LOCUS       sequence-471754-        6878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-EWS-FLI1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6878)
  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 6878)
  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
FEATURES             Location/Qualifiers
     source          1..6878
                     /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      3178..3187
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3191..3325
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3228..3247)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3282..3301
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3394..3751
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3394..3585
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3417..3437)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3602..3737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3771..3788)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3771..3787)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3780..3802)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4000..4455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4087..4106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4297..4318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4472..4491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4591..4613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4651..4669)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4737..4841
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4842..4910,4911..5702)
                     /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(5060..5079)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5873..6461
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6362..6381
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6615..6632
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6749..6770
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6785..6790,6791..6808,6809..6815)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6823..6839
                     /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     6828..6850
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6847..6863
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6847..6863
                     /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 agctactact gaacctgcag aagcttggcc
     3181 gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3241 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3301 caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat
     3361 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
     3421 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3481 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3541 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3601 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3661 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3721 aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc
     3781 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     3841 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     3901 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     3961 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4021 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4081 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4141 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4201 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4261 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4321 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     4381 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4441 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4501 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4561 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4621 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4681 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4741 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4801 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4861 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4921 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4981 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5041 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5101 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5161 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5221 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5281 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5341 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5401 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5461 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5521 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5581 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5641 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5701 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5761 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5821 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5881 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5941 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6001 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6061 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6121 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6181 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6241 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6301 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6361 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6421 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6481 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6541 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6601 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     6661 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     6721 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     6781 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     6841 ttcacacagg aaacagctat gacatgatta cgaattaa
//
