LOCUS       sequence-474568-        4665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knock-in msfGFP to NPM1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4665)
  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 4665)
  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: pUC19_msfGFP-NPM1-repair-template for U2OS
FEATURES             Location/Qualifiers
     source          1..4665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(1356..1358,1359..1361,1362..2072)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1401..1422)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2009..2030
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2668..2685)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2668..2684)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2677..2699)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2893..2912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3012..3034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3072..3090)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3158..3262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3263..3331,3332..4123)
                     /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(3481..3500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4294..4665,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactaact
      661 acatctttcc ttcctaacaa agaataagcc gcaattcact ctctctggca cctgaacttt
      721 ggggtaacga ttaactgcga tccgaagcct tcaggcaagt aaaaaattcc tgaagtgatt
      781 acgcctgttt ggaggcttgc agggcactag gggatgggga aaggtgaatc gaggtgctct
      841 ctggctcatt cgcagccggc taacccgcca tatcttacac agctaagggc tgccgacgcc
      901 attttgcagg gtgggctgcg cagactcttg gcgggaggcc ggcgcgcttg agcgggagag
      961 gacgtggaag ggttggaggt ggggagtgcg cgttacgact ggaaagcacg cgtgcgcaca
     1021 ggcgacagca gcggaggggt ggggccagtg tacgagtgcg cgtgctcggt gggagcccgc
     1081 ggagtacctg gaaggaggtg ggggcgaggt agaaaggagt ggggttgaaa agcgcttgcg
     1141 caggacggct acggtacggg ggtgggaggg cttcggagca cgcgcgcgga ggcgggactt
     1201 gggaagcgct cgcgagatct tcagggtcta tatataagcg cggggagcct gcgtcctttc
     1261 cctggtgtga ttccgtcctg cgcggttgtt ctctggagca gcgttctttt atctccgtcc
     1321 gccttctctc ctacctaagt gcgtgccgcc acccgatggt gagcaagggc gaggagctgt
     1381 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1441 gcgtgcgcgg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg aagttcatct
     1501 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1561 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1621 tgcccgaagg ctacgtccag gagcgcacca tctccttcaa ggacgacggc acctacaaga
     1681 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1741 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac ttcaacagcc
     1801 acaacgtcta tatcacggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc
     1861 gccacaacgt cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1921 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga
     1981 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2041 ggatcactct cggcatggac gagctgtaca aaggaggagg cggatccgga ggcggaggat
     2101 ccgaagattc gatggacatg gacatgagcc ccctgaggcc ccagaactat cttttcggta
     2161 actgctgggg gtagctggag cgaggccgag cggggcctgg tggcggtgag ggtgggggtg
     2221 aggggcggga atccggctgc aaccgggtct ggtggagacc gccagaccga cgggaggcct
     2281 gagcgggtgg gaagagctgc ctgagccctg gacctggagg gattggagcc gcggggggag
     2341 ccggtggcgt gaagggggag gagacgagcg gcctgaagcg tctggggcgt gagcggtccg
     2401 aggcccgggg cctgaggtaa agtggaaccg gccgcctgga agttcggcct ttgccgggga
     2461 tacgtggcgg agctccaggc gtggtttatt ttctaacggc ggtggttcat aggcttctga
     2521 tgccaggccc cgggttggtg gtcctggagg ctgggttcac cgggaagcat gggcctgctt
     2581 gttggagcgg gtagatgcac agcctggcgt tattctggcc tcagagcctg gagctgccgg
     2641 acgtagactg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga
     2701 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2761 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2821 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     2881 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     2941 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3001 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3061 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3121 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3181 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3241 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3301 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3361 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3421 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3481 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3541 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3601 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3661 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3721 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3781 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3841 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3901 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3961 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4021 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4081 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4141 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4201 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4261 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4321 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4381 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4441 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4501 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4561 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4621 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaact
//
