LOCUS       sequence-472218-        6344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-NPM1-TDP43HP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6344)
  AUTHORS   Zhang Y, Stoppelkamp I, Fernandez-Pernas P, Allram M, Charman M, 
            Magalhaes AP, Piedavent-Salomon M, Sommer G, Sung YC, Meyer K, Grams
            N, Halko E, Dongre S, Meierhofer D, Malszycki M, Ilik IA, Aktas T, 
            Kraushar ML, Vastenhouw N, Weitzman MD, Grebien F, Niskanen H, Hnisz
            D
  TITLE     Probing condensate microenvironments with a micropeptide killswitch.
  JOURNAL   Nature. 2025 Jun 4. doi: 10.1038/s41586-025-09141-5.
  PUBMED    40468084
REFERENCE   2  (bases 1 to 6344)
  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"; journalName: "Nature. 
            2025 Jun 4. doi: 10.1038/s41586-025-09141-5."
COMMENT     Sequence Label: pRK5_mEGFP-NPM1-TDP43HP
FEATURES             Location/Qualifiers
     source          1..6344
                     /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      934..943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(940..942,943..945,946..1656)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(985..1006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1246..1265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1593..1614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2644..2653
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2657..2791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2694..2713)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2748..2767
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2860..3217
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2860..3051
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2883..2903)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3068..3203
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3237..3254)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3237..3253)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3246..3268)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3466..3921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3553..3572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3763..3784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3938..3957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4057..4079
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4117..4135)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4203..4307
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4308..4376,4377..5168)
                     /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(4526..4545)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5339..5927
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5828..5847
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6081..6098
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6215..6236
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6251..6256,6257..6274,6275..6281)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6289..6305
                     /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     6294..6316
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6313..6329
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6313..6329
                     /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 gtcgccacca tggtgagcaa gggcgaggag
      961 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1021 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1081 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1141 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1201 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1261 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1321 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1381 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1441 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1501 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1561 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1621 gccgggatca ctctcggcat ggacgagctg tacaagaccg gcggcatgga agattcgatg
     1681 gacatggaca tgagccccct gaggccccag aactatcttt tcggttgtga actaaaggcc
     1741 gacaaagatt atcactttaa ggtggataat gatgaaaatg agcaccagtt atctttaaga
     1801 acggtcagtt taggggctgg tgcaaaggat gagttgcaca ttgttgaagc agaggcaatg
     1861 aattacgaag gcagtccaat taaagtaaca ctggcaactt tgaaaatgtc tgtacagcca
     1921 acggtttccc ttgggggctt tgaaataaca ccaccagtgg tcttaaggtt gaagtgtggt
     1981 tcagggccag tgcatattag tggacagcac ttagtagctg tggaggaaga tgcagagtca
     2041 gaagatgaag aggaggagga tgtgaaactc ttaagtatat ctggaaagcg gtctgcccct
     2101 ggaggtggta gcaaggttcc acagaaaaaa gtaaaacttg ctgctgatga agatgatgac
     2161 gatgatgatg aagaggatga tgatgaagat gatgatgatg atgattttga tgatgaggaa
     2221 gctgaagaaa aagcgccagt gaagaaatct atacgagata ctccagccaa aaatgcacaa
     2281 aagtcaaatc agaatggaaa agactcaaaa ccatcatcaa caccaagatc aaaaggacaa
     2341 gaatccttca agaaacagga aaaaactcct aaaacaccaa aaggacctag ttctgtagaa
     2401 gacattaaag caaaaatgca agcaagtata gaaaaaggtg gttctcttcc caaagtggaa
     2461 gccaaattca tcaattatgt gaagaattgc ttccggatga ctgaccaaga ggctattcaa
     2521 gatctctggc agtggaggaa gtctcttatt aatccagcca tgatggctgc cgcccaggca
     2581 gcactacaga gcagttgggg tatgatgggc atgttagcca gccagtgaac ctgcagaagc
     2641 ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2701 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2761 actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag caccatggcc
     2821 tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct
     2881 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2941 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3001 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3061 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3121 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3181 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag cttggcactg
     3241 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     3301 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     3361 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     3421 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     3481 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3541 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3601 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3661 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3721 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3781 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     3841 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3901 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     3961 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4021 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4081 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4141 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4201 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4261 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4321 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4381 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4441 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4501 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4561 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4621 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4681 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4741 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4801 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4861 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4921 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4981 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5041 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5101 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5161 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5221 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5281 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5341 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5401 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5461 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5521 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5581 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5641 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5701 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5761 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5821 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5881 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5941 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6001 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6061 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     6121 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6181 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     6241 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6301 aacaatttca cacaggaaac agctatgaca tgattacgaa ttaa
//
