LOCUS       sequence-471776-        8693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-YAP-MAMLD1-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8693)
  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 8693)
  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-YAP-MAMLD1-KS
FEATURES             Location/Qualifiers
     source          1..8693
                     /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      4993..5002
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5006..5140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5043..5062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5097..5116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5209..5566
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5209..5400
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5232..5252)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5417..5552
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5586..5603)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5586..5602)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5595..5617)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5815..6270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5902..5921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6112..6133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6287..6306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6406..6428
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6466..6484)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6552..6656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6657..6725,6726..7517)
                     /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(6875..6894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7688..8276
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8177..8196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8430..8447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8564..8585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8600..8605,8606..8623,8624..8630)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8638..8654
                     /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     8643..8665
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8662..8678
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8662..8678
                     /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 gcggcatgga tcccgggcag
     1681 cagccgccgc ctcaaccggc cccccagggc caagggcagc cgccttcgca gcccccgcag
     1741 gggcagggcc cgccgtccgg acccgggcaa ccggcacccg cggcgaccca ggcggcgccg
     1801 caggcacccc ccgccgggca tcagatcgtg cacgtccgcg gggactcgga gaccgacctg
     1861 gaggcgctct tcaacgccgt catgaacccc aagacggcca acgtgcccca gaccgtgccc
     1921 atgaggctcc ggaagctgcc cgactccttc ttcaagccgc cggagcccaa atcccactcc
     1981 cgacaggcca gtactgatgc aggcactgca ggagccctga ctccacagca tgttcgagct
     2041 cattcctctc cagcttctct gcagttggga gctgtttctc ctgggacact gacccccact
     2101 ggagtagtct ctggcccagc agctacaccc acagctcagc atcttcgaca gtcttctttt
     2161 gagatacctg atgatgtacc tctgccagca ggttgggaga tggcaaagac atcttctggt
     2221 cagagatact tcttaaatca catcgatcag acaacaacat ggcaggaccc caggaaggcc
     2281 atgctgtccc agatgaacgt cacagccccc accagtccac cagtgcagca gaatatgatg
     2341 aactcggctt caggtcctct tcctgatgga tgggaacaag ccatgactca ggatggagaa
     2401 atttactata taaaccataa gaacaagacc acctcttggc tagacccaag gcttgaccct
     2461 cgttttgcca tgaaccagag aatcagtcag agtgctccag tgaaacagcc accacccctg
     2521 gctccccaga gcccacaggg aggcgtcatg ggtggcagca actccaacca gcagcaacag
     2581 atgcgactgc agcaactgca gatggagaag gagaggctgc ggctgaaaca gcaagaactg
     2641 cttcggcagg caatgcggaa tatcaatccc agcacagcaa attctccaaa atgtcaggga
     2701 aagaagccct cgtggatgga ggaagaagat ttatcttttc tctacaagag cagcccagga
     2761 agaaagcatc agggaactgt taagaggaga caagaagaag accacttcca gtttccagac
     2821 atggctgatg ggggctaccc taataaaatt aagaggcctt gccttgaaga tgtcaccctt
     2881 gcaatgggcc caggtgctca tcctagtact gcttgtgcag aactgcaggt ccctccattg
     2941 acaataaatc ctagccctgc ggctatggga gtggctggcc agtcattact gctggagaat
     3001 aaccctatga atggcaacat catgggctca ccatttgtag taccacagac tacagaagtg
     3061 ggactgaaag ggcccactgt tccttactat gagaaaatca acagcgtgcc ggctgtagac
     3121 caggagcttc aagagctgct agaggagctc accaaaattc aagacccttc tccaaatgag
     3181 ctagatcttg agaagatact ggggacgaag ccagaagagc cactggtttt agatcatccc
     3241 caggcaaccc taagcacaac tcccaagcct tcggttcaga tgtcacactt ggagagcctg
     3301 gcttccagca aggagtttgc ttctagttgc agccaagtta ctggcatgtc acttcagatc
     3361 ccatcctcct ccacagggat cagctattcg attccttcca ccagtaagca gatagtgtca
     3421 ccgagttctt caatggcaca gtccaagagc caggtccagg ccatgctccc tgtcgctctg
     3481 ccccccttac cagtgcctca gtggcatcac gcccaccagc tgaaggcgtt ggcagccagc
     3541 aagcaggggt ctgctacaaa gcagcaaggg cccaccccca gttggtctgg tctgcctcct
     3601 ccaggactct ctccacctta ccgcccagtg ccatcaccac acccaccacc gctgccactg
     3661 ccaccaccac cacccccatt cagcccccag agcctcatgg tgtcctgcat gtcgtccaat
     3721 accttgtcgg gtagcactct ccgaggctct cccaatgcct tactgtcaag catgacgtcc
     3781 agcagcaatg ctgccctggg gcccgccatg ccctatgctc ctgagaagct ccccagccct
     3841 gctctcactc aacagccgca gttcggccct cagagctcca ttcttgccaa cctcatgtcc
     3901 tctaccatca aaacccctca aggacacctg atgtctgctc tgcctgccag caaccctggg
     3961 ccgtccccac cctatcgccc agagaagctc tctagcccag gcttgccaca gcagtccttc
     4021 accccacagt gttccctgat ccgaagcctc actcccacca gtaatcttct aagccagcaa
     4081 cagcagcagc agcagcagca gcagcaagca aatgtgatct ttaagcccat aagcagcaac
     4141 tcatccaaaa ccctgagcat gatcatgcag caggggatgg caagctccag cccaggagcc
     4201 acggagccat ttacttttgg caacaccaag cccttgtccc attttgtttc tgagccgggt
     4261 ccccagaaga tgccctccat gcctaccacc tctaggcagc cttccctgct ccactacctg
     4321 cagcagccga caccaacgca ggcctcctca gccactgcct cctccacggc cactgccacc
     4381 ttgcagctgc agcagcagca gcagcaacag cagcagcagc ctgaccattc ttcattcctt
     4441 ctgcagcaga tgatgcagca accccagcgt tttcagcgat cagtggcctc agattccatg
     4501 cctgctctgc ccagacaggg ctgttgccat ctgtttgcat ggacttctgc agctagctcg
     4561 gtgaagcccc agcatcaaca cgggaactct ttcactagca ggcaagatcc tcagcctgga
     4621 gacgtgtcac cgtctaacat tactcatgta gacaaagcct gcaagcttgg ggaagccagg
     4681 cacccccagg tcagcctcgg gcgacagccc ccgtcctgcc aggccctggg gagtgagtcc
     4741 ttcctgcccg gcagctcctt tgctcatgag ctggcccgag tcacctcctc gtacagcacc
     4801 tcagaggcag cgccctgggg cagctgggat ccgaaggcct ggaggcaggt gcccgctcca
     4861 ctactgccta gctgcgacgc cacagcaaga ggaacagaga tcaggtctta tggcaatgac
     4921 cccatgatga tgatgaataa gttggttcta gcgcagttct tcttctcctg tctatgaacc
     4981 tgcagaagct tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata
     5041 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5101 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc
     5161 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
     5221 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     5281 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     5341 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     5401 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     5461 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     5521 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc
     5581 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     5641 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     5701 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     5761 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     5821 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5881 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5941 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     6001 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     6061 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     6121 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     6181 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     6241 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     6301 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6361 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6421 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     6481 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     6541 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6601 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6661 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6721 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6781 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6841 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6901 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6961 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     7021 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     7081 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     7141 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     7201 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     7261 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     7321 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     7381 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7441 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7501 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7561 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7621 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7681 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7741 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7801 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7861 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7921 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7981 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     8041 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     8101 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     8161 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     8221 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     8281 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     8341 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     8401 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     8461 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     8521 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     8581 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     8641 tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat taa
//
