LOCUS       sequence-368363-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ribosomal stalling reporter: positive control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  AUTHORS   Kok G, Schene IF, Ilcken EF, Alcaraz PS, Mendes MI, Smith DEC, 
            Salomons G, Shehata S, Jans JJM, Maroofian R, Hoek TA, van Es RM, 
            Rehmann H, Nieuwenhuis EES, Vos HR, Fuchs SA
  TITLE     Isoleucine-to-valine substitutions support cellular physiology 
            during isoleucine deprivation.
  JOURNAL   Nucleic Acids Res. 2025 Jan 7;53(1):gkae1184. doi: 
            10.1093/nar/gkae1184.
  PUBMED    39657787
REFERENCE   2  (bases 1 to 7748)
  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: "Nucleic 
            Acids Res."; date: "2025-01-7"; volume: "53(1):gkae1184. doi"; 
            pages: " 10.1093/nar/gkae1184"
COMMENT     Sequence Label: pJET-CMV-GFP-P2A-K20-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        198..501
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(join(1057..1063,1064..1081,1082..1087))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1102..1123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1243..1260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1414..2002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1494..1513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2173..2964,2965..3033))
                     /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     2796..2815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3034..3138)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3530..3548
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        3719..4022
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4023..4226
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4177..4197
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(4373..4375,4376..4378,4379..5089)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4418..4439)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4679..4698)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             5105..5161
                     /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"
     CDS             5168..5233
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5669..5725
                     /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"
     CDS             5732..6439
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(5874..5892)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6142..6161
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    6572..6693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6609..6628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6663..6682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(6794..6812)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        7204..7308
                     /gene="bla"
                     /label=AmpR promoter
     promoter        7311..7667
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        7311..7501
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(7333..7353)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7518..7653
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg gttcactaaa cgaatcgaag
       61 gtatccggta gcggtgcgcg ccgaattcct gccgcccggg ggaatctcta agtagaggcg
      121 ggtttttcag agggtgtaaa aaccggtggg atgggggatc ccggtgatat ggaggtttcc
      181 taaaagaggt tttgttmcat ggtaatascg aygactaata cgtagatgta ctgccaagta
      241 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
      301 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
      361 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
      421 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
      481 tttaccgtaa gttatgtaac gcggaactcc caagcttatc gataaaattt tgaatttttg
      541 taatttgttt ttgtaattct ttagtttgta tgtctgttgc tattatgtct actattcttt
      601 cccctgcact gtacccccca atatctttct agaagatctc ctacaatatt ctcagctgcc
      661 atggaaaatc gatgttcttc ttttattctc tcaagatttt caggctgtat attaaaactt
      721 atattaagaa ctatgctaac cacctcatca ggaaccgttg taggtggcgt gggttttctt
      781 ggcaatcgac tctcatgaaa actacgagct aaatattcaa tatgttcctc ttgaccaact
      841 ttattctgca ttttttttga acgaggttta gagcaagctt caggaaactg agacaggaat
      901 tttattaaaa atttaaattt tgaagaaagt tcagggttaa tagcatccat tttttgcttt
      961 gcaagttcct cagcattctt aacaaaagac gtctcttttg acatgtttaa agtttaaacc
     1021 tcctgtgtga aattattatc cgctcataat tccacacatt atacgagccg gaagcataaa
     1081 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     1141 gccaattgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1201 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1861 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1981 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2041 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2101 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2161 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2221 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2281 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2341 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2401 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2461 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2521 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2581 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2641 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2701 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2761 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2821 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2881 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2941 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3001 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3061 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3121 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     3181 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccgcccc tgcagccgaa
     3241 ttatattatt tttgccaaat aatttttaac aaaagctctg aagtcttctt catttaaatt
     3301 cttagatgat acttcatctg gaaaattgtc ccaattagta gcatcacgct gtgagtaagt
     3361 tctaaaccat ttttttattg ttgtattatc tctaatctta ctactcgatg agttttcggt
     3421 attatctcta tttttaactt ggagcaggtt ccattcattg tttttttcat catagtgaat
     3481 aaaatcaact gctttaacac ttgtgcctga acaccatatc catccggcgt aatacgactc
     3541 actataggga gagcggccgc cagatckwsm rkattgtagg agatcttcta gaaagatatt
     3601 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     3661 gaattacaaa aacaaattac aaaaattcaa aattttatcg ataagcttgg gagttccgcg
     3721 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     3781 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     3841 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     3901 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     3961 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     4021 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     4081 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     4141 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     4201 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     4261 atccacgctg ttttgacctc catagaagac accgactcta gctagaggat cccccgggct
     4321 gcaggaattc ggcgcgccac gcgtaccgat agcttcgaat tctgcagtcg acatggtgag
     4381 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     4441 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     4501 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     4561 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     4621 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     4681 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     4741 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     4801 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     4861 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     4921 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4981 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tccttaagga
     5041 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggaggcag
     5101 cggcgccacc aacttttccc tgctcaagca ggccggcgac gtggaagaga atcccggccc
     5161 cggtactgac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     5221 tgacgatgac aaggccggat ctcctggtcc gacccccagt ggcactaacg tgggatcctc
     5281 actctctgac gaggacttca aggctgtttt tggcatgacc cgctctgcct ttgccaactt
     5341 gcccttgtgg aaacagcaga acctcaagaa ggagaaagga ctcttcggat cagggcgctc
     5401 tcccagcaaa gcagtggccg cccgggcggc gggatccact gtccggcaga ggaaaaatgc
     5461 cagctgtggg acaaggagtg caggccgcac aacctcggca ggcaccgggg ggatgtggcg
     5521 attctacaca gaagattcac ctgggctcaa agttggccct aggtcgaccg ccatggcgac
     5581 maaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5641 acccggggga tccggtaccg gaagcggagc tactaacttc agcctgctga agcaggctgg
     5701 agacgtggag gagaaccctg gacctctcga ggtgagcaag ggcgaggagg ataacatggc
     5761 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     5821 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     5881 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     5941 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     6001 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     6061 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     6121 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     6181 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     6241 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     6301 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     6361 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     6421 ggacgagctg tacaagtaaa ctggatccac cggatctaga taactgatca taatcagcca
     6481 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     6541 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     6601 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     6661 ttgtggtttg tccaaactca tcaatgtatc ttactcgagt ctagacagcg ctcaccggtc
     6721 gttaacatta agctagccta gtgccatctt gctgaaaaac tcgagccatc cggaagatct
     6781 ggcggccgct ctccctatag tgagtcgtat tacgccggat ggatatggtg ttcaggcaca
     6841 agtgttaaag cagttgattt tattcactat gatgaaaaaa acaatgaatg gaacctgctc
     6901 caagttaaaa atagagataa taccgaaaac tcatcgagta gtaagattag agataataca
     6961 acaataaaaa aatggtttag aacttactca cagcgtgatg ctactaattg ggacaatttt
     7021 ccagatgaag tatcatctaa gaatttaaat gaagaagact tcagagcttt tgttaaaaat
     7081 tatttggcaa aaataatata attcggctgc aggggcggcc tcgtgatacg cctattttta
     7141 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     7201 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     7261 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtan tgaggcggaa
     7321 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     7381 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     7441 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     7501 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     7561 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     7621 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     7681 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     7741 gcttgggt
//
