LOCUS       sequence-364929-        6122 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 6122)
  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 6122)
  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..6122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(442..448,449..466,467..472))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    487..508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(628..645)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(799..1387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(879..898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1558..2349,2350..2418))
                     /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     2181..2200
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2419..2523)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2591..2609
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     2915..2934
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2915..2933
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        3103..3406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3407..3610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3561..3581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3607..3631
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(3757..3759,3760..3762,3763..4473)
                     /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(3802..3823)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4063..4082)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4489..4545
                     /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             4552..4617
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5053..5109
                     /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             5116..5823
                     /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(5258..5276)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5526..5545
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5956..6077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5993..6012)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6047..6066
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ycccaatatc tttctagaag atctcctaca atattctcag ctgccatgga aaatcgatgt
       61 tcttctttta ttctctcaag attttcaggc tgtatattaa aacttatatt aagaactatg
      121 ctaaccacct catcaggaac cgttgtaggt ggcgtgggtt ttcttggcaa tcgactctca
      181 tgaaaactac gagctaaata ttcaatatgt tcctcttgac caactttatt ctgcattttt
      241 tttgaacgag gtttagagca agcttcagga aactgagaca ggaattttat taaaaattta
      301 aattttgaag aaagttcagg gttaatagca tccatttttt gctttgcaag ttcctcagca
      361 ttcttaacaa aagacgtctc ttttgacatg tttaaagttt aaacctcctg tgtgaaatta
      421 ttatccgctc ataattccac acattatacg agccggaagc ataaagtgta aagcctgggg
      481 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgccaa ttgctttcca
      541 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      601 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
      661 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
      721 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      781 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      841 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      901 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      961 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1021 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1081 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1141 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1201 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1261 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1321 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1381 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1441 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1501 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1561 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1621 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1681 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     1741 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1801 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1861 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1921 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1981 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2041 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2101 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2161 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2221 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2281 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2341 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2401 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2461 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     2521 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     2581 aacctataaa aataggcgta tcacgaggcc gcccctgcag ccgaattata ttatttttgc
     2641 caaataattt ttaacaaaag ctctgaagtc ttcttcattt aaattcttag atgatacttc
     2701 atctggaaaa ttgtcccaat tagtagcatc acgctgtgag taagttctaa accatttttt
     2761 tattgttgta ttatctctaa tcttactact cgatgagttt tcggtattat ctctattttt
     2821 aacttggagc aggttccatt cattgttttt ttcatcatag tgaataaaat caactgcttt
     2881 aacacttgtg cctgaacacc atatccatcc ggcgtaatac gactcactat agggagagcg
     2941 gccgccagat cttccggatg gctsgagttt ttcagmaaga trkyrsgggg tacagtgcag
     3001 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     3061 ttacaaaaat tcaaaatttt atcgataagc ttgggagttc cgcgttacat aacttacggt
     3121 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3181 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3241 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3301 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3361 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3421 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3481 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3541 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3601 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     3661 cctccataga agacaccgac tctagctaga ggatcccccg ggctgcagga attcggcgcg
     3721 ccacgcgtac cgatagcttc gaattctgca gtcgacatgg tgagcaaggg cgaggagctg
     3781 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3841 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3901 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3961 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     4021 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     4081 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     4141 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     4201 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     4261 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     4321 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     4381 agcaaagacc ccaacgagaa gcgcgatcac atggtcctta aggagttcgt gaccgccgcc
     4441 gggatcactc tcggcatgga cgagctgtac aagtccggag gcagcggcgc caccaacttt
     4501 tccctgctca agcaggccgg cgacgtggaa gagaatcccg gccccggtac tgactacaaa
     4561 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaaggcc
     4621 ggatctcctg gtccgacccc cagtggcact aacgtgggat cctcactctc tgacgaggac
     4681 ttcaaggctg tttttggcat gacccgctct gcctttgcca acttgccctt gtggaaacag
     4741 cagaacctca agaaggagaa aggactcttc ggatcagggc gctctcccag caaagcagtg
     4801 gccgcccggg cggcgggatc cactgtccgg cagaggaaaa atgccagctg tgggacaagg
     4861 agtgcaggcc gcacaacctc ggcaggcacc ggggggatgt ggcgattcta cacagaagat
     4921 tcacctgggc tcaaagttgg cccwaggkcs accsccawgg sgamaaaaaa aaaaaaaaaa
     4981 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaacccgg gggatccggt
     5041 accggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac
     5101 cctggacctc tcgaggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     5161 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     5221 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     5281 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     5341 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     5401 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     5461 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     5521 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     5581 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     5641 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     5701 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     5761 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     5821 taaactggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     5881 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     5941 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     6001 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     6061 ctcatcaatg tatcttactc gagtctagac agcgctcacc ggtcgttaac attmasykwr
     6121 cc
//
