LOCUS       sequence-247723-        6103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 3xFLAG tagged destabilized nanoLuciferase (nLuc) protein 
            from AUG start codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6103)
  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 6103)
  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: pcDNA3.1(+)/AUG-nLuc-3XFLAG-PEST
FEATURES             Location/Qualifiers
     source          1..6103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      943..952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             949..1461
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1462..1527
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1531..1650
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     primer_bind     complement(1694..1711)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1700..1924
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1970..2398
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2057..2076)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2267..2288
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2407..2427)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2412..2741
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2592..2727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2654..2673
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2808..3602
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2862..2881)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3472..3491
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3776..3897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3813..3832)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3867..3886
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3946..3962)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3946..3962)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3959..3981)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3970..3986
                     /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)."
     promoter        complement(join(3994..4000,4001..4018,4019..4024))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4039..4060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4177..4194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4348..4936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4428..4447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5107..5898,5899..5967))
                     /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     5730..5749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5968..6072)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggca atccggtact gttggtaaat aagccaccat ggtcttcaca
      961 ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt
     1021 gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa
     1081 aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat
     1141 gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct
     1201 gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt
     1261 acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc
     1321 aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg
     1381 atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg
     1441 ctgtgcgaac gcattctggc ggactacaaa gaccatgacg gtgattataa agatcatgac
     1501 atcgattaca aggatgacga tgacaagaat tctcacggct ttccgcctga ggttgaagag
     1561 caagccgccg gtacattgcc tatgtcctgc gcacaagaaa gcggtatgga ccggcaccca
     1621 gccgcttgtg cttcagctcg catcaacgtc taaggccgcg actctagagg gcccgtttaa
     1681 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1741 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1801 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1861 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1921 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     1981 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2041 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2101 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2161 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2221 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2281 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2341 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     2401 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2461 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2521 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2581 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2641 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     2701 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     2761 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     2821 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2881 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     2941 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3001 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3061 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3121 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3181 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3241 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3301 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     3361 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3421 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3481 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3541 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3601 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     3661 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3721 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     3781 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3841 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3901 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     3961 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4021 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4081 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4141 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4201 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4261 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4321 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4381 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4441 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4501 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4561 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4621 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4681 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4741 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4801 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4861 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4921 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4981 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5041 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5101 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5161 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5221 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5281 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5341 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5401 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5461 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5521 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5581 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5641 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5701 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5761 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5821 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5881 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5941 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6001 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6061 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
