LOCUS       sequence-487107-        9058 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  For overexpression of CFP-LacI-NUT-KS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9058)
  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 9058)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 24, 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: CFP-LacI-NUT-KS
FEATURES             Location/Qualifiers
     source          1..9058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      199..334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      367..376
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(373..375,376..378,379..1089)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(418..439)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(679..698)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1026..1047
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1127..1146)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2188..2208
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5846..5967
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5883..5902)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5937..5956
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5974..6429)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6111..6132)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6323..6342
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6456..6560
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6562..6919
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6562..6753
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     rep_origin      6770..6905
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6954..7748
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6954..7748
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7008..7027)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7618..7637
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7936..7955)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7980..8027
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      8356..8944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8845..8864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattac tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
       61 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
      121 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
      181 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
      241 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
      301 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctagcgcta
      361 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      421 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      481 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      541 ccctggccca ccctcgtgac caccctgacc tggggcgtgc agtgcttcag ccgctacccc
      601 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      661 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      721 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      781 atcctggggc acaagctgga gtacaactac atcagccaca acgtctatat caccgccgac
      841 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcagc
      901 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
      961 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1021 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1081 ctgtacaagt ccggactcag atccatggtg aaaccagtaa cgttatacga tgtcgcagag
     1141 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     1201 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     1261 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     1321 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     1381 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     1441 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     1501 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     1561 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     1621 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     1681 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     1741 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     1801 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     1861 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     1921 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     1981 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     2041 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     2101 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2161 caggtttccc gaagcagcct gaggcctcct aagaagaaga ggaaggttgg tgcaccgggt
     2221 tctgcgggtt ctgccgcagg tggatccgcg atcgccgcat ctgcattgcc gggaccggat
     2281 atgagcatga aacctagtgc cgccctgtct ccatcccctg cacttccctt tctcccacca
     2341 acttctgacc caccagacca cccacccagg gagccacctc cacagcccat catgccttca
     2401 gtattctctc cagacaaccc tctgatgctc tctgctttcc ccagctcact gttggtgaca
     2461 ggggacgggg gcccttgcct cagtggggct ggggctggca aggtcattgt caaagtcaag
     2521 acagaagggg ggtcagctga gccctctcaa actcagaact ttatccttac tcagactgcc
     2581 ctcaattcga ctgccccggg cactccctgt ggaggccttg agggtcctgc acctccattt
     2641 gtgacagcat ctaatgtgaa gaccattctg ccctctaagg ctgttggtgt cagccaggag
     2701 ggtcctccag gccttccgcc tcagcctcca ccaccagttg ctcaactggt ccccattgtg
     2761 cccctggaaa aagcttggcc agggccacat gggacaaccg gggaaggagg tcctgtggcc
     2821 actctatcca agccttccct aggtgaccgc tccaaaattt ccaaggacgt ttatgagaac
     2881 ttccgtcagt ggcagcgtta caaagccttg gcccggaggc acctatccca gagtcctgac
     2941 acagaagctc tttcctgttt tcttatccca gtgcttcgtt ccctggcccg gctgaagccc
     3001 actatgaccc tggaggaggg actgccattg gctgtgcagg agtgggagca caccagcaac
     3061 tttgaccgga tgatctttta tgagatggca gaaaggttca tggagtttga ggctgaggag
     3121 atgcagattc agaacacaca gctgatgaat gggtctcagg gcctgtctcc tgcaacccct
     3181 ttgaaacttg atcctctagg gcccctggcc tctgaggttt gccagcagcc agtgtacatt
     3241 ccgaagaagg cagcttctaa aacaagagct cctcgccggc gtcagcgtaa agcccagaga
     3301 cctcctgctc ctgaggcacc caaggagatc ccaccagaag ctgtgaagga gtatgttgac
     3361 atcatggaat ggctggtggg gactcacttg gccactgggg agtcagatgg aaaacaagag
     3421 gaagaagggc agcagcagga ggaggaaggg atgtatccag atccaggtct cctgagctac
     3481 atcaatgagc tgtgttctca gaaggtcttt gtctccaagg tggaggctgt cattcaccct
     3541 caatttctgg cagatctgct gtccccagaa aaacagagag atcccttggc cttaattgag
     3601 gagctagagc aagaagaagg actcactctt gcccagctgg tccagaagcg actcatggcc
     3661 ttggaagagg aggaagatgc agaggcgcct ccaagtttca gtggcgctca gttggactca
     3721 agtccttctg gttctgttga ggatgaagat ggggatgggc ggcttcggcc ctcacctggg
     3781 cttcaggggg ctgggggcgc cgcttgcctt ggaaaggttt cttcttcagg aaaacgggca
     3841 agagaagtgc atggtgggca ggagcaagcc ctagatagcc ccagagggat gcacagggat
     3901 gggaacactc tgccatcccc cagcagctgg gacctgcagc cagaacttgc agctccacag
     3961 ggaactccgg gacccttggg tgtggagagg agagggtctg ggaaggttat aaaccaggta
     4021 tctctacatc aggatggcca tctaggaggc gctgggcctc ctgggcactg cctggtggct
     4081 gataggactt cagaggctct gcccctttgt tggcagggag gcttccagcc tgagagcact
     4141 cccagtttgg atgctggact tgcagagctg gctcctctgc aaggacaagg gttagaaaag
     4201 caagtcctgg gattgcagaa aggacaacaa acagggggtc gtggagtgct tcctcaaggg
     4261 aaggagcctt tagcagtgcc ctgggaaggc tcttcaggag ccatgtgggg agatgacaga
     4321 ggtaccccca tggctcagag ttatgatcag aatccttccc ctagagcagc tggggagagg
     4381 gacgatgtct gtctcagccc aggagtttgg ctgagcagtg agatggatgc tgtaggcttg
     4441 gagctgcctg tacaaataga ggaggtcata gagagcttcc aagttgagaa gtgtgtaact
     4501 gagtatcagg aaggctgcca gggactgggc tccaggggca acatttccct gggtcctgga
     4561 gaaaccctag tacctgggga tacggagagc agtgtgattc cctgtggagg cacagttgcg
     4621 gcagctgccc tagaaaagag aaactattgc agcttgccag gacctttgag ggccaacagc
     4681 ccacccttga ggtccaaaga aaatcaagaa cagagctgtg aaaccgtagg gcatcccagt
     4741 gatctgtggg cagaaggctg cttcccattg ctagaaagtg gtgattccac actggggtct
     4801 tccaaagaaa cccttccacc cacatgccaa ggcaatctcc ttatcatggg gactgaggat
     4861 gcctcctcct tgcctgaagc cagtcaagag gcagggagca gaggcaattc cttttctcct
     4921 ctgttggaaa ccatagaacc tgtcaacata ctagatgtta aagatgactg tggcctccaa
     4981 ctaagggtca gcgaggacac ctgcccactg aatgttcatt cttatgaccc ccaaggagaa
     5041 ggcagggtgg atcctgatct gtccaagcct aaaaaccttg ctcctttaca agagagtcag
     5101 gagtcttaca caactgggac tcccaaagca acatcttctc accagggcct tggaagcact
     5161 ttgcctagaa ggggaaccag gaatgccata gttccgagag aaacttctgt tagtaaaaca
     5221 cacaggtcag cagacagggc caaaggaaag gagaaaaaga aaaaggaagc agaggaagag
     5281 gatgaggaac tctccaactt tgcttacctc ttggcctcta aacttagcct ctcaccaagg
     5341 gagcatcccc tcagtcctca ccatgcctca ggaggtcagg gcagccagag agcatcccac
     5401 ctgctccctg ctggagcaaa aggccccagc aaacttccat atcctgttgc caagtctggg
     5461 aagcgagctc tagctggagg tccagcccct actgaaaaga caccccactc aggagctcaa
     5521 cttggggtcc ccagggagaa acccctagct ctgggagtag ttcgaccctc acagcctcgt
     5581 aaaaggcggt gtgacagttt tgtcacgggc agaaggaaga aacgacgtcg tagccagatg
     5641 atgatgatga ataagttggt tctagcgcag ttcttcttct cctgtctatg acgtacgtct
     5701 agagggccct aggaattcga gcgccgtcga caagcttgcg gccgcaatca gccataccac
     5761 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     5821 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     5881 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5941 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     6001 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6061 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6121 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     6181 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6241 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6301 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6361 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6421 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6481 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6541 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     6601 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     6661 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     6721 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     6781 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     6841 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     6901 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     6961 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     7021 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     7081 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     7141 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     7201 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     7261 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     7321 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     7381 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     7441 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     7501 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     7561 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     7621 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     7681 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     7741 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     7801 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     7861 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     7921 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     7981 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     8041 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     8101 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     8161 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     8221 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     8281 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     8341 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     8401 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     8461 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     8521 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     8581 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     8641 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     8701 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     8761 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     8821 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     8881 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     8941 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     9001 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
