LOCUS       sequence-385269-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of mEGFP-SQSTM1 WT Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  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 6702)
  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-SQSTM1-WT
FEATURES             Location/Qualifiers
     source          1..6702
                     /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      3002..3011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3015..3149
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3052..3071)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3106..3125
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3218..3575
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3218..3409
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3241..3261)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3426..3561
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3595..3612)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3595..3611)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3604..3626)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3824..4279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3911..3930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4121..4142
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4296..4315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4415..4437
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4475..4493)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4561..4665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4666..4734,4735..5526)
                     /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(4884..4903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5697..6285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6186..6205
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6439..6456
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6573..6594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6609..6614,6615..6632,6633..6639)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6647..6663
                     /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     6652..6674
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6671..6687
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6671..6687
                     /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 ggcgtcgctc accgtgaagg
     1681 cctaccttct gggcaaggag gacgcggcgc gcgagattcg ccgcttcagc ttctgctgca
     1741 gccccgagcc tgaggcggaa gccgaggctg cggcgggtcc gggaccctgc gagcggctgc
     1801 tgagccgggt ggccgccctg ttccccgcgc tgcggcctgg cggcttccag gcgcactacc
     1861 gcgatgagga cggggacttg gttgcctttt ccagtgacga ggaattgaca atggccatgt
     1921 cctacgtgaa ggatgacatc ttccgaatct acattaaaga gaaaaaagag tgccggcggg
     1981 accaccgccc accgtgtgct caggaggcgc cccgcaacat ggtgcacccc aatgtgatct
     2041 gcgatggctg caatgggcct gtggtaggaa cccgctacaa gtgcagcgtc tgcccagact
     2101 acgacttgtg tagcgtctgc gagggaaagg gcttgcaccg ggggcacacc aagctcgcat
     2161 tccccagccc cttcgggcac ctgtctgagg gcttctcgca cagccgctgg ctccggaagg
     2221 tgaaacacgg acacttcggg tggccaggat gggaaatggg tccaccagga aactggagcc
     2281 cacgtcctcc tcgtgcaggg gaggcccgcc ctggccccac ggcagaatca gcttctggtc
     2341 catcggagga tccgagtgtg aatttcctga agaacgttgg ggagagtgtg gcagctgccc
     2401 ttagccctct gggcattgaa gttgatatcg atgtggagca cggagggaaa agaagccgcc
     2461 tgacccccgt ctctccagag agttccagca cagaggagaa gagcagctca cagccaagca
     2521 gctgctgctc tgaccccagc aagccgggtg ggaatgttga gggcgccacg cagtctctgg
     2581 cggagcagat gaggaagatc gccttggagt ccgaggggcg ccctgaggaa cagatggagt
     2641 cggataactg ttcaggagga gatgatgact ggacccatct gtcttcaaaa gaagtggacc
     2701 cgtctacagg tgaactccag tccctacaga tgccagaatc cgaagggcca agctctctgg
     2761 acccctccca ggagggaccc acagggctga aggaagctgc cttgtaccca catctcccgc
     2821 cagaggctga cccgcggctg attgagtccc tctcccagat gctgtccatg ggcttctctg
     2881 atgaaggcgg ctggctcacc aggctcctgc agaccaagaa ctatgacatc ggagcggctc
     2941 tggacaccat ccagtattca aagcatcccc cgccgttgtg aagtcgacct gcagaagctt
     3001 ggccgccatg gcccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3061 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3121 tcatcaatgt atcttatcat gtctggatcg ggaattaatt cggcgcagca ccatggcctg
     3181 aaataacctc tgaaagagga acttggttag gtaccttctg aggcggaaag aaccagctgt
     3241 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3301 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3361 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3421 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3481 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     3541 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct gttaacagct tggcactggc
     3601 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     3661 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     3721 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     3781 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     3841 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3901 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3961 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4021 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4081 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4141 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4201 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4261 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4321 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4381 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4441 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4501 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4561 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4621 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4681 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4741 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4801 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4861 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4921 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4981 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5041 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5101 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5161 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5221 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5281 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5341 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5401 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5461 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5521 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5581 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5641 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5701 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5761 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5821 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5881 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5941 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6001 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6061 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6121 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6181 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6241 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6301 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6361 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6421 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     6481 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     6541 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     6601 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     6661 caatttcaca caggaaacag ctatgacatg attacgaatt aa
//
