LOCUS       sequence-318021-        5698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Luciferase reporter for ribosome pausing control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5698)
  AUTHORS   Han P, Shichino Y, Schneider-Poetsch T, Mito M, Hashimoto S, Udagawa
            T, Kohno K, Yoshida M, Mishima Y, Inada T, Iwasaki S
  TITLE     Genome-wide Survey of Ribosome Collision.
  JOURNAL   Cell Rep. 2020 May 5;31(5):107610. doi: 
            10.1016/j.celrep.2020.107610.
  PUBMED    32375038
REFERENCE   2  (bases 1 to 5698)
  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: "Cell Rep.";
            date: "2020-05-5"; volume: "31(5):107610. doi"; pages: " 
            10.1016/j.celrep.2020.107610"
COMMENT     Sequence Label: psiCHECK2-2A-3xFLAG-SBP-2A
FEATURES             Location/Qualifiers
     source          1..5698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..421
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        64..255
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(87..107)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      272..407
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          491..623
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     606..625
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        668..686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             708..1640
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1650..1706
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1707..1772
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1773..1883
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             1905..1961
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1962..3611
                     /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="ADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             2918..2926
                     /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    3655..3776
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3666..3685)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3720..3739
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3909..4013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4014..4082,4083..4874)
                     /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(4232..4251)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5045..5633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5534..5553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acagatctgc gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac
       61 cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
      121 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
      181 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      241 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
      301 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
      361 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
      421 aaagcttgat tcttctgaca caacagtctc gaacttaagc tgcagaagtt ggtcgtgagg
      481 cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg
      541 cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat
      601 ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta
      661 gagtacttaa tacgactcac tataggtttc cttcctcttt tggcaacatg gcttccaagg
      721 tgtacgaccc cgagcaacgc aaacgcatga tcactgggcc tcagtggtgg gctcgctgca
      781 agcaaatgaa cgtgctggac tccttcatca actactatga ttccgagaag cacgccgaga
      841 acgccgtgat ttttctgcat ggtaacgctg cctccagcta cctgtggagg cacgtcgtgc
      901 ctcacatcga gcccgtggct agatgcatca tccctgatct gatcggaatg ggtaagtccg
      961 gcaagagcgg gaatggctca tatcgcctcc tggatcacta caagtacctc accgcttggt
     1021 tcgagctgct gaaccttcca aagaaaatca tctttgtggg ccacgactgg ggggcttgtc
     1081 tggcctttca ctactcctac gagcaccaag acaagatcaa ggccatcgtc catgctgaga
     1141 gtgtcgtgga cgtgatcgag tcctgggacg agtggcctga catcgaggag gatatcgccc
     1201 tgatcaagag cgaagagggc gagaaaatgg tgcttgagaa taacttcttc gtcgagacca
     1261 tgctcccaag caagatcatg cggaaactgg agcctgagga gttcgctgcc tacctggagc
     1321 cattcaagga gaagggcgag gttagacggc ctaccctctc ctggcctcgc gagatccctc
     1381 tcgttaaggg aggcaagccc gacgtcgtcc agattgtccg caactacaac gcctaccttc
     1441 gggccagcga cgatctgcct aagatgttca tcgagtccga ccctgggttc ttttccaacg
     1501 ctattgtcga gggagctaag aagttcccta acaccgagtt cgtgaaggtg aagggcctcc
     1561 acttcagcca ggaggacgct ccagatgaaa tgggtaagta catcaagagc ttcgtggagc
     1621 gcgtgctgaa gaacgagcag ggaagcggag ctactaactt cagcctgctg aagcaggctg
     1681 gagacgtgga ggagaaccct ggacctgact acaaagacca tgacggtgat tataaagatc
     1741 atgacatcga ttacaaggat gacgatgaca aggacgagaa aaccaccggc tggcggggag
     1801 gccacgtggt ggaagggctg gcaggcgagc tggaacagct gcgggccaga ctggaacacc
     1861 acccccaggg ccagagagag cctagcggcg gaggaggaag cggagctact aacttcagcc
     1921 tgctgaagca ggctggagac gtggaggaga accctggacc tgccgatgct aagaacatta
     1981 agaagggccc tgctcccttc taccctctgg aggatggcac cgctggcgag cagctgcaca
     2041 aggccatgaa gaggtatgcc ctggtgcctg gcaccattgc cttcaccgat gcccacattg
     2101 aggtggacat cacctatgcc gagtacttcg agatgtctgt gcgcctggcc gaggccatga
     2161 agaggtacgg cctgaacacc aaccaccgca tcgtggtgtg ctctgagaac tctctgcagt
     2221 tcttcatgcc agtgctgggc gccctgttca tcggagtggc cgtggcccct gctaacgaca
     2281 tttacaacga gcgcgagctg ctgaacagca tgggcatttc tcagcctacc gtggtgttcg
     2341 tgtctaagaa gggcctgcag aagatcctga acgtgcagaa gaagctgcct atcatccaga
     2401 agatcatcat catggactct aagaccgact accagggctt ccagagcatg tacacattcg
     2461 tgacatctca tctgcctcct ggcttcaacg agtacgactt cgtgccagag tctttcgaca
     2521 gggacaaaac cattgccctg atcatgaaca gctctgggtc taccggcctg cctaagggcg
     2581 tggccctgcc tcatcgcacc gcctgtgtgc gcttctctca cgcccgcgac cctattttcg
     2641 gcaaccagat catccccgac accgctattc tgagcgtggt gccattccac cacggcttcg
     2701 gcatgttcac caccctgggc tacctgattt gcggctttcg ggtggtgctg atgtaccgct
     2761 tcgaggagga gctgttcctg cgcagcctgc aagactacaa aattcagtct gccctgctgg
     2821 tgccaaccct gttcagcttc ttcgctaaga gcaccctgat cgacaagtac gacctgtcta
     2881 acctgcacga gattgcctct ggcggcgccc cactgtctaa ggaggtgggc gaagccgtgg
     2941 ccaagcgctt tcatctgcca ggcatccgcc agggctacgg cctgaccgag acaaccagcg
     3001 ccattctgat taccccagag ggcgacgaca agcctggcgc cgtgggcaag gtggtgccat
     3061 tcttcgaggc caaggtggtg gacctggaca ccggcaagac cctgggagtg aaccagcgcg
     3121 gcgagctgtg tgtgcgcggc cctatgatta tgtccggcta cgtgaataac cctgaggcca
     3181 caaacgccct gatcgacaag gacggctggc tgcactctgg cgacattgcc tactgggacg
     3241 aggacgagca cttcttcatc gtggaccgcc tgaagtctct gatcaagtac aagggctacc
     3301 aggtggcccc agccgagctg gagtctatcc tgctgcagca ccctaacatt ttcgacgccg
     3361 gagtggccgg cctgcccgac gacgatgccg gcgagctgcc tgccgccgtc gtcgtgctgg
     3421 aacacggcaa gaccatgacc gagaaggaga tcgtggacta tgtggccagc caggtgacaa
     3481 ccgccaagaa gctgcgcggc ggagtggtgt tcgtggacga ggtgcccaag ggcctgaccg
     3541 gcaagctgga cgcccgcaag atccgcgaga tcctgatcaa ggctaagaaa ggcggcaaga
     3601 tcgccgtgta ataattctag agtcggggcg gccggccgct tcgagcagac atgataagat
     3661 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3721 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3781 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     3841 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccaggtggc acttttcggg
     3901 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3961 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4021 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4081 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4141 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4201 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4261 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4321 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4381 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4441 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4501 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4561 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4621 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4681 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4741 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4801 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4861 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4921 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4981 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5041 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5101 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5161 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5221 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5281 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5341 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5401 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5461 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5521 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5581 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5641 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atggctcg
//
