LOCUS       sequence-247796-        6157 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express 3xFLAG tagged highly destabilized nanoLuciferase (nLuc) 
            protein from CUG start codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6157)
  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 6157)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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(+)/CUG-nLuc-3XFLAG-CL1/PEST
FEATURES             Location/Qualifiers
     source          1..6157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..440
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        441..644
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     595..615
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     689..708
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        689..707
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             778..1287
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             1288..1353
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1360..1407
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             1411..1530
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     primer_bind     complement(1574..1591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1580..1804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1850..2278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1937..1956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2147..2168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2287..2307)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2292..2621
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2472..2607
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2534..2553
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2688..3482
                     /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(2742..2761)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3352..3371
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3656..3777
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3693..3712)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3747..3766
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3826..3842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3826..3842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3839..3861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3850..3866
                     /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(3874..3880,3881..3898,3899..3904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3919..3940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4057..4074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4228..4816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4308..4327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4987..5778,5779..5847))
                     /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     5610..5629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5848..5952)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6027..6046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
       61 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      121 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      181 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      241 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      301 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      361 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      421 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      481 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      541 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      601 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      661 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      721 gctagcgttt aaacttaagc ttggcaatcc ggtactgttg gtaaataagc caccctggtc
      781 ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa
      841 gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg
      901 atccaaagga ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc
      961 ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg
     1021 taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac
     1081 ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc
     1141 gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag
     1201 cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc
     1261 tggcggctgt gcgaacgcat tctggcggac tacaaagacc atgacggtga ttataaagat
     1321 catgacatcg attacaagga tgacgatgac aagaattctg cttgcaagaa ctggttcagt
     1381 agcttaagcc actttgtgat ccaccttaac agccacggct ttccgcctga ggttgaagag
     1441 caagccgccg gtacattgcc tatgtcctgc gcacaagaaa gcggtatgga ccggcaccca
     1501 gccgcttgtg cttcagctcg catcaacgtc taaggccgcg actctagagg gcccgtttaa
     1561 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1621 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1681 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1741 gacggcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1801 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     1861 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1921 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1981 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2041 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2101 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2161 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2221 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     2281 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2341 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2401 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2461 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2521 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     2581 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     2641 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     2701 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2761 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     2821 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     2881 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     2941 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3001 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3061 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3121 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3181 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     3241 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3301 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3361 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3421 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3481 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     3541 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3601 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     3661 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3721 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3781 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     3841 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3901 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3961 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4021 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4081 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4141 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4201 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4261 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4321 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4381 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4441 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4501 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4561 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4621 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4681 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4741 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4801 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4861 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4921 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4981 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5041 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5101 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5161 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5221 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5281 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5341 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5401 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5461 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5521 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5581 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5641 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5701 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5761 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5821 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5881 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5941 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     6001 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6061 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     6121 acaacaaggc aaggcttgac cgacaattgc atgaaga
//
