LOCUS       sequence-247773-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 3xFLAG tagged highly destabilized nanoLuciferase (nLuc) 
            protein from CrPV IRES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  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 7200)
  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: pcDNA3.1D/mEGFP + CrPV IGR IRES-nLuc-3XFLAG-CL...
FEATURES             Location/Qualifiers
     source          1..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        87..466
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        467..670
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     621..641
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     715..734
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        715..733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(764..766,767..769,770..1483)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(809..830)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1070..1089)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1417..1438
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1719..2231
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2232..2297
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2304..2351
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             2355..2474
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     CDS             2550..2591
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2601..2618
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2641..2658)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2647..2871
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2917..3345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3004..3023)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3214..3235
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3354..3374)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3359..3688
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3539..3674
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3601..3620
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3755..4549
                     /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(3809..3828)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4419..4438
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4725..4846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4762..4781)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4816..4835
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4895..4911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4895..4911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4908..4930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4919..4935
                     /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(4943..4949,4950..4967,4968..4973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4988..5009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5126..5143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5297..5885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5377..5396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6056..6847,6848..6916))
                     /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     6679..6698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6917..7021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7096..7115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
       61 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      121 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      181 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      241 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      301 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      361 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      421 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      481 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      541 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      601 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      661 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      721 gactcactat agggagaccc aagctggcta gttaagcttc accatggtga gcaagggcga
      781 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
      841 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
      901 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
      961 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1021 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1081 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1141 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1201 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1261 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1321 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1381 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1441 cgccgccggg atcactctcg gcatggacga gctgtacaag taataataag gatccagtac
     1501 ccttcaccaa agcaaaaatg tgatcttgct tgtaaataca attttgagag gttaataaat
     1561 tacaagtagt gctatttttg tatttaggtt agctatttag ctttacgttc caggatgcct
     1621 agtggcagcc ccacaatatc caggaagccc tctctgcggt ttttcagatt aggtagtcga
     1681 aaaacctaag aaatttacct gctacatttc aagataccat ggtcttcaca ctcgaagatt
     1741 tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt gaacagggag
     1801 gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa aggattgtcc
     1861 tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat gaaggtctga
     1921 gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc
     1981 atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca
     2041 tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca
     2101 ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg
     2161 acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac
     2221 gcattctggc ggactacaaa gaccatgacg gtgattataa agatcatgac atcgattaca
     2281 aggatgacga tgacaagaat tctgcttgca agaactggtt cagtagctta agccactttg
     2341 tgatccacct taacagccac ggctttccgc ctgaggttga agagcaagcc gccggtacat
     2401 tgcctatgtc ctgcgcacaa gaaagcggta tggaccggca cccagccgct tgtgcttcag
     2461 ctcgcatcaa cgtctaaaag ggtcaagaca attctgcaga tatccagcac agtggcggcc
     2521 gctcgagtct agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc
     2581 tcgattctac gcgtaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag
     2641 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2701 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2761 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2821 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2881 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2941 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3001 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3061 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3121 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3181 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3241 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3301 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3361 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3421 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3481 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3541 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3601 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3661 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     3721 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3781 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3841 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3901 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     3961 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4021 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4081 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4141 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4201 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4261 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4321 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4381 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4441 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4501 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4561 gggttcgcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4621 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4681 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4741 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4801 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4861 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4921 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4981 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5041 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5101 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5161 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5221 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5281 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5341 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5401 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5461 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5521 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5581 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5641 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5701 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5761 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5821 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5881 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5941 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6001 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6061 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6121 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6181 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6241 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6301 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6361 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6421 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6481 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6541 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6601 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6661 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6721 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6781 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6841 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6901 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6961 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7021 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7081 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7141 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
//
