LOCUS       sequence-247703-        6001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 3xFLAG tagged nanoLuciferase (nLuc) from CUG start codon 
            from mRNA with BAG1 5' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6001)
  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 6001)
  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(+)/BAG1 5′ UTR CUG-nLuc-3xFLAG
FEATURES             Location/Qualifiers
     source          1..6001
                     /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"
     CDS             973..1482
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             1483..1548
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1592..1609)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1598..1822
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1868..2296
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1955..1974)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2165..2186
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2305..2325)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2310..2639
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2490..2625
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2552..2571
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2706..3500
                     /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(2760..2779)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3370..3389
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3674..3795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3711..3730)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3765..3784
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3844..3860)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3844..3860)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3857..3879)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3868..3884
                     /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(3892..3898,3899..3916,3917..3922))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3937..3958
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4075..4092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4246..4834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4326..4345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5005..5796,5797..5865))
                     /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     5628..5647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5866..5970)
                     /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 ggaggccggg gcggggctgg
      901 gaagtagtcg ggcggggttg tgagacgccg cgctcagctt ccatcgctgg gcggtcaaca
      961 agtgcgggcc tggtcttcac actcgaagat ttcgttgggg actggcgaca gacagccggc
     1021 tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg
     1081 gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct gaagatcgac
     1141 atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa
     1201 atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct gcactatggc
     1261 acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg gccgtatgaa
     1321 ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg gaacggcaac
     1381 aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg agtaaccatc
     1441 aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cggactacaa agaccatgac
     1501 ggtgattata aagatcatga catcgattac aaggatgacg atgacaagta aggccgcgac
     1561 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     1621 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     1681 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     1741 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     1801 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     1861 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1921 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1981 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2041 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2101 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2161 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2221 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2281 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2341 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2401 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2461 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     2521 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     2581 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     2641 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     2701 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     2761 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     2821 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     2881 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     2941 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3001 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3061 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3121 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3181 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     3241 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3301 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3361 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     3421 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     3481 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     3541 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3601 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3661 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3721 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3781 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     3841 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3901 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3961 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4021 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4081 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4141 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4201 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4261 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4321 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4381 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4441 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4501 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4561 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4621 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4681 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4741 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4801 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4861 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4921 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4981 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5041 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5101 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5161 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5221 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5281 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5341 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5401 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5461 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5521 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5581 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5641 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5701 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5761 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5821 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5881 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5941 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6001 c
//
