LOCUS       sequence-422284-        8495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP-tagged IARS1 Trp435Cys.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8495)
  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 8495)
  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-IARS1[W435C]-GFP
FEATURES             Location/Qualifiers
     source          1..8495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        372..675
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        676..879
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     830..850
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     876..900
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      4857..4866
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4863..4865,4866..4868,4869..5579)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4863..4865,4866..4868,4869..5579)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4908..4929)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5169..5188)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5516..5537
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5585..5641
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    5765..5886
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5802..5821)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5856..5875
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(join(6327..6333,6334..6351,6352..6357))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    6372..6393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6513..6530)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6684..7272)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6764..6783)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7443..8234,8235..8303))
                     /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     8066..8085
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8304..8408)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8476..8494
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
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 gactcactat agggagagcg gccgccagat cttccggatg gctcgagttt
      361 ttcagcaaga tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      421 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      481 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      541 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      601 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      661 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      721 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      781 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      841 ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc
      901 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccgact ctagctagag
      961 gatcccccgg gctgcaggaa ttcggcgcgc cacgcgtacc gatagcttcg aattctgcag
     1021 tcgacatgct tcaacaagtt ccagaaaaca taaattttcc tgctgaagaa gagaaaatct
     1081 tggagttttg gactgaattt aattgttttc aggaatgctt aaagcaatca aaacataaac
     1141 caaaatttac cttctatgat ggtcctcctt ttgcaactgg actgcctcac tatggacata
     1201 tacttgcggg tacaattaaa gatatagtta caagatatgc tcaccagagt gggtttcatg
     1261 ttgacagaag atttggatgg gattgccatg gcttacctgt ggaatatgaa attgataaga
     1321 cactgggaat cagaggacca gaggatgtgg ccaaaatggg gattacagag tataacaatc
     1381 agtgccgagc aattgtgatg agatattctg ctgagtggaa gtctactgtt agcagacttg
     1441 gccgatggat tgactttgac aatgactata aaactctgta tccacaattc atggaatcag
     1501 tctggtgggt cttcaaacaa ctctatgata aaggccttgt ttatagaggt gtgaaagtca
     1561 tgcccttctc tacggcatgt aacactccac tttccaactt cgagtcacac cagaattata
     1621 aggatgttca agatccttca gtatttgtaa ctttcccttt ggaagaagat gaaactgtat
     1681 ctttagttgc ttggacaacc actccctgga ctctacctag taaccttgct gtgtgtgtta
     1741 atccagaaat gcaatatgtg aaaattaaag atgttgccag aggacgatta ctcattttaa
     1801 tggaagccag attgtcagcc ctctataaat tggagagtga ctatgagatc cttgaaagat
     1861 ttcctggtgc ctatcttaaa ggcaagaagt acaggcccct gtttgactat ttcctgaagt
     1921 gtaaagagaa tggcgctttc actgtgcttg ttgacaacta tgtgaaggaa gaagaaggca
     1981 caggggttgt ccaccaagct ccttacttcg gtgctgagga ctatcgggtc tgtatggact
     2041 ttaacattat tcggaaagac tcactccctg tttgccctgt ggatgcttca ggctgcttca
     2101 caacggaggt gacagatttc gcaggacagt atgtgaagga tgctgacaaa agtatcatca
     2161 ggactttgaa ggaacaaggc cgacttctgg ttgccaccac cttcactcac agctaccctt
     2221 tttgctggag atcagacact cctctaattt acaaagcagt gcccagctgg tttgtgcgag
     2281 tggagaacat ggtggaccag ctcctaagga acaatgacct gtgctactgc gtcccagagt
     2341 tggtacgaga aaaacgattt ggaaattggc tgaaagatgc acgtgactgg acaatttcca
     2401 gaaacagata ctggggcacc cccatcccac tgtgggtcag cgatgacttt gaggaggtgg
     2461 tatgcattgg gtcagtggcg gaacttgaag aactgtcagg agcaaagatc tcagatctcc
     2521 acagagagag tgttgaccac ctgaccattc cttcacgctg tgggaaggga tccttgcacc
     2581 gcatctctga agtgtttgac tgttggtttg agagtggcag catgccctat gctcaggttc
     2641 attacccgtt tgaaaacaag agggagtttg aggatgcttt tcctgcagat ttcattgccg
     2701 agggcatcga ccaaaccaga ggatggtttt ataccctgct ggtgctggcc acggccctct
     2761 ttggacaacc gcctttcaag aacgtaattg tgaatgggct tgtcctggca agtgatggcc
     2821 aaaaaatgag caaacggaaa aagaattatc cagatccagt ttccatcatc cagaagtatg
     2881 gtgctgatgc cctcagatta tatctgatta actcccctgt ggtgagagca gaaaacctcc
     2941 gctttaaaga agagggtgtg cgggacgtcc ttaaggatgt actgctccca tggtacaatg
     3001 cctatcgctt cttaatccag aacgttctga ggctccagaa ggaggaagaa atagaatttc
     3061 tctacaatga gaacacggtt agagaaagcc ccaacattac agaccggtgg atcctgtcct
     3121 tcatgcagtc tctcattggc ttctttgaga ctgaaatggc agcttatagg ctttatactg
     3181 tggtgcctcg cctggtcaag tttgtagata ttctgaccaa ttggtatgtt agaatgaacc
     3241 gcagaagatt aaagggtgaa aatgggatgg aggattgtgt catggcccta gaaaccttgt
     3301 ttagtgttct gctttctctt tgcagactta tggctcccta cacacctttt ctcactgaat
     3361 tgatgtacca gaatctaaag gtgctgattg accctgtttc tgttcaggac aaggacacac
     3421 tcagcattca ctacctcatg ctgccccgtg ttcgagaaga attgattgac aagaaaacag
     3481 agagtgcagt atctcagatg cagtctgtga ttgaacttgg aagagtgatc agagaccgaa
     3541 aaactattcc cataaagtat cctttgaaag aaattgtggt tatccatcaa gatccagaag
     3601 ctcttaaaga tatcaagtct ttggagaagt atatcattga ggaactcaat gttcgaaaag
     3661 ttacactgtc tacagataaa aacaagtatg gcattcggct aagggcagaa ccagatcaca
     3721 tggtcctggg gaagcgtctg aagggagcct ttaaggcagt gatgacgtcc atcaagcagt
     3781 tgagcagtga ggagctggag cagttccaga agactgggac cattgttgtg gaaggccatg
     3841 aattgcacga tgaagacatc cgcctcatgt acacctttga tcaggccaca ggtgggactg
     3901 cgcaatttga agcacactca gatgctcagg ctttggtcct cttagatgtc actcctgacc
     3961 agtcaatggt agatgaagga atggctcggg aagtcatcaa tcgcatacag aaacttcgca
     4021 aaaagtgcaa tctggttcca actgatgaaa tcacagtgta ctataaagca aagtctgaag
     4081 gaacatatct gaatagtgtt attgaaagcc acacagagtt catatttacc accataaagg
     4141 ctcccttgaa accatatcca gtttctccat cggataaagt ccttattcaa gaaaaaacac
     4201 agttgaaggg atctgaactg gaaattacac tcaccagagg atcttccctt cctggtcctg
     4261 cttgtgcata tgtcaatctt aacatttgtg caaatggcag tgaacaaggt ggagtattgc
     4321 tcctggaaaa tccaaaaggt gacaataggt tggacctttt aaagctgaag agtgttgtca
     4381 ctagcatttt tggtgtgaaa aatacagagc tggctgtctt ccatgatgaa acagaaatac
     4441 aaaaccaaac tgacttactg agtcttagtg gaaaaacact ttgtgtgact gcaggatcgg
     4501 ctccctctct gatcaacagt tctagtactc ttctttgtca gtatatcaac ctacagctcc
     4561 tgaatgcaaa gccacaagag tgtttaatgg ggacagtggg cactctcctg cttgaaaacc
     4621 cacttgggca gaatggactc acccaccaag gtcttctgta tgaagcagcc aaggtgtttg
     4681 gccttcggag caggaagcta aagctgtttc tgaatgagac ccaaacgcag gaaattacag
     4741 aagacatccc cgtgaagact ttgaatatga agactgtgta tgtttctgtg ttaccaacaa
     4801 cagcagactt ctccggactc agatctcgag ctcaagcttc gaattctgca gtcgacgcca
     4861 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     4921 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     4981 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     5041 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     5101 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     5161 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     5221 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     5281 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     5341 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     5401 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     5461 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     5521 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     5581 actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc
     5641 caccggatct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc
     5701 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     5761 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5821 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5881 atcttaacgc gtatctttct agaagatctc ctacaatatt ctcagctgcc atggaaaatc
     5941 gatgttcttc ttttattctc tcaagatttt caggctgtat attaaaactt atattaagaa
     6001 ctatgctaac cacctcatca ggaaccgttg taggtggcgt gggttttctt ggcaatcgac
     6061 tctcatgaaa actacgagct aaatattcaa tatgttcctc ttgaccaact ttattctgca
     6121 ttttttttga acgaggttta gagcaagctt caggaaactg agacaggaat tttattaaaa
     6181 atttaaattt tgaagaaagt tcagggttaa tagcatccat tttttgcttt gcaagttcct
     6241 cagcattctt aacaaaagac gtctcttttg acatgtttaa agtttaaacc tcctgtgtga
     6301 aattattatc cgctcataat tccacacatt atacgagccg gaagcataaa gtgtaaagcc
     6361 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gccaattgct
     6421 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6481 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6541 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6601 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6661 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6721 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6781 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6841 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6901 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6961 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     7021 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7081 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     7141 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     7201 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     7261 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     7321 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     7381 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     7441 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     7501 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7561 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7621 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7681 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7741 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7801 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7861 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7921 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7981 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     8041 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     8101 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     8161 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     8221 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     8281 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     8341 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     8401 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     8461 acattaacct ataaaaatag gcgtatcacg aggcc
//
