LOCUS       sequence-474382-        4493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knock-in msfGFP to SRRM2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4493)
  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 4493)
  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-SRRM2-repair-template
FEATURES             Location/Qualifiers
     source          1..4493
                     /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(1312..1314,1315..1317,1318..2031)
                     /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(1357..1378)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1965..1986
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2496..2513)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2496..2512)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2505..2527)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2721..2740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2840..2862
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2900..2918)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2986..3090
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3091..3159,3160..3951)
                     /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(3309..3328)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4122..4493,1..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 tcgacgctgg
      661 ccctgtgtgg tgtgaggttt ggtggtcagg acctgggcgg gtggtcctga gtgctggccc
      721 gtgtgcttga aagggtgtgg tgctattggg tcctactgtg ttccccaggt cccgcagccc
      781 ccggaagcca atagactccc tcagggactc tcggtccctc agctactcgc ctgtggagcg
      841 tcgccgtccc tcgccccagc cctcaccacg ggaccagcag aggtaaggcc aactgcaggt
      901 gtcagcaccc agcctgtctg gccgccactg ctttctacaa agaagaaagc tttgcggttg
      961 tggctatgtg gtgcctgagg tggtgccacc ctgtggcctg atgtctgtcc tgtgttgcag
     1021 cagcagcagt gagcggggtt cccggagagg ccagcgtggg gacagccgct cccccagcca
     1081 caagcgcagg agggagacac ctagccctcg gcccatgaga caccgctcct ccaggtgcgt
     1141 gtcctggaag gctgatgccc ccttccggga gccagttgtg gtggtgggtg gcggccccat
     1201 tttgggagtg gcccagaaac tggccttgag ggctggggtg ggaactccct gttgaactat
     1261 atcttctctt gcaggtctcc aggaggaggc ggatccggag gcggaggatc catggtgagc
     1321 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1381 aacggccaca agttcagcgt gcgcggcgag ggcgagggcg atgccaccaa cggcaagctg
     1441 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1501 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1561 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc cttcaaggac
     1621 gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1681 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1741 tacaacttca acagccacaa cgtctatatc acggccgaca agcagaagaa cggcatcaag
     1801 gcgaacttca agatccgcca caacgtcgag gacggcagcg tgcagctcgc cgaccactac
     1861 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1921 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1981 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaata aattgtcttt
     2041 gggggattcc accacaccca atgctctgga gccacaagga gtgtcccttc ttccccagca
     2101 gagccgtggg agggtccttg tctgctctcc tttgaacctt ggcagccctt ggatggaggg
     2161 ctccctttcc ctcccctttt ttttttcttt gttcctgtga aatgttaatc tccgtgagtt
     2221 cttcctggtt catgtgttct ggggggtttg gggtgggagg gaatgcagat gggagttggg
     2281 ggaggggagg atacagttca ggatacccca gcctggagtc agggccaggg aggcatggcc
     2341 ccacttgtat ccagaagttc ccaggggtga ttgtgatggt ggttgggact ggaggttgta
     2401 taaggtgttc ttggaaggaa ggggcaggag ttggaattag ttggtcccta ctgtccccca
     2461 tgaggttgtg aacccctggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt
     2521 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2581 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2641 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2701 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     2761 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2821 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2881 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2941 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3001 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3061 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3121 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3181 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3241 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3301 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3361 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3421 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3481 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3541 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3601 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3661 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3721 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3781 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3841 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3901 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3961 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4021 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4081 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4141 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4201 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4261 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4321 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4381 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4441 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga act
//
