LOCUS       sequence-464748-        6415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of bicistronic reporter with TOP motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6415)
  AUTHORS   Shichino Y, Yamaguchi T, Kashiwagi K, Mito M, Takahashi M, Ito T, 
            Ingolia NT, Kuba K, Iwasaki S
  TITLE     eIF4A1 enhances LARP1-mediated translational repression during 
            mTORC1 inhibition.
  JOURNAL   Nat Struct Mol Biol. 2024 Oct;31(10):1557-1566. doi: 
            10.1038/s41594-024-01321-7. Epub 2024 May 21.
  PUBMED    38773334
REFERENCE   2  (bases 1 to 6415)
  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"; doi: 
            "10.1038/s41594-024-01321-7"; journalName: "Nat Struct Mol Biol"; 
            date: "2024-10"; volume: "31"; issue: "10"; pages: "1557-1566"
COMMENT     Sequence Label: psiCHECK2-Eef2TOP-RLCP-HCVIRES-FL
FEATURES             Location/Qualifiers
     source          1..6415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1153..1162
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1159..2091
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             2098..2145
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             2149..2268
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     CDS             2674..4326
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /db_xref="GI:42718124"
                     /protein_id="AAS38485.1"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             3633..3641
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4370..4491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4381..4400)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4435..4454
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4624..4728
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4729..4797,4798..5589)
                     /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(4947..4966)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5760..6348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6249..6268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tttgagacag ggtttctctg tgtagccctg gctgtcctgg aactcactct gtagaccagc
      121 ctggcctcga actcagaaat tggcctgcct ctgtctctgc tgggattaaa ggcatgcacc
      181 accaacgccc cgcctttttg tttgttttta agatagggtt tcttgattta gccttgtctg
      241 tcatggaact cactctgtag accaggttag ccttgaacaa agatctcctg tctttgcctt
      301 ctaaatgctg ggattaaagg cgtgcaccac tactgtctgc cttttgtttt ggttttatca
      361 taattgttgc atttttgccc gcaggaggat caggaattca aggacaacct ctacagccaa
      421 cccagttgag ggccagcctg ttctatcgag atcctgtctc cggacaaaaa aagtttcatg
      481 agagcgtggt gtgttggcac agtcttttag tccgagcact gacgcagtag attaaggcag
      541 atcttcatga tttggtgcgg tcctgggctg catccaaaga ccttgtttcc aaaacaaaca
      601 aacgaaaaag tgtaataaaa atacaagcaa ggttttgcag aaataaatct gtggcctcaa
      661 gggtcccttt ctccagcttt tcccagaggt gtcaaattac ataacagtct caaacccagg
      721 aaattgaact ttggcacaac ccactgaccg gtccgaattt caaagtcttt ttcctattga
      781 cctacaaggt tttcaagaat catgttgtaa gcaactgtgt tctgaggaat ctatgtttaa
      841 aaacccaccc gtggatcttg gcccagggtc cagagactga gctagccacg ccccggccgc
      901 gccgcagcca ctcccacggc agttcaagtg ttaagtccca aagaccgcgc tctgtgcatg
      961 cgcagacccg tccacagctg gctcctagcc aacccggccg gacgagcacc cggcgccgtc
     1021 acgtgacgca cccaaccggc gtcgacctat aaaaggccgg gcgttgacgt cagcggtctc
     1081 ttccgccgca gccgccgcca tcgtcggcgc gcttccctgt tcacctctga ctctgagaat
     1141 ccgtcgccat ccgccaccat ggcttccaag gtgtacgacc ccgagcaacg caaacgcatg
     1201 atcactgggc ctcagtggtg ggctcgctgc aagcaaatga acgtgctgga ctccttcatc
     1261 aactactatg attccgagaa gcacgccgag aacgccgtga tttttctgca tggtaacgct
     1321 gcctccagct acctgtggag gcacgtcgtg cctcacatcg agcccgtggc tagatgcatc
     1381 atccctgatc tgatcggaat gggtaagtcc ggcaagagcg ggaatggctc atatcgcctc
     1441 ctggatcact acaagtacct caccgcttgg ttcgagctgc tgaaccttcc aaagaaaatc
     1501 atctttgtgg gccacgactg gggggcttgt ctggcctttc actactccta cgagcaccaa
     1561 gacaagatca aggccatcgt ccatgctgag agtgtcgtgg acgtgatcga gtcctgggac
     1621 gagtggcctg acatcgagga ggatatcgcc ctgatcaaga gcgaagaggg cgagaaaatg
     1681 gtgcttgaga ataacttctt cgtcgagacc atgctcccaa gcaagatcat gcggaaactg
     1741 gagcctgagg agttcgctgc ctacctggag ccattcaagg agaagggcga ggttagacgg
     1801 cctaccctct cctggcctcg cgagatccct ctcgttaagg gaggcaagcc cgacgtcgtc
     1861 cagattgtcc gcaactacaa cgcctacctt cgggccagcg acgatctgcc taagatgttc
     1921 atcgagtccg accctgggtt cttttccaac gctattgtcg agggagctaa gaagttccct
     1981 aacaccgagt tcgtgaaggt gaagggcctc cacttcagcc aggaggacgc tccagatgaa
     2041 atgggtaagt acatcaagag cttcgtggag cgcgtgctga agaacgagca gaattctgct
     2101 tgcaagaact ggttcagtag cttaagccac tttgtgatcc accttaacag ccacggcttc
     2161 cctcccgagg tggaggagca ggccgccggc accctgccca tgagctgcgc ccaggagagc
     2221 ggcatggata gacaccctgc tgcttgcgcc agcgccagga tcaacgtcta attctaggcg
     2281 atcgctcgag gccagccccc tgatgggggc gacactccac catgaatcac tcccctgtga
     2341 ggaactactg tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag
     2401 cctccaggac cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg
     2461 gaattgccag gacgaccggg tcctttcttg gagttacccg ctcaatgcct ggagatttgg
     2521 gcgtgccccc gcaagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact
     2581 gcctgatagg gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg
     2641 aatcctaaac ctcaaagaaa aaccaaacgt aacatggccg atgctaagaa cattaagaag
     2701 ggccctgctc ccttctaccc tctggaggat ggcaccgctg gcgagcagct gcacaaggcc
     2761 atgaagaggt atgccctggt gcctggcacc attgccttca ccgatgccca cattgaggtg
     2821 gacatcacct atgccgagta cttcgagatg tctgtgcgcc tggccgaggc catgaagagg
     2881 tacggcctga acaccaacca ccgcatcgtg gtgtgctctg agaactctct gcagttcttc
     2941 atgccagtgc tgggcgccct gttcatcgga gtggccgtgg cccctgctaa cgacatttac
     3001 aacgagcgcg agctgctgaa cagcatgggc atttctcagc ctaccgtggt gttcgtgtct
     3061 aagaagggcc tgcagaagat cctgaacgtg cagaagaagc tgcctatcat ccagaagatc
     3121 atcatcatgg actctaagac cgactaccag ggcttccaga gcatgtacac attcgtgaca
     3181 tctcatctgc ctcctggctt caacgagtac gacttcgtgc cagagtcttt cgacagggac
     3241 aaaaccattg ccctgatcat gaacagctct gggtctaccg gcctgcctaa gggcgtggcc
     3301 ctgcctcatc gcaccgcctg tgtgcgcttc tctcacgccc gcgaccctat tttcggcaac
     3361 cagatcatcc ccgacaccgc tattctgagc gtggtgccat tccaccacgg cttcggcatg
     3421 ttcaccaccc tgggctacct gatttgcggc tttcgggtgg tgctgatgta ccgcttcgag
     3481 gaggagctgt tcctgcgcag cctgcaagac tacaaaattc agtctgccct gctggtgcca
     3541 accctgttca gcttcttcgc taagagcacc ctgatcgaca agtacgacct gtctaacctg
     3601 cacgagattg cctctggcgg cgccccactg tctaaggagg tgggcgaagc cgtggccaag
     3661 cgctttcatc tgccaggcat ccgccagggc tacggcctga ccgagacaac cagcgccatt
     3721 ctgattaccc cagagggcga cgacaagcct ggcgccgtgg gcaaggtggt gccattcttc
     3781 gaggccaagg tggtggacct ggacaccggc aagaccctgg gagtgaacca gcgcggcgag
     3841 ctgtgtgtgc gcggccctat gattatgtcc ggctacgtga ataaccctga ggccacaaac
     3901 gccctgatcg acaaggacgg ctggctgcac tctggcgaca ttgcctactg ggacgaggac
     3961 gagcacttct tcatcgtgga ccgcctgaag tctctgatca agtacaaggg ctaccaggtg
     4021 gccccagccg agctggagtc tatcctgctg cagcacccta acattttcga cgccggagtg
     4081 gccggcctgc ccgacgacga tgccggcgag ctgcctgccg ccgtcgtcgt gctggaacac
     4141 ggcaagacca tgaccgagaa ggagatcgtg gactatgtgg ccagccaggt gacaaccgcc
     4201 aagaagctgc gcggcggagt ggtgttcgtg gacgaggtgc ccaagggcct gaccggcaag
     4261 ctggacgccc gcaagatccg cgagatcctg atcaaggcta agaaaggcgg caagatcgcc
     4321 gtgtaataat tctagagtcg gggcggccgg ccgcttcgag cagacatgat aagatacatt
     4381 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     4441 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4501 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4561 taaaacctct acaaatgtgg taaaatcgat aaggatccag gtggcacttt tcggggaaat
     4621 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4681 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4741 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4801 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4861 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4921 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4981 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5041 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5101 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5161 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5221 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5281 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5341 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5401 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5461 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5521 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5581 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5641 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5701 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5761 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5821 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5881 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5941 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6001 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6061 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6121 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6181 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6241 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6301 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6361 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatggc tcgac
//
