LOCUS       sequence-471719-        4909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knock-in msfGFP to TCOF1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4909)
  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 4909)
  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-TCOF1-repair-template
FEATURES             Location/Qualifiers
     source          1..4909
                     /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(1548..1550,1551..1553,1554..2264)
                     /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(1593..1614)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2201..2222
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2912..2929)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2912..2928)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2921..2943)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3137..3156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3256..3278
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3316..3334)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3402..3506
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3507..3575,3576..4367)
                     /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(3725..3744)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4538..4909,1..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 cctcttcccc
      661 acctcacccc accccacctg gacagcccag cagcgcagac taggggttct gggtttaggg
      721 ttcccaggca atagcagggt gttttctcat gcctagactc atgcagtgcc ctctgccagt
      781 cctctccttt gcacccagca atttcgggtg tctttatgcc tcagcttaga tatctctctg
      841 gggttcggtg ctcctccttg gggcgcccat aggctgcatc tgtccctgac aaggcacaca
      901 agatattgaa ctatagttgc ttggttacgg acagtcgtct ctgtgagctt tgagggcaac
      961 tgccatgtat tatgcatctt ttgaatcccc ctagcctagc cgggtgctag cccaagttag
     1021 gtgcttatta cagatttgtt cagttagcga atgaatcaat gaataaactg gcaattcgtg
     1081 tctggttcat gggcaccaaa aaccgtgcga aatggataaa tgagcaggta atggcagtac
     1141 aaagttcgga tctagagaaa gatccagatt agggagttca gagatgagta aaacgcagac
     1201 ccagccatca gagaattaac aaggaagata aatctcttca agaagccagc cggaaggatt
     1261 aaaagagagt cgtccaagtc ccggccggcc ccgggggcgg gtccctgtct agccccgcat
     1321 gctccgcgcc aatgggcggt attgttgatc acgagtctcc gcccccttcg ccttgagggg
     1381 cggggccggc cactcccgga gaggggacta cgtttcccgg cgcgccgcgc ggccggagcg
     1441 aaagaggagc cggaagtggg gcgcgcgagg tctaagggcg cgagaaaagt ggcgggcggg
     1501 gactaaggcg gggcgtgcag gtagccggcc ggccgggggt cgcgggtatg gtgagcaagg
     1561 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1621 gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     1681 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1741 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1801 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctccttc aaggacgacg
     1861 gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1921 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1981 acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc atcaaggcga
     2041 acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac cactaccagc
     2101 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     2161 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     2221 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaaggagga ggcggatccg
     2281 gaggcggagg atccgccgag gccaggaagc ggcgggagct acttcccctg atctaccacc
     2341 atctgctgcg ggctggctat gtgcgtgcgg cgcgggaagt gaaggagcag agcggccagg
     2401 taagcgttcg tgggccgtgt gcgagggccg cgtgcaagat gtggagatca gcggcccgcg
     2461 gcccgcgccc cgtccccagg cgacccggca ggcgcccgga gccgggtccc gcagtgctcg
     2521 acggcgcggc caggggtacc ggaggagccg caatctctgc cttcccactg cgacttcagt
     2581 tcccctgggc ctcactttcc tcatccgggc agtgggttgg gccagaaagt gtgctcgcag
     2641 gggccgaact ttgtaattcc cgcttccctt acctccacgc ccttcctggt agcgggtatt
     2701 ttaagtttcc taagtctccc gccacgtggc taggctctgc gcggcccccc ctggggcaag
     2761 gaggttgctg cgagtctcgg gggtggattg cgaccagccc ctcgggggag cgcctggaaa
     2821 agggacccta cagcctctta gttgagctgg aactcaggcc tttagcgatt gtctgtactg
     2881 agacggtcag ctttgtaccg agctcgaatt cactggccgt cgttttacaa cgtcgtgact
     2941 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3001 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3061 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3121 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     3181 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3241 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3301 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3361 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3421 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3481 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3541 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3601 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3661 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3721 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3781 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3841 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3901 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3961 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     4021 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     4081 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4141 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4201 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4261 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4321 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4381 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4441 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4501 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4561 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4621 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4681 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4741 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4801 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4861 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaact
//
