LOCUS       sequence-474438-        6314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of ALFA-EGFP-NPM1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  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 6314)
  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_ALFA-EGFP-NPM1
FEATURES             Location/Qualifiers
     source          1..6314
                     /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"
     CDS             945..983
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             join(996..998,999..1709)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(996..998,999..1709)
                     /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(1038..1059)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1299..1318)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1646..1667
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2614..2623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2627..2761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2664..2683)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2718..2737
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2830..3187
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2830..3021
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2853..2873)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3038..3173
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3207..3224)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3207..3223)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3216..3238)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3436..3891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3523..3542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3733..3754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3908..3927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4027..4049
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4087..4105)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4173..4277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4278..4346,4347..5138)
                     /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(4496..4515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5309..5897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5798..5817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6051..6068
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6185..6206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6221..6226,6227..6244,6245..6251)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6259..6275
                     /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     6264..6286
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6283..6299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6283..6299
                     /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 gccttccaga ctcgaagagg
      961 aattgagaag gagattgacc gaacctggaa gcggagtgag caagggcgag gagctgttca
     1021 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1081 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1141 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1201 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1261 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1321 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1381 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1441 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1501 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1561 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     1621 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1681 tcactctcgg catggacgag ctgtacaaga tggaagattc gatggacatg gacatgagcc
     1741 ccctgaggcc ccagaactat cttttcggtt gtgaactaaa ggccgacaaa gattatcact
     1801 ttaaggtgga taatgatgaa aatgagcacc agttatcttt aagaacggtc agtttagggg
     1861 ctggtgcaaa ggatgagttg cacattgttg aagcagaggc aatgaattac gaaggcagtc
     1921 caattaaagt aacactggca actttgaaaa tgtctgtaca gccaacggtt tcccttgggg
     1981 gctttgaaat aacaccacca gtggtcttaa ggttgaagtg tggttcaggg ccagtgcata
     2041 ttagtggaca gcacttagta gctgtggagg aagatgcaga gtcagaagat gaagaggagg
     2101 aggatgtgaa actcttaagt atatctggaa agcggtctgc ccctggaggt ggtagcaagg
     2161 ttccacagaa aaaagtaaaa cttgctgctg atgaagatga tgacgatgat gatgaagagg
     2221 atgatgatga agatgatgat gatgatgatt ttgatgatga ggaagctgaa gaaaaagcgc
     2281 cagtgaagaa atctatacga gatactccag ccaaaaatgc acaaaagtca aatcagaatg
     2341 gaaaagactc aaaaccatca tcaacaccaa gatcaaaagg acaagaatcc ttcaagaaac
     2401 aggaaaaaac tcctaaaaca ccaaaaggac ctagttctgt agaagacatt aaagcaaaaa
     2461 tgcaagcaag tatagaaaaa ggtggttctc ttcccaaagt ggaagccaaa ttcatcaatt
     2521 atgtgaagaa ttgcttccgg atgactgacc aagaggctat tcaagatctc tggcagtgga
     2581 ggaagtctct ttgagtcgac ctgcagaagc ttggccgcca tggcccaact tgtttattgc
     2641 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2701 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     2761 cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt
     2821 aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     2881 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2941 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3001 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3061 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3121 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3181 ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa
     3241 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     3301 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     3361 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3421 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3481 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3541 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3601 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3661 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3721 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     3781 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3841 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3901 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3961 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4021 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     4081 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4141 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4201 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4261 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4321 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4381 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4441 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4501 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4561 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4621 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4681 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4741 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4801 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4861 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4921 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4981 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5041 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5101 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5161 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5221 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5281 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5341 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5401 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5461 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5521 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5581 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5641 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5701 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5761 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5821 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5881 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5941 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6001 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6061 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6121 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     6181 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     6241 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca
     6301 tgattacgaa ttaa
//
