LOCUS       sequence-471774-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-SS18-SSX2-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6902)
  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 6902)
  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-KS
FEATURES             Location/Qualifiers
     source          1..6902
                     /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      3202..3211
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3215..3349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3252..3271)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3306..3325
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3418..3775
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3418..3609
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3441..3461)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3626..3761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3795..3812)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3795..3811)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3804..3826)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4024..4479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4111..4130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4321..4342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4496..4515)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4615..4637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4675..4693)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4761..4865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4866..4934,4935..5726)
                     /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(5084..5103)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5897..6485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6386..6405
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6639..6656
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6773..6794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6809..6814,6815..6832,6833..6839)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6847..6863
                     /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     6852..6874
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6871..6887
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6871..6887
                     /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 gatgacgaga tgatgatgat gaataagttg gttctagcgc agttcttctt ctcctgtcta
     3181 taatgaacct gcagaagctt ggccgccatg gcccaacttg tttattgcag cttataatgg
     3241 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3301 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg ggaattaatt
     3361 cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg
     3421 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3481 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3541 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3601 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3661 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3721 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3781 gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     3841 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     3901 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     3961 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4021 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4081 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4141 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4201 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4261 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4321 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     4381 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4441 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     4501 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4561 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4621 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4681 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4741 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4801 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4861 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4921 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4981 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5041 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5101 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5161 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5221 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5281 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5341 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5401 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5461 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5521 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5581 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5641 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5701 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5761 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5821 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5881 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5941 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6001 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6061 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6121 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6181 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6241 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6301 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6361 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6421 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6481 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6541 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     6601 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6661 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     6721 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     6781 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     6841 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt
     6901 aa
//
