LOCUS       sequence-385251-        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-PHOX2B WT Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6324)
  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 6324)
  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-PHOX2B-WT
FEATURES             Location/Qualifiers
     source          1..6324
                     /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      2624..2633
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2637..2771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2674..2693)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2728..2747
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2840..3197
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2840..3031
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2863..2883)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3048..3183
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3217..3234)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3217..3233)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3226..3248)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3446..3901
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3533..3552)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3743..3764
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3918..3937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4037..4059
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4097..4115)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4183..4287
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4288..4356,4357..5148)
                     /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(4506..4525)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5319..5907
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5808..5827
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6061..6078
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6195..6216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6231..6236,6237..6254,6255..6261)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6269..6285
                     /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     6274..6296
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6293..6309
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6293..6309
                     /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 gtacaagatg gagtactcct
     1681 atcttaactc aagcgcttat gaaagttgca tggcaggtat ggatacaagt agtctcgcca
     1741 gcgcgtacgc cgacttttcc agctgttctc aagcttccgg gtttcaatac aatcctatcc
     1801 ggacaacatt cggagcaaca agtgggtgtc caagtctgac acccgggagt tgtagtttag
     1861 gaacactgag agatcatcaa tcaagcccat atgcagccgt gccatataag ctgtttactg
     1921 atcatggtgg gcttaatgag aaacggaaac aaagaagaat tcggactacg tttacaagcg
     1981 cacaacttaa ggaactcgag cgcgtgtttg ccgaaaccca ttatccagat atttatacgc
     2041 gagaagaact cgctttaaag atagatctga ccgaagcccg cgttcaagtt tggtttcaaa
     2101 atcggcgggc aaaattccgg aaacaagaac gggctgccgc cgcagctgca gcagctgcaa
     2161 agaatggaag ctcagggaag aaaagcgata gtagtcgcga tgatgaatcc aaggaagcta
     2221 aaagtacaga tccagattct acaggcggac ccggccctaa ccctaatcca actccatcat
     2281 gtggtgctaa cggtggtgga ggaggtggcc cgtcacctgc aggcgcccct ggcgcagctg
     2341 gacctggtgg gcctggcgga gagccaggga aaggtggggc cgccgccgca gctgctgctg
     2401 ccgctgctgc tgcagcagcc gcagcggcag ctgctgcggg cggacttgcc gcagcaggcg
     2461 ggcccggtca gggatgggcc ccaggtccag gaccaataac aagtataccc gactcactcg
     2521 gcggcccatt tgcttccgtt ctgtcaagct tgcagaggcc aaatggcgcg aaggcagctc
     2581 ttgtcaaatc ttctatgttt tagagtcgac ctgcagaagc ttggccgcca tggcccaact
     2641 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2701 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     2761 atgtctggat cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag
     2821 gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2881 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2941 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3001 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3061 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3121 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3181 gcctaggctt ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg
     3241 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3301 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3361 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3421 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3481 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3541 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3601 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3661 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3721 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3781 atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3841 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     3901 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     3961 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4021 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4081 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4141 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4201 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4261 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4321 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4381 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4441 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4501 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4561 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4621 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4681 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4741 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4801 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4861 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4921 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4981 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5041 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5101 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5161 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5221 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5281 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5341 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5401 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5461 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5521 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5581 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5641 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5701 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5761 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5821 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5881 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5941 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6001 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6061 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     6121 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     6181 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     6241 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     6301 agctatgaca tgattacgaa ttaa
//
