LOCUS       sequence-471713-        7892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of mEGFP-PAX-FOXO1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7892)
  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 7892)
  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_mEGFP-PAX-FOXO1
FEATURES             Location/Qualifiers
     source          1..7892
                     /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      934..943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(940..942,943..945,946..1656)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(985..1006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1246..1265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1593..1614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      4192..4201
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4205..4339
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4242..4261)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4296..4315
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4408..4765
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4408..4599
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4431..4451)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4616..4751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4785..4802)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4785..4801)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4794..4816)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5014..5469
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5101..5120)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5311..5332
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5486..5505)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5605..5627
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5665..5683)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5751..5855
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5856..5924,5925..6716)
                     /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(6074..6093)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6887..7475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7376..7395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7629..7646
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7763..7784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7799..7804,7805..7822,7823..7829)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7837..7853
                     /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     7842..7864
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7861..7877
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7861..7877
                     /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 gtcgccacca tggtgagcaa gggcgaggag
      961 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1021 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1081 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1141 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1201 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1261 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1321 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1381 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1441 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1501 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     1561 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1621 gccgggatca ctctcggcat ggacgagctg tacaagaccg gcggcatgac cacgctggcc
     1681 ggcgctgtgc ccaggatgat gcggccgggc ccggggcaga actacccgcg tagcgggttc
     1741 ccgctggaag tgtccactcc cctcggccag ggccgcgtca accagctcgg cggcgttttt
     1801 atcaacggca ggccgctgcc caaccacatc cgccacaaga tcgtggagat ggcccaccac
     1861 ggcatccggc cctgcgtcat ctcgcgccag ctgcgcgtgt cccacggctg cgtctccaag
     1921 atcctgtgca ggtaccagga gactggctcc atacgtcctg gtgccatcgg cggcagcaag
     1981 cccaagcagg tgacaacgcc tgacgtggag aagaaaattg aggaatacaa aagagagaac
     2041 ccgggcatgt tcagctggga aatccgagac aaattactca aggacgcggt ctgtgatcga
     2101 aacaccgtgc cgtcagtgag ttccatcagc cgcatcctga gaagtaaatt cgggaaaggt
     2161 gaagaggagg aggccgactt ggagaggaag gaggcagagg aaagcgagaa gaaggccaaa
     2221 cacagcatcg acggcatcct gagcgagcga gcctcagcac cccaatcaga tgaaggctct
     2281 gatattgact ctgaaccaga tttaccacta aagaggaaac agcgcagaag ccgaaccacc
     2341 ttcacagcag aacagctgga ggaactggag cgtgcttttg agagaactca ttaccctgac
     2401 atttatacta gggaggaact ggcccagagg gcgaagctca ccgaggcccg agtacaggtc
     2461 tggtttagca accgccgtgc aagatggagg aagcaagctg gggccaatca actgatggct
     2521 ttcaaccatc tcattcccgg ggggttccct cccactgcca tgccgacctt gccaacgtac
     2581 cagctgtcgg agacctctta ccagcccaca tctattccac aagctgtgtc agatcccagc
     2641 agcaccgttc acagacctca accgcttcct ccaagcactg tacaccaaag cacgattcct
     2701 tccaacccag acagcagctc tgcctactgc ctccccagca ccaggcatgg attttccagc
     2761 tatacagaca gctttgtgcc tccgtcgggg ccctccaacc ccatgaaccc caccattggc
     2821 aatggcctct cacctcagaa ttcaattcgt cataatctgt ccctacacag caagttcatt
     2881 cgtgtgcaga atgaaggaac tggaaaaagt tcttggtgga tgctcaatcc agagggtggc
     2941 aagagcggga aatctcctag gagaagagct gcatccatgg acaacaacag taaatttgct
     3001 aagagccgaa gccgagctgc caagaagaaa gcatctctcc agtctggcca ggagggtgct
     3061 ggggacagcc ctggatcaca gttttccaaa tggcctgcaa gccctggctc tcacagcaat
     3121 gatgactttg ataactggag tacatttcgc cctcgaacta gctcaaatgc tagtactatt
     3181 agtgggagac tctcacccat tatgaccgaa caggatgatc ttggagaagg ggatgtgcat
     3241 tctatggtgt acccgccatc tgccgcaaag atggcctcta ctttacccag tctgtctgag
     3301 ataagcaatc ccgaaaacat ggaaaatctt ttggataatc tcaaccttct ctcatcacca
     3361 acatcattaa ctgtttcgac ccagtcctca cctggcacca tgatgcagca gacgccgtgc
     3421 tactcgtttg cgccaccaaa caccagtttg aattcaccca gcccaaacta ccaaaaatat
     3481 acatatggcc aatccagcat gagccctttg ccccagatgc ctatacaaac acttcaggac
     3541 aataagtcga gttatggagg tatgagtcag tataactgtg cgcctggact cttgaaggag
     3601 ttgctgactt ctgactctcc tccccataat gacattatga caccagttga tcctggggta
     3661 gcccagccca acagccgggt tctgggccag aacgtcatga tgggccctaa ttcggtcatg
     3721 tcaacctatg gcagccaggc atctcataac aaaatgatga atcccagctc ccatacccac
     3781 cctggacatg ctcagcagac atctgcagtt aacgggcgtc ccctgcccca cacggtaagc
     3841 accatgcccc acacctcggg tatgaaccgc ctgacccaag tgaagacacc tgtacaagtg
     3901 cctctgcccc accccatgca gatgagtgcc ctggggggct actcctccgt gagcagctgc
     3961 aatggctatg gcagaatggg ccttctccac caggagaagc tcccaagtga cttggatggc
     4021 atgttcattg agcgcttaga ctgtgacatg gaatccatca ttcggaatga cctcatggat
     4081 ggagatacat tggattttaa ctttgacaat gtgttgccca accaaagctt cccacacagt
     4141 gtcaagacaa cgacacatag ctgggtgtca ggctgaacct gcagaagctt ggccgccatg
     4201 gcccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4261 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4321 atcttatcat gtctggatcg ggaattaatt cggcgcagca ccatggcctg aaataacctc
     4381 tgaaagagga acttggttag gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg
     4441 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4501 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4561 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4621 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4681 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4741 ttttggaggc ctaggctttt gcaaaaagct gttaacagct tggcactggc cgtcgtttta
     4801 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4861 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4921 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4981 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     5041 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     5101 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5161 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5221 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5281 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5341 tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5401 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5461 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5521 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5581 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5641 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5701 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5761 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5821 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5881 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5941 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6001 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6061 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6121 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6181 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6241 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6301 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6361 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6421 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6481 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6541 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6601 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6661 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6721 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6781 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6841 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6901 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6961 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7021 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7081 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7141 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7201 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7261 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7321 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7381 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7441 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7501 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7561 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7621 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     7681 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7741 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7801 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     7861 caggaaacag ctatgacatg attacgaatt aa
//
