LOCUS       sequence-471687-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of msfGFP-HMGB1-Shuffled 10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  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 6064)
  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-Shuffled 10
FEATURES             Location/Qualifiers
     source          1..6064
                     /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      2364..2373
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2377..2511
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2414..2433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2468..2487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2580..2937
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2580..2771
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2603..2623)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2788..2923
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2957..2974)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2957..2973)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2966..2988)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3186..3641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3273..3292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3483..3504
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3658..3677)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3777..3799
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3837..3855)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3923..4027
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4028..4096,4097..4888)
                     /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(4246..4265)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5059..5647
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5548..5567
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5801..5818
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5935..5956
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5971..5976,5977..5994,5995..6001)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6009..6025
                     /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     6014..6036
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6033..6049
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6033..6049
                     /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 gatgttcatg aataagttct tggttctagc gcagttctcc tgtctataat
     2341 ctagagtcgc ctgcagaagc ttggccgcca tggcccaact tgtttattgc agcttataat
     2401 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2461 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat cgggaattaa
     2521 ttcggcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc
     2581 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2641 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2761 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2821 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2881 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2941 ctgttaacag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     3001 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     3061 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     3121 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     3181 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3241 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3301 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3361 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     3421 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3481 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga
     3541 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3601 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     3661 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     3721 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3781 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     3841 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     3901 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3961 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4021 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4081 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4141 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4201 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4261 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4321 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4381 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4441 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4501 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4561 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4621 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4681 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4741 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4801 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4861 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4921 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4981 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5041 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5101 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5161 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5221 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5281 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5341 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5401 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5461 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5521 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5581 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5641 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5701 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5761 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5821 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5881 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     5941 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     6001 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
     6061 ttaa
//
