LOCUS       sequence-471723-        6060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of msfGFP-HMGB1-Mut-C16toA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6060)
  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 6060)
  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: pRK5_msfGFP-HMGB1-Mut-C16toA
FEATURES             Location/Qualifiers
     source          1..6060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(939..941,942..944,945..1652)
                     /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
                     EFVTAAGITLGMDELY"
     primer_bind     complement(984..1005)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1592..1613
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2360..2369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2373..2507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2410..2429)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2464..2483
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2576..2933
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2576..2767
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2599..2619)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2784..2919
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2953..2970)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2953..2969)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2962..2984)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3182..3637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3269..3288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3479..3500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3654..3673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3773..3795
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3833..3851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3919..4023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4024..4092,4093..4884)
                     /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(4242..4261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5055..5643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5544..5563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5797..5814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5931..5952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5967..5972,5973..5990,5991..5997)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6005..6021
                     /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     6010..6032
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6029..6045
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6029..6045
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgtaccg gtgccaccat ggtgagcaag ggcgaggagc
      961 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1021 tcagcgtgcg cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     1081 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1141 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1201 ccatgcccga aggctacgtc caggagcgca ccatctcctt caaggacgac ggcacctaca
     1261 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1321 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca
     1381 gccacaacgt ctatatcacg gccgacaagc agaagaacgg catcaaggcg aacttcaaga
     1441 tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1501 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     1561 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1621 ccgggatcac tctcggcatg gacgagctgt acacggcgat gggcaaagga gatcctaaga
     1681 agccgagagg caaaatgtca tcatatgcat tttttgtgca aacttgtcgg gaggagcata
     1741 agaagaagca cccagatgct tcagtcaact tctcagagtt ttctaagaag tgctcagaga
     1801 ggtggaagac catgtctgct aaagagaaag gaaaatttga agatatggca aaagcggaca
     1861 aggcccgtta tgaaagagaa atgaaaacct atatccctcc caaaggggag acaaaaaaga
     1921 agttcaagga tcccaatgca cccaagaggc ctccttcggc cttcttcctc ttctgctctg
     1981 agtatcgccc aaaaatcaaa ggagaacatc ctggcctgtc cattggtgat gttgcgaaga
     2041 aactgggaga gatgtggaat aacactgctg cagatgacaa gcagccttat gaaaagaagg
     2101 ctgcgaagct gaaggaaaaa tatgaaaagg atattgctgc atatcgagct aaaggaaagc
     2161 ctgatgcagc aaaaaaggga gttgtcaagg ctgaaaaaag caagaaaagg aagaggagga
     2221 agatgaggaa gatgaagagg atgaggagga ggaggaagat gaagaagatg aagatgaaga
     2281 agaagatgat gatgatgaat aagttggttc tagcgcagtt cttcttctcc gcgctataaa
     2341 gtcgacctgc agaagcttgg ccgccatggc ccaacttgtt tattgcagct tataatggtt
     2401 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2461 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcggg aattaattcg
     2521 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag
     2581 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2641 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2701 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2761 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2821 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     2881 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctgt
     2941 taacagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     3001 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     3061 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     3121 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     3181 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3241 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3301 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3361 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     3421 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3481 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     3541 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3601 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     3661 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3721 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3781 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3841 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3901 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3961 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4021 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4081 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4141 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4201 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4261 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4321 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4381 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4441 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4501 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4561 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4621 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4681 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4741 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4801 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4861 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4921 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4981 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5041 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5101 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5161 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5221 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5281 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5341 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5401 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5461 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5521 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5581 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5641 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5701 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5761 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5821 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5881 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     5941 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     6001 ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattaa
//
