LOCUS       sequence-364879-        7310 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Ribosomal stalling reporter: 15x isoleucine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7310)
  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 7310)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-I15-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..7310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        493..796
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        797..1000
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     951..971
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     997..1021
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1147..1149,1150..1152,1153..1863)
                     /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(1192..1213)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1453..1472)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1879..1935
                     /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             1942..2007
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2479..2535
                     /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             2560..3267
                     /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(2702..2720)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2970..2989
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3400..3521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(join(4008..4014,4015..4032,4033..4038))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    4053..4074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4194..4211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4365..4953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4445..4464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5124..5915,5916..5984))
                     /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     5747..5766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5985..6089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6481..6500
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6481..6499
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6600..6721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(7119..7223)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gackcasway aksgattttc catggcagct gagrawwwtg taggagatct
      361 tctagaaaga tattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
      421 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt atcgataagc
      481 ttgggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      541 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      601 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      661 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      721 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      781 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      841 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      901 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      961 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
     1021 cgcctggaga cgccatccac gctgttttga cctccataga agacaccgac tctagctaga
     1081 ggatcccccg ggctgcagga attcggcgcg ccacgcgtac cgatagcttc gaattctgca
     1141 gtcgacatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1201 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1261 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1321 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1381 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1441 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1501 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1561 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1621 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1681 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1741 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1801 atggtcctta aggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1861 aagtccggag gcagcggcgc caccaacttt tccctgctca agcaggccgg cgacgtggaa
     1921 gagaatcccg gccccggtac tgactacaaa gaccatgacg gtgattataa agatcatgac
     1981 atcgattaca aggatgacga tgacaaggcc ggatctcctg gtccgacccc cagtggcact
     2041 aacgtgggat cctcactctc tgacgaggac ttcaaggctg tttttggcat gacccgctct
     2101 gcctttgcca acttgccctt gtggaaacag cagaacctca agaaggagaa aggactcttc
     2161 ggatcagggc gctctcccag caaagcagtg gccgcccggg cggcgggatc cactgtccgg
     2221 cagaggaaaa atgccagctg tgggacaagg agtgcaggcc gcacaacctc ggcaggcacc
     2281 ggggggatgt ggcgattcta cacagaagat tcacctgggc tcaaagttgg ccctaggtcg
     2341 accgccatgg cgaccatgat tatcggcagt atcagcatca tctcaggtat tggcggatct
     2401 agtgggggta ttggcggggg cggcataatc ggtggaggca ttatcatatc tattatcata
     2461 ggcaccggtg gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag
     2521 gagaaccctg gacctgaatt catggcccag tccaagcacg tgagcaaggg cgaggaggat
     2581 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     2641 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2701 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     2761 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     2821 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     2881 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     2941 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     3001 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     3061 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     3121 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     3181 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     3241 ggcggcatgg acgagctgta caagtaaact ggatccaccg gatctagata actgatcata
     3301 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3361 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     3421 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3481 cattctagtt gtggtttgtc caaactcatc aatgtatctt actcgagtct agactatgtc
     3541 tactattctt tcccctgcac tgtacccccc aatatctttc tagaagatct cctacaatat
     3601 tctcagctgc catggaaaat cgatgttctt cttttattct ctcaagattt tcaggctgta
     3661 tattaaaact tatattaaga actatgctaa ccacctcatc aggaaccgtt gtaggtggcg
     3721 tgggttttct tggcaatcga ctctcatgaa aactacgagc taaatattca atatgttcct
     3781 cttgaccaac tttattctgc attttttttg aacgaggttt agagcaagct tcaggaaact
     3841 gagacaggaa ttttattaaa aatttaaatt ttgaagaaag ttcagggtta atagcatcca
     3901 ttttttgctt tgcaagttcc tcagcattct taacaaaaga cgtctctttt gacatgttta
     3961 aagtttaaac ctcctgtgtg aaattattat ccgctcataa ttccacacat tatacgagcc
     4021 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4081 ttgcgctcac tgccaattgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4141 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4801 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5401 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6121 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggccgccc
     6181 ctgcagccga attatattat ttttgccaaa taatttttaa caaaagctct gaagtcttct
     6241 tcatttaaat tcttagatga tacttcatct ggaaaattgt cccaattagt agcatcacgc
     6301 tgtgagtaag ttctaaacca tttttttatt gttgtattat ctctaatctt actactcgat
     6361 gagttttcgg tattatctct atttttaact tggagcaggt tccattcatt gtttttttca
     6421 tcatagtgaa taaaatcaac tgctttaaca cttgtgcctg aacaccatat ccatccggcg
     6481 taatacgact cactataggg agagcggccg ccagatcttc cggatggctc gagtttttca
     6541 gcaagatggc actaggctag cttaatgtta acgaccggtg agcgctgtct agactcgagt
     6601 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     6661 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     6721 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     6781 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcagt tatctagatc
     6841 cggtggatcc agtttacttg tacagacact acttgaagca ctcaaggcaa gctttattga
     6901 ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac
     6961 cagagagacc cagtacaagc aaaaagcaga tcttgtcttc gttgggagtg aattagccct
     7021 tctagtcccc ccttttcttt taaaaagtgg atgagatcta cagctgcttt gtaagtcatc
     7081 ggtcccaacg gtaccgaggc ggggggttga atactcatac tcttcctctt tcaatattat
     7141 tgaagcattt atcggggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7201 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     7261 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
//
