LOCUS       sequence-385271-        6303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-PHOX2B Mutant (S207Afs*102) Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6303)
  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 6303)
  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-MUT
FEATURES             Location/Qualifiers
     source          1..6303
                     /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      2603..2612
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2616..2750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2653..2672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2707..2726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2819..3176
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2819..3010
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2842..2862)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3027..3162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3196..3213)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3196..3212)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3205..3227)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3425..3880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3512..3531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3722..3743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3897..3916)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4016..4038
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4076..4094)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4162..4266
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4267..4335,4336..5127)
                     /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(4485..4504)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5298..5886
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5787..5806
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6040..6057
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6174..6195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6210..6215,6216..6233,6234..6240)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6248..6264
                     /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     6253..6275
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6272..6288
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6272..6288
                     /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 gagtacagct
     1681 atttgaacag ctccgcttat gaatcatgca tggccggaat ggatacatct tccttagcca
     1741 gtgcatacgc cgattttagc agctgttccc aagcttctgg gtttcaatac aatccaattc
     1801 ggactacgtt cggtgcaacc tcaggttgtc caagcttaac tcctgggagc tgttcattgg
     1861 gaacattgcg ggatcatcaa tccagcccat atgctgctgt gccctataag ctgtttacag
     1921 atcatggcgg acttaatgaa aagcgcaaac aaaggcggat tcggacaacc tttacttccg
     1981 ctcaactgaa ggaactcgag cgagtgtttg ctgaaaccca ttatccagat atttatacac
     2041 gcgaagaact tgcgctcaaa attgatttaa ccgaagcacg ggtgcaagtt tggtttcaaa
     2101 ataggagagc aaaattcagg aaacaagaac gtgcggccgc cgcagctgct gcagctgcaa
     2161 agaatggaag ctctgggaag aaaagcgatt ccagcagaga tgatgaatca aaggaagcta
     2221 aatctaccga tcccgattct acaggtggac ctggccctaa cccgaatcct actcctgcag
     2281 ctggccgcat ggaagctgcc gctgcaggtc ctgcgagatt ggaactgagg ggcagaagag
     2341 gtcccggagc aagggaagcc aatccagccc gagctgctca acaacaaagg cgccgaccaa
     2401 ggcaaagaag aaggagacaa agacaaagac gccaattgga agcatggctc cgattgggtg
     2461 ctttagataa agccggcctg cccgctcccg cacctagtcc accctcacga atcaggcttg
     2521 gagtgcccag tcccgcttca tacctcagaa gcaaggatcc cactgtaccg aaaccccctt
     2581 gaagtcgacc tgcagaagct tggccgccat ggcccaactt gtttattgca gcttataatg
     2641 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2701 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat
     2761 tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct
     2821 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2881 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2941 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3001 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3061 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     3121 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3181 tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     3241 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     3301 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     3361 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     3421 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3481 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3541 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3601 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     3661 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3721 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     3781 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3841 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     3901 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     3961 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4021 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     4081 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     4141 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4201 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4261 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4321 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4381 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4441 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4501 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4561 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4621 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4681 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4741 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4801 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4861 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4921 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4981 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     5041 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     5101 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5161 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5221 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5281 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5341 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5401 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5461 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5521 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5581 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5641 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5701 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5761 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5821 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5881 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5941 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6001 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6061 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     6121 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     6181 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     6241 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat
     6301 taa
//
