LOCUS       sequence-472212-        6317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-NPM1-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6317)
  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 6317)
  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-NPM1-KS
FEATURES             Location/Qualifiers
     source          1..6317
                     /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      2617..2626
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2630..2764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2667..2686)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2721..2740
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2833..3190
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2833..3024
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2856..2876)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3041..3176
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3210..3227)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3210..3226)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3219..3241)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3439..3894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3526..3545)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3736..3757
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3911..3930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4030..4052
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4090..4108)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4176..4280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4281..4349,4350..5141)
                     /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(4499..4518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5312..5900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5801..5820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6054..6071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6188..6209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6224..6229,6230..6247,6248..6254)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6262..6278
                     /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     6267..6289
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6286..6302
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6286..6302
                     /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 agattcgatg
     1681 gacatggaca tgagccccct gaggccccag aactatcttt tcggttgtga actaaaggcc
     1741 gacaaagatt atcactttaa ggtggataat gatgaaaatg agcaccagtt atctttaaga
     1801 acggtcagtt taggggctgg tgcaaaggat gagttgcaca ttgttgaagc agaggcaatg
     1861 aattacgaag gcagtccaat taaagtaaca ctggcaactt tgaaaatgtc tgtacagcca
     1921 acggtttccc ttgggggctt tgaaataaca ccaccagtgg tcttaaggtt gaagtgtggt
     1981 tcagggccag tgcatattag tggacagcac ttagtagctg tggaggaaga tgcagagtca
     2041 gaagatgaag aggaggagga tgtgaaactc ttaagtatat ctggaaagcg gtctgcccct
     2101 ggaggtggta gcaaggttcc acagaaaaaa gtaaaacttg ctgctgatga agatgatgac
     2161 gatgatgatg aagaggatga tgatgaagat gatgatgatg atgattttga tgatgaggaa
     2221 gctgaagaaa aagcgccagt gaagaaatct atacgagata ctccagccaa aaatgcacaa
     2281 aagtcaaatc agaatggaaa agactcaaaa ccatcatcaa caccaagatc aaaaggacaa
     2341 gaatccttca agaaacagga aaaaactcct aaaacaccaa aaggacctag ttctgtagaa
     2401 gacattaaag caaaaatgca agcaagtata gaaaaaggtg gttctcttcc caaagtggaa
     2461 gccaaattca tcaattatgt gaagaattgc ttccggatga ctgaccaaga ggctattcaa
     2521 gatctctggc agtggaggaa gtctcttatg atgatgatga ataagttggt tctagcgcag
     2581 ttcttcttct cctgtctatg aacctgcaga agcttggccg ccatggccca acttgtttat
     2641 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2701 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2761 gatcgggaat taattcggcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg
     2821 gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     2881 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2941 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3001 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3061 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3121 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3181 cttttgcaaa aagctgttaa cagcttggca ctggccgtcg ttttacaacg tcgtgactgg
     3241 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3301 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3361 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3421 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3481 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3541 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3601 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3661 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3721 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3781 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3841 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3901 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3961 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4021 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4081 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4141 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4201 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4261 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4321 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4381 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4441 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4501 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4561 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4621 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4681 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4741 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4801 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4861 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4921 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4981 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5041 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5101 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5161 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5221 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5281 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5341 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5401 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5461 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5521 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5581 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5641 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5701 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5761 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5821 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5881 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5941 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6001 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6061 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6121 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6181 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     6241 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6301 acatgattac gaattaa
//
