LOCUS       sequence-471777-        6848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-SS18-SSX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6848)
  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 6848)
  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
FEATURES             Location/Qualifiers
     source          1..6848
                     /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      3148..3157
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3161..3295
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3198..3217)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3252..3271
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3364..3721
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3364..3555
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3387..3407)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3572..3707
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3741..3758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3741..3757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3750..3772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3970..4425
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4057..4076)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4267..4288
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4442..4461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4561..4583
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4621..4639)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4707..4811
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4812..4880,4881..5672)
                     /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(5030..5049)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5843..6431
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6332..6351
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6585..6602
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6719..6740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6755..6760,6761..6778,6779..6785)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6793..6809
                     /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     6798..6820
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6817..6833
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6817..6833
                     /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 gatgacgagt aaacctgcag aagcttggcc gccatggccc aacttgttta ttgcagctta
     3181 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3241 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcgggaa
     3301 ttaattcggc gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac
     3361 cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3421 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3541 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3601 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3661 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3721 aaagctgtta acagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3781 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3841 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     3901 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     3961 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4021 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4081 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4141 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4201 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4261 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     4321 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4381 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4441 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4501 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4561 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     4621 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4681 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4741 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4801 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4861 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4921 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4981 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5041 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5101 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5161 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5221 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5281 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5341 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5401 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5461 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5521 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5581 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5641 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5701 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5761 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5821 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5881 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5941 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6001 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6061 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6121 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6181 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6241 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6301 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6361 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6421 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6481 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6541 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6601 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6661 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6721 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     6781 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta
     6841 cgaattaa
//
