LOCUS       sequence-471775-        7406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-NONO-TFE3-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7406)
  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 7406)
  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-NONO-TFE3-KS
FEATURES             Location/Qualifiers
     source          1..7406
                     /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      3706..3715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3719..3853
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3756..3775)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3810..3829
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3922..4279
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3922..4113
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3945..3965)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4130..4265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4299..4316)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4299..4315)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4308..4330)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4528..4983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4615..4634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4825..4846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5000..5019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5119..5141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5179..5197)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5265..5369
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5370..5438,5439..6230)
                     /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(5588..5607)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6401..6989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6890..6909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7143..7160
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7277..7298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7313..7318,7319..7336,7337..7343)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7351..7367
                     /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     7356..7378
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7375..7391
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7375..7391
                     /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 gcggcatgca gagtaataaa
     1681 acttttaact tggagaagca aaaccatact ccaagaaagc atcatcaaca tcaccaccag
     1741 cagcagcacc accagcagca acagcagcag ccgccaccac cgccaatacc tgcaaatggg
     1801 caacaggcca gcagccaaaa tgaaggcttg actattgacc tgaagaattt tagaaaacca
     1861 ggagagaaga ccttcaccca acgaagccgt ctttttgtgg gaaatcttcc tcccgacatc
     1921 actgaggaag aaatgaggaa actatttgag aaatatggaa aggcaggcga agtcttcatt
     1981 cataaggata aaggatttgg ctttatccgc ttggaaaccc gaaccctagc ggagattgcc
     2041 aaagtggagc tggacaatat gccactccgt ggaaagcagc tgcgtgtgcg ctttgcctgc
     2101 catagtgcat cccttacagt tcgaaacctt cctcagtatg tgtccaacga actgctggaa
     2161 gaagcctttt ctgtgtttgg ccaggtagag agggctgtag tcattgtgga tgatcgagga
     2221 aggccctcag gaaaaggcat tgttgagttc tcagggaagc cagctgctcg gaaagctctg
     2281 gacagatgca gtgaaggctc cttcctgcta accacatttc ctcgtcctgt gactgtggag
     2341 cccatggacc agttagatga tgaagaggga cttccagaga agctggttat aaaaaaccag
     2401 caatttcaca aggaacgaga gcagccaccc agatttgcac agcctggctc ctttgagtat
     2461 gaatatgcca tgcgctggaa ggcactcatt gagatggaga agcagcagca ggaccaagtg
     2521 gaccgcaaca tcaaggaggc tcgtgagaag ctggagatgg agatggaagc tgcacgccat
     2581 gagcaccagg tcatgctaat gagacaggat ttgatgaggc gccaagaaga acttcggagg
     2641 atggaagagc tgcacaacca agaggtgcaa aaacgaaagc aactggagct caggcaggag
     2701 gaagagcgca ggcgccgtga agaagagatg cggcggcagc aagaagaaat gatgcggcga
     2761 cagcaggaag gattcaaggg aaccttccct gatgcgctgc ctgtgtcagg gaatctgctt
     2821 gatgtgtaca gtagtcaagg cgtggccaca ccagccatca ctgtcagcaa ctcctgccca
     2881 gctgagctgc ccaacatcaa acgggagatc tctgagaccg aggcaaaggc ccttttgaag
     2941 gaacggcaga agaaagacaa tcacaaccta attgagcgtc gcaggcgatt caacattaac
     3001 gacaggatca aggaactggg cactctcatc cctaagtcca gtgacccgga gatgcgctgg
     3061 aacaagggca ccatcctgaa ggcctctgtg gattatatcc gcaagctgca gaaggagcag
     3121 cagcgctcca aagacctgga gagccggcag cgatccctgg agcaggccaa ccgcagcctg
     3181 cagctccgaa ttcaggaact agaactgcag gcccagatcc atggcctgcc agtacctccc
     3241 actccagggc tgctttcctt ggccacgact tcggcttctg acagcctcaa gccagagcag
     3301 ctggacattg aggaggaggg caggccaggc gcagcaacgt tccatgtagg ggggggacct
     3361 gcccagaatg ctccccatca gcagccccct gcaccgccct cagatgccct tctggacctg
     3421 cactttccca gcgaccacct gggggacctg ggagacccct tccacctggg gctggaggac
     3481 attctgatgg aggaggagga gggggtggtg ggaggactgt cggggggtgc cctgtcccca
     3541 ctgcgggctg cctccgatcc cctgctctct tcagtgtccc ctgctgtctc caaggccagc
     3601 agccgccgca gcagcttcag catggaagag gagtccatga tgatgatgaa taagttggtt
     3661 ctagcgcagt tcttcttctc ctgtctatga acctgcagaa gcttggccgc catggcccaa
     3721 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3781 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3841 tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag
     3901 aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     3961 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4021 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4081 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4141 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4201 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4261 aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt
     4321 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4381 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4441 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4501 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4561 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4621 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4681 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4741 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4801 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4861 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4921 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4981 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5041 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5101 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5161 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5221 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5281 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5341 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5401 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5461 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5521 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5581 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5641 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5701 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5761 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5821 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5881 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5941 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6001 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6061 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6121 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6181 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6241 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6301 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6361 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6421 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6481 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6541 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6601 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6661 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6721 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6781 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6841 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6901 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6961 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7021 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     7081 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     7141 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     7201 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     7261 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     7321 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     7381 acagctatga catgattacg aattaa
//
