LOCUS       sequence-429913-        7550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP P2A RFP reporter with inert EJC immediately following stop 
            codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7550)
  AUTHORS   Inglis AJ, Guna A, Galvez-Merchan A, Pal A, Esantsi TK, Keys HR, 
            Frenkel EM, Oania R, Weissman JS, Voorhees RM
  TITLE     Coupled protein quality control during nonsense-mediated mRNA decay.
  JOURNAL   J Cell Sci. 2023 May 15;136(10):jcs261216. doi: 10.1242/jcs.261216. 
            Epub 2023 May 23.
  PUBMED    37218462
REFERENCE   2  (bases 1 to 7550)
  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: "J Cell 
            Sci."; date: "2023-05-15"; volume: "136(10):jcs261216. doi"; pages: 
            " 10.1242/jcs.261216. Epub 2023 May 23"
COMMENT     Sequence Label: inert_EJC
FEATURES             Location/Qualifiers
     source          1..7550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(23..79)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(892..916)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    926..944
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    947..965
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(967..1170)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(996..1016)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1171..1550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1722..1741
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1816..1920
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1921..1989,1990..2781)
                     /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     complement(2139..2158)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2952..3540
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3441..3460
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3694..3711
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3828..3849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3864..3869,3870..3887,3888..3894)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3902..3918
                     /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)."
     primer_bind     3907..3929
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3926..3942
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3926..3942
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3991..4112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4002..4021)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4056..4075
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4242..5264)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(4242..5264)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     protein_bind    complement(5272..5319)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     5451..5470
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5603..5827
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5816..5833
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 tcgccatgtt ggaaccggtg ccaggtccag ggttctcctc cacgtctcca gcctgcttca
       61 gcaggctgaa gttagtagct ccgcttccgg cggatccgcc ggttatgccg gcggcagtga
      121 cgaactcttt caagaccata tgatcgcgct tttcattcgg atctttagaa agagcggatt
      181 gtgtgctcaa atagtgatta tctggcaaga ggacgggacc gtctcctatc ggtgtattct
      241 gttggtaatg gtcggcaagc tgtactgatc cgtcctcgat attatgccgg atcttaaaat
      301 tgactttgat gccattcttc tgtttatctg ccatgatata gacgttgtgt gaattgtaat
      361 tatattcgag tttatggccc aggatgttac cgtcttcctt gaagtcgatt cccttcagct
      421 ctattcgatt cacgagcgtg tctccttcaa atttaacctc agcccgcgtt ttataattac
      481 catcgtcttt gaaaaagatc gtcctttcct gtacgtaccc ctcaggcatt gcactcttga
      541 aaaaatcgtg ctgcttcata tggtctgggt atctgctaaa acattgcacg ccgtaggtaa
      601 gcgttgttac gagcgtcggc cagggtacgg gcagtttccc ggtagtgcag ataaatttca
      661 aggtaagttt accgtaagtg gcgtctccct ctccttcacc gctcactgaa aatttatggc
      721 cattcacgtc tccgtccaat tcaaccagga tggggacgac acccgtaaaa agttcttcac
      781 ctttagacac catggtaccg taagcttaag tttaaacgct agagtccgga ggctggatcg
      841 gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg
      901 gttcactaaa cgagctcgtc gacgatctct atcactgata gggagatctc tatcactgat
      961 agggagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa
     1021 tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact
     1081 cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg
     1141 cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat
     1201 gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca
     1261 tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc
     1321 caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg
     1381 cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca
     1441 gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac
     1501 taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata
     1561 tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca
     1621 attgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac
     1681 ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt
     1741 actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact
     1801 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1861 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1921 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1981 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2041 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2101 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2161 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2221 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2281 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2341 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2401 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2461 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2521 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2581 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2641 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2701 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2761 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2821 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2881 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2941 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3001 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3061 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     3121 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3181 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3241 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3301 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3361 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3421 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3481 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3541 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3601 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3661 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3721 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     3781 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     3841 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     3901 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
     3961 tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca
     4021 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     4081 ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc cagcatgaga
     4141 tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt
     4201 tcatagaagg cggcggtgga atcgaaatct cgtagtacgt gctattcctt tgccctcgga
     4261 cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag
     4321 acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg
     4381 attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc
     4441 tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca
     4501 agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc
     4561 ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag
     4621 tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg
     4681 tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc
     4741 tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga
     4801 tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg
     4861 aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catggcctcc
     4921 gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc
     4981 tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact
     5041 tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa
     5101 ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa
     5161 gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg
     5221 atcagaaact tctcgacaga cgtcgcggtg agttcaggct ttttggccaa ggaagttcct
     5281 atactttcta gagaatagga acttcggaat aggaacttct aggtacgtga accatcaccc
     5341 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5401 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5461 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5521 acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag
     5581 ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg
     5641 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     5701 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     5761 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     5821 ggcacagtcg aggctgatca gcgggtttaa acgggccctc tagactcgag ttagtgatac
     5881 ttgtgggcca gggcattagc cacaccagcc accactttct gataggcagc ctgcactggt
     5941 ggggtgaatt ctttgccaaa gtgatgggcc agcacacaga ccagcacgtt gcccaggagc
     6001 tgtgggagga agataagagg tatgaacatg attagcaaaa gggcctagct tggactcaga
     6061 ataatccagc cttatcccaa ccataaaata aaagcagaat ggtagctgga ttgtagctgc
     6121 tattagcaat atgaaacctc ttacatcagt tacaatttat atgcagaaat atttatatgc
     6181 agaaatattg ctattgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     6241 agaggcatga tacattgtat cattattgcc ctgaaagaaa gagattaggg aaagtattag
     6301 aaataagata aacaaaaaag tatattaaaa gaagaaagca ttttttaaaa ttacaaatgc
     6361 aaaattaccc tgatttggtc aatatgtgta cacatattaa aacattacac tttaacccat
     6421 aaatatgtat aatgattatg tatcaattaa aaataaaaga aaataaagta gggagattat
     6481 gaatatgcaa ataagcacac atatattcca aatagtaatg tactaggcag actgtgtaaa
     6541 gttttttttt aagttactta atgtatctca gagatatttc cttttgttat acacaatgtt
     6601 aaggcattaa gtataatagt aaaaattgcg gagaagaaaa aaaaagaaag caagaattaa
     6661 acaaaagaaa acaattgtta tgaacagcaa ataaaagaaa ctaaaacgat cctgagactt
     6721 ccacactgat gcaatcattc gtctgtttcc cattctaaac tgtaccctgt tacttctccc
     6781 cttcctatga catgaactta accatagaaa agaaggggaa agaaaacatc aagggtccca
     6841 tagactcacc ctgaagttct caggatccac gtgcagcttg tcacccgcgg ccgcttaccc
     6901 tgttgaatgg cgtccctctg cgcgttcgta ttgctccact attgtgtaat cttcgttgtg
     6961 actggtaatg tccaatttaa tattgacatt gtacgcgcct ggcaattgga caggcttttt
     7021 agctttgtac gtcgttttaa cttccgcgtc ataatggcct ccgtctttga gtttcagccg
     7081 ctgcttaatc tcacccttaa gtgcgccgtc ctctgggtac atccgctcgc tactggcttc
     7141 ccaacccata gttttcttct gcatgactgg gccatcgctt gggaagttgg ttccgcgaag
     7201 tttcacctta tatataaact ctccatcttg caaagaagag tcctgagtca ctgtaacaac
     7261 gcccccatct tcaaagttca tgacgcgctc ccatttgaac ccctctggaa aactcagttt
     7321 aaggtaatca ggaatgtctg cgggatgttt gacatacgcc ttggaaccat acataaactg
     7381 ggggctaaga atgtcccatg cgaaaggaag tggtccacct ttagtaacct tcaatttagc
     7441 ggtctgtgtt ccttcatatg gtcgaccctc cccctctccc tcgatttcga actcgtggcc
     7501 gtttacagat ccctccatgt gaactttgaa cctcatgaat tccttgataa
//
