LOCUS       sequence-471755-        6851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-SS18-SSX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  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 6851)
  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-SS18-SSX2
FEATURES             Location/Qualifiers
     source          1..6851
                     /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      3151..3160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3164..3298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3201..3220)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3255..3274
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3367..3724
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3367..3558
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3390..3410)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3575..3710
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3744..3761)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3744..3760)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3753..3775)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3973..4428
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4060..4079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4270..4291
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4445..4464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4564..4586
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4624..4642)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4710..4814
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4815..4883,4884..5675)
                     /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(5033..5052)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5846..6434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6335..6354
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6588..6605
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6722..6743
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6758..6763,6764..6781,6782..6788)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6796..6812
                     /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     6801..6823
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6820..6836
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6820..6836
                     /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 gcggcatgtc tgtggctttc
     1681 gcggccccga ggcagcgagg caagggggag atcactcccg ctgcgattca gaagatgttg
     1741 gatgacaata accatcttat tcagtgtata atggactctc agaataaagg aaagacctca
     1801 gagtgttctc agtatcagca gatgttgcac acaaacttgg tataccttgc tacaatagca
     1861 gattctaatc aaaatatgca gtctctttta ccagcaccac ccacacagaa tatgcctatg
     1921 ggtcctggag ggatgaatca gagcggccct cccccacctc cacgctctca caacatgcct
     1981 tcagatggaa tggtaggtgg gggtcctcct gcaccgcaca tgcagaacca gatgaacggc
     2041 cagatgcctg ggcctaacca tatgcctatg cagggacctg gacccaatca actcaatatg
     2101 acaaacagtt ccatgaatat gccttcaagt agccatggat ccatgggagg ttacaaccat
     2161 tctgtgccat catcacagag catgccagta cagaatcaga tgacaatgag tcagggacaa
     2221 ccaatgggaa actatggtcc cagaccaaat atgagtatgc agccaaacca aggtccaatg
     2281 atgcatcagc agcctccttc tcagcaatac aatatgccac agggaggcgg acagcattac
     2341 caaggacagc agccacctat gggaatgatg ggtcaagtta accaaggcaa tcatatgatg
     2401 ggtcagagac agattcctcc ctatagacct cctcaacagg gcccaccaca gcagtactca
     2461 ggccaggaag actattacgg ggaccaatac agtcatggtg gacaaggtcc tccagaaggc
     2521 atgaaccagc aatattaccc tgatggtcat aatgattacg gttatcagca accgtcgtat
     2581 cctgaacaag gctacgatag gccttatgag gattcctcac aacattacta cgaaggagga
     2641 aattcacagt atggccaaca gcaagatgca taccagggac cacctccaca acagggatat
     2701 ccaccccagc agcagcagta cccagggcag caaggttacc caggacagca gcagggctac
     2761 ggtccttcac agggtggtcc aggtcctcag tatcctaact acccacaggg acaaggtcag
     2821 cagtatggag gatatagacc aacacagcct ggaccaccac agccacccca gcagaggcct
     2881 tatggatatg accagatcat gcccaagaag ccagcagagg aaggaaatga ttcggaggaa
     2941 gtgccagaag catctggccc acaaaatgat gggaaagagc tgtgcccccc gggaaaacca
     3001 actacctctg agaagattca cgagagatct ggacccaaaa ggggggaaca tgcctggacc
     3061 cacagactgc gtgagagaaa acagctggtg atttatgaag agatcagcga ccctgaggaa
     3121 gatgacgagt aatgaacctg cagaagcttg gccgccatgg cccaacttgt ttattgcagc
     3181 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3241 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcgg
     3301 gaattaattc ggcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg
     3361 taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3421 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3481 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3541 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3601 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3661 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3721 caaaaagctg ttaacagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3781 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3841 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3901 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3961 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4021 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4081 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4141 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4201 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4261 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     4321 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4381 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4441 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4501 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4561 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4621 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4681 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4741 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4801 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4861 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4921 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4981 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5041 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5101 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5161 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5221 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5281 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5341 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5401 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5461 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5521 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5581 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5641 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5701 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5761 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5821 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5881 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5941 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6001 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6061 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6121 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6181 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6241 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6301 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6361 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6421 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6481 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6541 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6601 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6661 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6721 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     6781 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga
     6841 ttacgaatta a
//
