LOCUS       sequence-476326-        5623 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of CFP-LacI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5623)
  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 5623)
  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: CFP-LacI
FEATURES             Location/Qualifiers
     source          1..5623
                     /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    2411..2532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2448..2467)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2502..2521
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2539..2994)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2676..2697)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2888..2907
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3021..3125
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3127..3484
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3127..3318
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     rep_origin      3335..3470
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3519..4313
                     /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             3519..4313
                     /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(3573..3592)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4183..4202
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4501..4520)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4545..4592
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4921..5509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5410..5429
                     /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 atcgcttaac gtacgtctag agggcccgaa
     2281 ttcgagcgcc gtcgacaagc ttgcggccgc aatcagccat accacatttg tagaggtttt
     2341 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2401 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2461 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2521 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2581 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2641 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2701 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2761 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2821 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2881 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2941 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3001 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3061 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3121 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3181 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3241 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3301 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3361 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3421 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3481 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3541 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3601 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3661 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     3721 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3781 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3841 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3901 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3961 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4021 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4081 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4141 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4201 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4261 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4321 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4381 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4441 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     4501 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     4561 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     4621 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     4681 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     4741 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     4801 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4861 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4921 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4981 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5041 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5101 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5161 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5221 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5281 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5341 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5401 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5461 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5521 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5581 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
