LOCUS       sequence-364881-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ribosomal stalling reporter: empty.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  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 6062)
  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-K0-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..6062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(72..90)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        158..262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(263..331,332..1123)
                     /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(481..500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1294..1882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1783..1802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2036..2053
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2173..2194
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2209..2214,2215..2232,2233..2239)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     enhancer        2795..3098
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3099..3302
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3253..3273
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3299..3323
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(3449..3451,3452..3454,3455..4165)
                     /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(3494..3515)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3755..3774)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4181..4237
                     /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             4244..4309
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4685..4741
                     /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             4748..5455
                     /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(4890..4908)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5158..5177
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5588..5709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5625..5644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5679..5698
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5809..5828)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5810..5828)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 aaatgaagaa gacttcagag cttttgttaa aaattatttg gcaaaaataa tataattcgg
       61 ctgcaggggc ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
      121 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      181 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      241 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      301 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      361 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      421 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      481 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
      541 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
      601 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
      661 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
      721 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
      781 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
      841 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
      901 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
      961 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1021 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1081 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1141 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1201 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1261 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1321 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1381 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1441 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1501 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1561 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1621 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1681 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1741 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1801 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1861 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     1921 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     1981 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2041 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2101 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcaa ttggcagtga
     2161 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     2221 gcttccggct cgtataatgt gtggaattat gagcggataa taatttcaca caggaggttt
     2281 aaactttaaa catgtcaaaa gagacgtctt ttgttaagaa tgctgaggaa cttgcaaagc
     2341 aaaaaatgga tgctattaac cctgaacttt cttcaaaatt taaattttta ataaaattcc
     2401 tgtctcagtt tcctgaagct tgctctaaac ctcgttcaaa aaaaatgcag aataaagttg
     2461 gtcaagagga acatattgaa tatttagctc gtagttttca tgagagtcga ttgccaagaa
     2521 aacccacgcc acctacaacg gttcctgatg aggtggttag catagttctt aatataagtt
     2581 ttaatataca gcctgaaaat cttgagagaa taaaagaaga acatcgattt tccatggcag
     2641 ctgagaatat tgtaggagat cttctagaaa gatattgggg ggtacagtgc aggggaaaga
     2701 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2761 attcaaaatt ttatcgataa gcttgggagt tccgcgttac ataacttacg gtaaatggcc
     2821 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     2881 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     2941 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     3001 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     3061 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     3121 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     3181 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     3241 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     3301 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     3361 gaagacaccg actctagcta gaggatcccc cgggctgcag gaattcggcg cgccacgcgt
     3421 accgatagct tcgaattctg cagtcgacat ggtgagcaag ggcgaggagc tgttcaccgg
     3481 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     3541 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     3601 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     3661 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3721 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3781 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3841 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3901 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3961 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     4021 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     4081 ccccaacgag aagcgcgatc acatggtcct taaggagttc gtgaccgccg ccgggatcac
     4141 tctcggcatg gacgagctgt acaagtccgg aggcagcggc gccaccaact tttccctgct
     4201 caagcaggcc ggcgacgtgg aagagaatcc cggccccggt actgactaca aagaccatga
     4261 cggtgattat aaagatcatg acatcgatta caaggatgac gatgacaagg ccggatctcc
     4321 tggtccgacc cccagtggca ctaacgtggg atcctcactc tctgacgagg acttcaaggc
     4381 tgtttttggc atgacccgct ctgcctttgc caacttgccc ttgtggaaac agcagaacct
     4441 caagaaggag aaaggactct tcggatcagg gcgctctccc agcaaagcag tggccgcccg
     4501 ggcggcggga tccactgtcc ggcagaggaa aaatgccagc tgtgggacaa ggagtgcagg
     4561 ccgcacaacc tcggcaggca ccggggggat gtggcgattc tacacagaag attcacctgg
     4621 gctcaaagtt ggccctaggt cgaccgccat ggcgaccccc gggggatccg gtaccggaag
     4681 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     4741 tctcgaggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     4801 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     4861 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     4921 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     4981 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     5041 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     5101 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     5161 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     5221 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     5281 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     5341 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     5401 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaactgg
     5461 atccaccgga tctagataac tgatcataat cagccatacc acatttgtag aggttttact
     5521 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     5581 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5641 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5701 tgtatcttac tcgagtctag acagcgctca ccggtcgtta acattaagct agcctagtgc
     5761 catcttgctg aaaaactcga gccatccgga agatctggcg gccgctctcc ctatagtgag
     5821 tcgtattacg ccggatggat atggtgttca ggcacaagtg ttaaagcagt tgattttatt
     5881 cactatgatg aaaaaaacaa tgaatggaac ctgctccaag ttaaaaatag agataatacc
     5941 gaaaactcat cgagtagtaa gattagagat aatacaacaa taaaaaaatg gtttagaact
     6001 tactcacagc gtgatgctac taattgggac aattttccag atgaagtatc atctaagaat
     6061 tt
//
