LOCUS       sequence-487105-        9007 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  For overexpression of CFP-LacI-NUT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9007)
  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 9007)
  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
FEATURES             Location/Qualifiers
     source          1..9007
                     /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    5795..5916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5832..5851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5886..5905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5923..6378)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6060..6081)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6272..6291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6405..6509
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6511..6868
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6511..6702
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     rep_origin      6719..6854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6903..7697
                     /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             6903..7697
                     /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(6957..6976)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7567..7586
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7885..7904)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7929..7976
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      8305..8893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8794..8813
                     /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 tagccagtga
     5641 cgtacgtcta gagggcccta ggaattcgag cgccgtcgac aagcttgcgg ccgcaatcag
     5701 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     5761 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     5821 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5881 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     5941 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     6001 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     6061 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     6121 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     6181 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6241 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     6301 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6361 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6421 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6481 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     6541 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     6601 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     6661 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     6721 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6781 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     6841 cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc
     6901 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     6961 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     7021 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     7081 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     7141 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     7201 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     7261 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     7321 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     7381 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     7441 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     7501 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     7561 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     7621 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     7681 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     7741 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     7801 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     7861 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     7921 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
     7981 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     8041 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     8101 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
     8161 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     8221 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     8281 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     8341 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     8401 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     8461 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     8521 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     8581 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     8641 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     8701 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     8761 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     8821 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     8881 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     8941 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     9001 catgcat
//
