LOCUS       sequence-474403-        5377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of EGFP-control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5377)
  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 5377)
  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_EGFP-control
FEATURES             Location/Qualifiers
     source          1..5377
                     /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..1658)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(939..941,942..944,945..1655)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(984..1005)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1245..1264)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1592..1613
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1677..1686
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1690..1824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1727..1746)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1781..1800
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1893..2250
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1893..2084
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1916..1936)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2101..2236
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2270..2287)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2270..2286)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2279..2301)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2499..2954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2586..2605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2796..2817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2971..2990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3090..3112
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3150..3168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3236..3340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3341..3409,3410..4201)
                     /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(3559..3578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4372..4960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4861..4880
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5114..5131
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5248..5269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5284..5289,5290..5307,5308..5314)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5322..5338
                     /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     5327..5349
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5346..5362
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5346..5362
                     /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 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1081 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1141 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1201 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1261 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1321 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1381 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     1441 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1501 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     1561 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1621 ccgggatcac tctcggcatg gacgagctgt acaagtgagt cgacctgcag aacttggccg
     1681 ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1741 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     1801 aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata
     1861 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     1921 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1981 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2041 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2101 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2161 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     2221 ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg
     2281 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2341 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2401 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     2461 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     2521 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2581 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2641 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2701 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2761 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2821 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     2881 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2941 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3001 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3061 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3121 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3181 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3241 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3301 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3361 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3421 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3481 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3541 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3601 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3661 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3721 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3781 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3841 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3901 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3961 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4021 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4081 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4141 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4201 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4261 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4321 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4381 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4441 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4501 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4561 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4621 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4681 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4741 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4801 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4861 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4921 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4981 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5041 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5101 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     5161 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5221 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     5281 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5341 tcacacagga aacagctatg acatgattac gaattaa
//
