LOCUS       sequence-474402-        4899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knock-in msfGFP to HP1a locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4899)
  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 4899)
  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-HP1a-repair-template
FEATURES             Location/Qualifiers
     source          1..4899
                     /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(1233..1235,1236..1238,1239..1949)
                     /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(1278..1299)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1886..1907
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2902..2919)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2902..2918)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2911..2933)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3127..3146)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3246..3268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3306..3324)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3392..3496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3497..3565,3566..4357)
                     /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(3715..3734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4528..4899,1..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 caataaagac ctctatttac ctcagaggaa
      661 aaagtttttg ttttgttttg tttttgtttt tgagacaggg tttctctgta tagccctggc
      721 tgtcctagaa ctcactttgt agaccaggct gaccttgaac tcagaaatct gcctgcatct
      781 tcctcccaag tgctgggatg aaaggtgtgt gctaccacgc ccatcgagga aaaagatttt
      841 ggttccttta attgaaagtt tcccacattt ctctttgagt tcctggctca gaagtagata
      901 cagaaatggt caggaataac gcccatgtta ttttctcttt gggaatataa aattaaagag
      961 ttaaataatt attctactta aaaattttga tctcttcagc tgaagagaag ttgttgttag
     1021 agaaatgtgg attgacccat tttaaagtta aatttgtaac cttgtcttat aattgtagat
     1081 caaaaagaag ggtaagtgac tcactggtgt tgtatcctct gttccatcaa gaagtgacgt
     1141 tttaaccctc aggtagattg aaccatttca tctttccccc tttcaggaac ctggtggcca
     1201 tactccttca ggtggatcaa caaccacacg acatggtgag caagggcgag gagctgttca
     1261 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1321 tgcgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca
     1381 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1441 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1501 ccgaaggcta cgtccaggag cgcaccatct ccttcaagga cgacggcacc tacaagaccc
     1561 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1621 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca
     1681 acgtctatat cacggccgac aagcagaaga acggcatcaa ggcgaacttc aagatccgcc
     1741 acaacgtcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1801 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     1861 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1921 tcactctcgg catggacgag ctgtacaagg gaggaggcgg atccggaggc ggaggatccg
     1981 gaaagaagac caagaggaca gccgacagct cttcttcaga ggatgaggag gaatatgtgg
     2041 tggaaaaggt gttggacagg cgcatggtta aggggcaagt ggaatatctg ttgaagtgga
     2101 aaggcttttc tgagtaagtg ctttcttttt cagtctttag aactctgtag aggtagaaaa
     2161 ctttgattta cttgcccatg agccacagtc tgtctttttc ccagttacat actttcatga
     2221 gaatcaagag aggcagcaga ccacatagtc tccctattgt ggagctgata ttttattgtt
     2281 cttattttga ggggtacttt aaggaattgg ttgggaacac agtcttggga gccagatatc
     2341 ctgggaccag attcttgttc caccccgtaa gatacaggtg aagacattga ttaatttctt
     2401 ttattattat gacttaacat tttttgttgt tgttgttgtt ttgtttgttt attttttatg
     2461 ttttttgaga cagggttttt ctgtgtagcc ttggctgtcc tggaactccc tcagtggacc
     2521 aggctgactt tgaactcagg agatcagcct gccaagtgtt gggattaaag gcatgcacca
     2581 ctatacagag ctaaattttt tttctgattt tatgtgtata actgtttggc ttgcatgcac
     2641 tgtgtgtatg ttgtctggta cccacagtcc aggcatcaga tcagctagaa gaggagttac
     2701 agatagttgt aagctgccgt gtgaatgctg gaaatcagac cacagttctc tgggagagca
     2761 gacagtgctc ttaaacaccg agccatttct tcagcccatt ggttaatttc ataatcttat
     2821 ctccctccct agtaaagctc taataaaata gggataagag gttggtatgt atagcttagc
     2881 ccgggtaccg agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2941 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3001 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3061 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     3121 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3181 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3241 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     3301 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3361 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3421 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3481 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3541 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3601 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3661 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3721 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3781 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3841 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3901 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3961 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4021 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4081 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4141 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4201 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4261 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4321 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4381 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4441 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4501 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4561 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4621 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4681 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4741 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4801 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4861 cacacagccc agcttggagc gaacgaccta caccgaact
//
