LOCUS       sequence-471785-        6929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-EWS-FLI1-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6929)
  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 6929)
  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-EWS-FLI1-KS
FEATURES             Location/Qualifiers
     source          1..6929
                     /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      3229..3238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3242..3376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3279..3298)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3333..3352
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3445..3802
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3445..3636
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3468..3488)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3653..3788
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3822..3839)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3822..3838)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3831..3853)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4051..4506
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4138..4157)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4348..4369
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4523..4542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4642..4664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4702..4720)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4788..4892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4893..4961,4962..5753)
                     /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(5111..5130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5924..6512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6413..6432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6666..6683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6800..6821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6836..6841,6842..6859,6860..6866)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6874..6890
                     /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     6879..6901
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6898..6914
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6898..6914
                     /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 gcggcatggc gtccacggat
     1681 tacagtacct atagccaagc tgcagcgcag cagggctaca gtgcttacac cgcccagccc
     1741 actcaaggat atgcacagac cacccaggca tatgggcaac aaagctatgg aacctatgga
     1801 cagcccactg atgtcagcta tacccaggct cagaccactg caacctatgg gcagaccgcc
     1861 tatgcaactt cttatggaca gcctcccact ggttatacta ctccaactgc cccccaggca
     1921 tacagccagc ctgtccaggg gtatggcact ggtgcttatg ataccaccac tgctacagtc
     1981 accaccaccc aggcctccta tgcagctcag tctgcatatg gcactcagcc tgcttatcca
     2041 gcctatgggc agcagccagc agccactgca cctacaagac cgcaggatgg aaacaagccc
     2101 actgagacta gtcaacctca atctagcaca gggggttaca accagcccag cctaggatat
     2161 ggacagagta actacagtta tccccaggta cctgggagct accccatgca gccagtcact
     2221 gcacctccat cctaccctcc taccagctat tcctctacac agccgactag ttatgatcag
     2281 agcagttact ctcagcagaa cacctatggg caaccgagca gctatggaca gcagagtagc
     2341 tatggtcaac aaagcagcta tgggcagcag cctcccacta gttacccacc ccaaactgga
     2401 tcctacagcc aagctccaag tcaatatagc caacagagca gcagctacgg gcagcagaac
     2461 ccttcttatg actcagtcag aagaggagca tggggcaata acatgaattc tggcctcaac
     2521 aaaagtcctc cccttggagg ggcacaaacg atcagtaaga atacagagca acggccccag
     2581 ccagatccgt atcagatcct gggcccgacc agcagtcgcc tagccaaccc tggaagcggg
     2641 cagatccagc tgtggcaatt cctcctggag ctgctctccg acagcgccaa cgccagctgt
     2701 atcacctggg aggggaccaa cggggagttc aaaatgacgg accccgatga ggtggccagg
     2761 cgctggggcg agcggaaaag caagcccaac atgaattacg acaagctgag ccgggccctc
     2821 cgttattact atgataaaaa cattatgacc aaagtgcacg gcaaaagata tgcttacaaa
     2881 tttgacttcc acggcattgc ccaggctctg cagccacatc cgaccgagtc gtccatgtac
     2941 aagtaccctt ctgacatctc ctacatgcct tcctaccatg cccaccagca gaaggtgaac
     3001 tttgtccctc cccatccatc ctccatgcct gtcacttcct ccagcttctt tggagccgca
     3061 tcacaatact ggacctcccc cacgggggga atctacccca accccaacgt cccccgccat
     3121 cctaacaccc acgtgccttc acacttaggc agctactaca tgatgatgat gaataagttg
     3181 gttctagcgc agttcttctt ctcctgtcta tgaacctgca gaagcttggc cgccatggcc
     3241 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3301 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3361 ttatcatgtc tggatcggga attaattcgg cgcagcacca tggcctgaaa taacctctga
     3421 aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca
     3481 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3541 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3601 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3661 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3721 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3781 tggaggccta ggcttttgca aaaagctgtt aacagcttgg cactggccgt cgttttacaa
     3841 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3901 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3961 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4021 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4081 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4141 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4201 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4261 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4321 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4381 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4441 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     4501 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     4561 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4621 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4681 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4741 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4801 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4861 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4921 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4981 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5041 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5101 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5161 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5221 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5281 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5341 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5401 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5461 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5521 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5581 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5641 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5701 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5761 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5821 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5881 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5941 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6001 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6061 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6121 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6181 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6241 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6301 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6361 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6421 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6481 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6541 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     6601 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     6661 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     6721 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     6781 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     6841 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     6901 gaaacagcta tgacatgatt acgaattaa
//
