LOCUS       sequence-385234-        6633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-CALR WT Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6633)
  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 6633)
  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-WT
FEATURES             Location/Qualifiers
     source          1..6633
                     /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      2933..2942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2946..3080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2983..3002)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3037..3056
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3149..3506
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3149..3340
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3172..3192)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3357..3492
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3526..3543)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3526..3542)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3535..3557)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3755..4210
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3842..3861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4052..4073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4227..4246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4346..4368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4406..4424)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4492..4596
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4597..4665,4666..5457)
                     /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(4815..4834)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5628..6216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6117..6136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6370..6387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6504..6525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6540..6545,6546..6563,6564..6570)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6578..6594
                     /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     6583..6605
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6602..6618
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6602..6618
                     /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 ggcagcagag aaacaaatga aggacaaaca ggacgaggag cagaggctta
     2761 aggaggagga agaagacaag aaaaggaagg aagaagaaga agccgaagat aaagaagacg
     2821 acgaagataa ggacgaagac gaagaagacg aagaagataa agaagaggac gaagaagagg
     2881 acgtgcctgg gcaagctaaa gatgaattgt gaagtcgacc tgcagaagct tggccgccat
     2941 ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3001 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3061 tatcttatca tgtctggatc gggaattaat tcggcgcagc accatggcct gaaataacct
     3121 ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt
     3181 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3241 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3301 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3361 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3421 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3481 tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt
     3541 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     3601 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     3661 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3721 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     3781 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3841 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3901 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3961 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4021 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4081 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4141 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4201 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4261 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4321 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4381 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4441 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4501 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4561 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4621 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4681 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4741 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4801 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4861 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4921 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4981 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5041 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5101 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5161 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5221 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5281 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5341 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5401 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5461 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5521 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5581 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5641 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5701 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5761 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5821 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5881 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5941 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6001 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6061 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6121 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6181 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6241 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6301 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6361 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6421 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     6481 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     6541 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     6601 acaggaaaca gctatgacat gattacgaat taa
//
