LOCUS       sequence-471790-        6899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-SS18-SSX1-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6899)
  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 6899)
  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-SSX1-KS
FEATURES             Location/Qualifiers
     source          1..6899
                     /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      3199..3208
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3212..3346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3249..3268)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3303..3322
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3415..3772
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3415..3606
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3438..3458)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3623..3758
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3792..3809)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3792..3808)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3801..3823)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4021..4476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4108..4127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4318..4339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4493..4512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4612..4634
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4672..4690)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4758..4862
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4863..4931,4932..5723)
                     /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(5081..5100)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5894..6482
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6383..6402
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6636..6653
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6770..6791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6806..6811,6812..6829,6830..6836)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6844..6860
                     /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     6849..6871
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6868..6884
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6868..6884
                     /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 acgaaaatga ttcgaaggga
     2941 gtgtcagaag catctggccc acaaaacgat gggaaacaac tgcacccccc aggaaaagca
     3001 aatatttctg agaagattaa taagagatct ggacccaaaa gggggaaaca tgcctggacc
     3061 cacagactgc gtgagagaaa gcagctggtg atttatgaag agatcagtga ccctgaggaa
     3121 gatgacgaga tgatgatgat gaataagttg gttctagcgc agttcttctt ctcctgtcta
     3181 taaacctgca gaagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta
     3241 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3301 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga attaattcgg
     3361 cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg
     3421 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3481 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3541 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3601 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3661 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3721 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctgtt
     3781 aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     3841 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     3901 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     3961 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     4021 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4081 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4141 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4201 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     4261 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4321 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     4381 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4441 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     4501 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     4561 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4621 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4681 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4741 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4801 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4861 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4921 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4981 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5041 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5101 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5161 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5221 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5281 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5341 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5401 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5461 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5521 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5581 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5641 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5701 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5761 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5821 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5881 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5941 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6001 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6061 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6121 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6181 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6241 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6301 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6361 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6421 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6481 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6541 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6601 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6661 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6721 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6781 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6841 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaattaa
//
