LOCUS       sequence-247785-        6024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 3xFLAG tagged nanoLuciferase (nLuc) from AUG start codon 
            from mRNA with hairpin in 5' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  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 6024)
  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(+)/HP + AUG-nLuc-3XFLAG
FEATURES             Location/Qualifiers
     source          1..6024
                     /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      987..996
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             993..1505
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1506..1571
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1615..1632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1621..1845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1891..2319
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1978..1997)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2188..2209
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2328..2348)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2333..2662
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2513..2648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2575..2594
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2729..3523
                     /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(2783..2802)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3393..3412
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3697..3818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3734..3753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3788..3807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3867..3883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3867..3883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3880..3902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3891..3907
                     /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(3915..3921,3922..3939,3940..3945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3960..3981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4098..4115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4269..4857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4349..4368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5028..5819,5820..5888))
                     /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     5651..5670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5889..5993)
                     /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 aagcttggct tggggcgcgt ggtggcggct gcagccgcca ccacgcgccc
      961 cggaatccgg tactgttggt aaataagcca ccatggtctt cacactcgaa gatttcgttg
     1021 gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt
     1081 ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg
     1141 gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg
     1201 accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact
     1261 ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg
     1321 actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa
     1381 cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct
     1441 ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc
     1501 tggcggacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
     1561 acgatgacaa gtaaggccgc gactctagag ggcccgttta aacccgctga tcagcctcga
     1621 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     1681 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     1741 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     1801 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     1861 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     1921 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1981 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2041 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2101 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2161 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2221 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2281 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     2341 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2401 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2461 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     2521 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2581 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     2641 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     2701 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     2761 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     2821 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     2881 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     2941 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3001 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3061 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3121 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3181 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3241 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3301 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3361 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3421 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     3481 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     3541 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     3601 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     3661 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     3721 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3781 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     3841 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3901 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3961 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4021 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4081 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4141 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4201 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4261 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4321 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4381 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4441 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4501 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4561 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4621 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4681 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4741 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4801 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4861 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4921 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4981 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5041 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5101 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5161 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5221 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5281 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5341 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5401 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5461 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5521 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5581 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5641 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5701 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5761 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5821 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5881 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5941 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6001 ccccgaaaag tgccacctga cgtc
//
