LOCUS       sequence-252785-        4707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 3xFLAG tagged firefly luciferase (FFLuc) from GUG start 
            codon (AUG at codon 30 mutated to AAA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4707)
  AUTHORS   Kearse MG, Goldman DH, Choi J, Nwaezeapu C, Liang D, Green KM, 
            Goldstrohm AC, Todd PK, Green R, Wilusz JE
  TITLE     Ribosome queuing enables non-AUG translation to be resistant to 
            multiple protein synthesis inhibitors.
  JOURNAL   Genes Dev. 2019 Jun 6. pii: gad.324715.119. doi: 
            10.1101/gad.324715.119.
  PUBMED    31171704
REFERENCE   2  (bases 1 to 4707)
  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: "Genes Dev. 
            2019 Jun 6. pii: gad.324715.119. doi: 10.1101/gad.324715.119."
COMMENT     Sequence Label: pGL4.13/GUG, AAA(30)-FFLuc-3XFLAG
FEATURES             Location/Qualifiers
     source          1..4707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        106..463
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        106..297
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(129..149)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      314..449
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             502..2148
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAKKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             2149..2214
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2261..2382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2272..2291)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2326..2345
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2630..2647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2801..3389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2881..2900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3589..4380,4381..4449))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    4554..4602
                     /note="synthetic polyadenylation signal"
     misc_feature    4616..4707
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4656..4675
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcagatct gcgcagcacc
       61 atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa
      121 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
      181 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
      241 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
      301 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
      361 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
      421 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg gcaatccggt
      481 actgttggta aagccaccgt ggaagatgcc aaaaacatta agaagggccc agcgccattc
      541 tacccactcg aagacgggac cgccggcgag cagctgcaca aagccaaaaa gcgctacgcc
      601 ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat tacctacgcc
      661 gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg gctgaataca
      721 aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc cgtgttgggt
      781 gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga gcgcgagctg
      841 ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa agggctgcaa
      901 aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat catggatagc
      961 aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca tttgccaccc
     1021 ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac catcgccctg
     1081 atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc gcaccgcacc
     1141 gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat catccccgac
     1201 accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac cacgctgggc
     1261 tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga gctattcttg
     1321 cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact atttagcttc
     1381 ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga gatcgccagc
     1441 ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt ccacctacca
     1501 ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat cacccccgaa
     1561 ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc taaggtggtg
     1621 gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg cgtccgtggc
     1681 cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct catcgacaag
     1741 gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca cttcttcatc
     1801 gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc agccgaactg
     1861 gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg cctgcccgac
     1921 gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa aaccatgacc
     1981 gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa gctgcgcggt
     2041 ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga cgcccgcaag
     2101 atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgga ctacaaagac
     2161 catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga caagtaataa
     2221 ttctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt
     2281 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     2341 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     2401 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     2461 tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag ccttcaaccc
     2521 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     2581 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg
     2641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3241 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3541 agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt accagtgctt
     3601 gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag tggcctgact
     3661 ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca gcgcagcaat
     3721 gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc agccagcagg
     3781 gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt ctatgagctg
     3841 ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag ttgtggccat
     3901 tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca actctggttc
     3961 ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag tcagctcctt
     4021 agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca tggtaatggc
     4081 agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg tgaccggcga
     4141 gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct cttgcccggc
     4201 gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca tcatcgggaa
     4261 tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca gttcgatata
     4321 gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg tttcggggtg
     4381 tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac gaaaatgttg
     4441 gatgctcata ctcgtccttt ttcaatatta ttgaagcatt tatcagggtt actagtacgt
     4501 ctctcaagga taagtaagta atattaaggt acgggaggta ttggacaggc cgcaataaaa
     4561 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat
     4621 acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg
     4681 caagtgcagg tgccagaaca tttctct
//
