LOCUS       sequence-474415-        6311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of V5-EGFP-NPM1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6311)
  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 6311)
  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_V5-EGFP-NPM1
FEATURES             Location/Qualifiers
     source          1..6311
                     /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      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             942..983
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(993..995,996..1706)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(993..995,996..1706)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1035..1056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1296..1315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1643..1664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2611..2620
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2624..2758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2661..2680)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2715..2734
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2827..3184
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2827..3018
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2850..2870)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3035..3170
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3204..3221)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3204..3220)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3213..3235)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3433..3888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3730..3751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3905..3924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4024..4046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4084..4102)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4170..4274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4275..4343,4344..5135)
                     /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(4493..4512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5306..5894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5795..5814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6048..6065
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6182..6203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6218..6223,6224..6241,6242..6248)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6256..6272
                     /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     6261..6283
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6280..6296
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6280..6296
                     /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 gtgccaccat gggcaagccc atccccaacc
      961 ccctgctggg cctggacagc accggaagcg gagtgagcaa gggcgaggag ctgttcaccg
     1021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1201 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1441 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1561 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1621 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1681 ctctcggcat ggacgagctg tacaagatgg aagattcgat ggacatggac atgagccccc
     1741 tgaggcccca gaactatctt ttcggttgtg aactaaaggc cgacaaagat tatcacttta
     1801 aggtggataa tgatgaaaat gagcaccagt tatctttaag aacggtcagt ttaggggctg
     1861 gtgcaaagga tgagttgcac attgttgaag cagaggcaat gaattacgaa ggcagtccaa
     1921 ttaaagtaac actggcaact ttgaaaatgt ctgtacagcc aacggtttcc cttgggggct
     1981 ttgaaataac accaccagtg gtcttaaggt tgaagtgtgg ttcagggcca gtgcatatta
     2041 gtggacagca cttagtagct gtggaggaag atgcagagtc agaagatgaa gaggaggagg
     2101 atgtgaaact cttaagtata tctggaaagc ggtctgcccc tggaggtggt agcaaggttc
     2161 cacagaaaaa agtaaaactt gctgctgatg aagatgatga cgatgatgat gaagaggatg
     2221 atgatgaaga tgatgatgat gatgattttg atgatgagga agctgaagaa aaagcgccag
     2281 tgaagaaatc tatacgagat actccagcca aaaatgcaca aaagtcaaat cagaatggaa
     2341 aagactcaaa accatcatca acaccaagat caaaaggaca agaatccttc aagaaacagg
     2401 aaaaaactcc taaaacacca aaaggaccta gttctgtaga agacattaaa gcaaaaatgc
     2461 aagcaagtat agaaaaaggt ggttctcttc ccaaagtgga agccaaattc atcaattatg
     2521 tgaagaattg cttccggatg actgaccaag aggctattca agatctctgg cagtggagga
     2581 agtctctttg agtcgacctg cagaagcttg gccgccatgg cccaacttgt ttattgcagc
     2641 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2701 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcgg
     2761 gaattaattc ggcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg
     2821 taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2881 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2941 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3001 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3061 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3121 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3181 caaaaagctg ttaacagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3241 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3301 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3361 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3421 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3481 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3721 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     3781 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3841 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3901 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3961 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4021 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4081 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4141 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4201 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4261 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4321 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4381 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4441 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4501 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4561 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4621 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4681 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4741 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4801 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4861 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4921 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4981 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5041 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5101 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5161 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5221 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5281 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5341 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5401 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5461 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5521 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5581 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5641 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5701 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5761 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5821 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5881 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5941 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6001 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6061 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6121 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6181 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     6241 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga
     6301 ttacgaatta a
//
