452,4453..5244)
                     /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(4602..4621)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5415..6003
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5904..5923
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6157..6174
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6291..6312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6327..6332,6333..6350,6351..6357)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6365..6381
                     /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     6370..6392
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6389..6405
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6389..6405
                     /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 gcatcttcca ggttgtgcac
     1681 ctgctatggc cgacggttct tttagcctgg ctgggcatct tctgcggtca cctggtggaa
     1741 gtacttcccg gttgcattcc atagaagcga tattaggttt cacaaaagat gatggcatat
     1801 tgggaacatt tcctgccgaa agaggggcaa gaggagcaaa agaaagggac cgccggttag
     1861 gagcccgccc tgcatgtcca aaagctccag aagagggatc tgaaccttct cccccacctg
     1921 ctcccgcacc agctccagaa tatgaggcac cccgccctta ttgtccaaaa gaacctggag
     1981 aagcccgccc ttcacccggc cttcctgttg gacctgcaac aggagaggcc aagctcagtg
     2041 aagaagagca accgaagaag aaacaccgac gtaatcgtac tacctttact acctatcaac
     2101 tccacgaatt ggaacgggct tttgaaaagt ctcattatcc agatgtttat tccagagaag
     2161 aacttgcggg gaaagttaat ctgcccgaag ttcgagtaca agtctggttt caaaatagga
     2221 gagcaaaatg gagaaggcaa gaaaagctcg aggtcagcag catgaaactt caagatagcc
     2281 ctttactgag ttttagtagg agtccccctt cagctacttt atcaccactg ggagccggtc
     2341 caggatctgg tggtggacct gcaggcggtg ccctgccttt agaaagttgg ttaggcccac
     2401 cccttccagg tggcggagca accgccttgc aatccttacc cggatttggg cccccagccc
     2461 aaagtttacc agcttcttat acacctcctc cacccccacc cccttttctc aattcaccac
     2521 ctcttgggcc tggattgcag cccctggccc cgccaccccc aagctatcca tgtggaccgg
     2581 ggtttggcga taaatttcct ctcgatgaag ctgatccaag gaattcttct attgccgccc
     2641 tcaggctcaa ggcaaaagaa catattcaag ctataggaaa accttggcaa gcgctttgaa
     2701 gtcgacctgc agaagcttgg ccgccatggc ccaacttgtt tattgcagct tataatggtt
     2761 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2821 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcggg aattaattcg
     2881 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag
     2941 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3001 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3061 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3121 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3181 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3241 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctgt
     3301 taacagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     3361 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     3421 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     3481 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     3541 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3601 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3661 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3721 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     3781 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3841 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     3901 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3961 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     4021 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4081 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4141 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4201 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4261 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4321 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4381 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4441 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4501 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4561 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4621 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4681 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4741 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4801 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4861 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4921 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4981 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5041 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5101 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5161 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5221 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5281 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5341 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5401 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5461 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5521 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5581 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5641 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5701 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5761 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5821 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5881 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5941 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6001 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6061 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     6121 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     6181 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     6241 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     6301 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     6361 ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattaa
//
