LOCUS       sequence-471709-        6065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of msfGFP-HMGB1-Shuffled 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6065)
  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 6065)
  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 2
FEATURES             Location/Qualifiers
     source          1..6065
                     /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      2365..2374
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2378..2512
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2415..2434)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2469..2488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2581..2938
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2581..2772
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2604..2624)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2789..2924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2958..2975)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2958..2974)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2967..2989)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3187..3642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3274..3293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3484..3505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3659..3678)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3778..3800
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3838..3856)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3924..4028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4029..4097,4098..4889)
                     /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(4247..4266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5060..5648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5549..5568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5802..5819
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5936..5957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5972..5977,5978..5995,5996..6002)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6010..6026
                     /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     6015..6037
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6034..6050
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6034..6050
                     /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 agaagtgttt catgttgaat aagttcctag ttatgcagat gtccttcatg ctagcgtaat
     2341 ctagagtcga cctgcagaag cttggccgcc atggcccaac ttgtttattg cagcttataa
     2401 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2461 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcgggaatta
     2521 attcggcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt
     2581 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2641 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2701 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2761 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2821 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2881 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2941 gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3001 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3061 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     3121 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     3181 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3241 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3301 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3361 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3421 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3481 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg
     3541 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3601 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3661 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3721 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3781 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3841 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3901 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3961 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4021 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4081 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4141 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4201 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4261 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4321 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4381 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4441 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4501 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4561 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4621 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4681 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4741 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4801 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4861 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4921 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4981 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5041 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5101 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5161 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5221 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5281 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5341 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5401 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5461 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5521 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5581 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5641 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5701 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5761 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5821 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5881 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5941 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     6001 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     6061 attaa
//
