LOCUS       sequence-471796-        7355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-NONO-TFE3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7355)
  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 7355)
  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
FEATURES             Location/Qualifiers
     source          1..7355
                     /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      3655..3664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3668..3802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3705..3724)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3759..3778
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3871..4228
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3871..4062
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3894..3914)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4079..4214
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4248..4265)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4248..4264)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4257..4279)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4477..4932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4564..4583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4774..4795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4949..4968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5068..5090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5128..5146)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5214..5318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5319..5387,5388..6179)
                     /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(5537..5556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6350..6938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6839..6858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7092..7109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7226..7247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7262..7267,7268..7285,7286..7292)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7300..7316
                     /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     7305..7327
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7324..7340
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7324..7340
                     /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 gagtcctgaa cctgcagaag cttggccgcc
     3661 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3721 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3781 tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac
     3841 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt
     3901 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3961 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     4021 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4081 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4141 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4201 cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt
     4261 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4321 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4381 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4441 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4501 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4561 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4621 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4681 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4741 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4801 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     4861 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4921 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4981 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5041 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5101 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5161 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5221 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5281 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5341 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5401 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5461 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5521 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5581 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5641 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5701 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5761 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5821 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5881 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5941 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6001 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6061 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6121 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6181 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6241 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6301 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6361 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6421 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6481 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6541 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6601 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6661 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6721 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6781 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6841 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6901 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6961 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7021 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     7081 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     7141 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     7201 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     7261 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     7321 acacaggaaa cagctatgac atgattacga attaa
//
