LOCUS       sequence-385209-        6669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-CALR Mutant (D387Rfs*44) Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6669)
  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 6669)
  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-CALR-MUT
FEATURES             Location/Qualifiers
     source          1..6669
                     /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      2969..2978
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2982..3116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3019..3038)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3073..3092
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3185..3542
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3185..3376
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3208..3228)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3393..3528
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3562..3579)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3562..3578)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3571..3593)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3791..4246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3878..3897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4088..4109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4263..4282)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4382..4404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4442..4460)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4528..4632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4633..4701,4702..5493)
                     /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(4851..4870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5664..6252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6153..6172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6406..6423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6540..6561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6576..6581,6582..6599,6600..6606)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6614..6630
                     /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     6619..6641
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6638..6654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6638..6654
                     /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 gctgctatcc gtgccgctgc
     1681 tgctcggcct cctcggcctg gccgtcgccg agcctgccgt ctacttcaag gagcagtttc
     1741 tggacggaga cgggtggact tcccgctgga tcgaatccaa acacaagtca gattttggca
     1801 aattcgttct cagttccggc aagttctacg gtgacgagga gaaagataaa ggtttgcaga
     1861 caagccagga tgcacgcttt tatgctctgt cggccagttt cgagcctttc agcaacaaag
     1921 gccagacgct ggtggtgcag ttcacggtga aacatgagca gaacatcgac tgtgggggcg
     1981 gctatgtgaa gctgtttcct aatagtttgg accagacaga catgcacgga gactcagaat
     2041 acaacatcat gtttggtccc gacatctgtg gccctggcac caagaaggtt catgtcatct
     2101 tcaactacaa gggcaagaac gtgctgatca acaaggacat ccgttgcaag gatgatgagt
     2161 ttacacacct gtacacactg attgtgcggc cagacaacac ctatgaggtg aagattgaca
     2221 acagccaggt ggagtccggc tccttggaag acgattggga cttcctgcca cccaagaaga
     2281 taaaggatcc tgatgcttca aaaccggaag actgggatga gcgggccaag atcgatgatc
     2341 ccacagactc caagcctgag gactgggaca agcccgagca tatccctgac cctgatgcta
     2401 agaagcccga ggactgggat gaagagatgg acggagagtg ggaaccccca gtgattcaga
     2461 accctgagta caagggtgag tggaagcccc ggcagatcga caacccagat tacaagggca
     2521 cttggatcca cccagaaatt gacaaccccg agtattctcc cgatcccagt atctatgcct
     2581 atgataactt tggcgtgctg ggcctggacc tctggcaggt caagtctggc accatctttg
     2641 acaacttcct catcaccaac gatgaggcat acgctgagga gtttggcaac gagacgtggg
     2701 gcgtaacaaa ggccgccgaa aagcagatga aagataagca agatgaagaa caaagactga
     2761 aagaagaaga agaggataag aagagaaagg aagaagaaga agctgaagat aaggaacgca
     2821 tgatgagaac taaaatgcgt atgcgccgga tgcggcgaac taggaggaag atgaggagga
     2881 agatgtcccc ggccaggcca aggacgagct gtagagaggc ctgcctccag ggctggactg
     2941 aggcctgaag tcgacctgca gaagcttggc cgccatggcc caacttgttt attgcagctt
     3001 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3061 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga
     3121 attaattcgg cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta
     3181 ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3241 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3301 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3361 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3421 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3481 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3541 aaaagctgtt aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3601 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3661 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3721 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     3781 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3841 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3901 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3961 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4021 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4081 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     4141 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4201 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4261 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4321 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4381 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4441 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4501 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4561 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4621 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4681 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4741 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4801 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4861 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4921 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4981 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5041 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5101 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5161 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5221 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5281 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5341 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5401 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5461 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5521 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5581 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5941 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6001 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6061 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6121 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6181 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6241 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6301 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6361 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6421 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6481 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6541 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6601 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt
     6661 acgaattaa
//
