LOCUS       sequence-345397-        8107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes His-MBP-TEV-ybbR-WT eRF3∆165N.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8107)
  AUTHORS   Lawson MR, Lessen LN, Wang J, Prabhakar A, Corsepius NC, Green R, 
            Puglisi JD
  TITLE     Mechanisms that ensure speed and fidelity in eukaryotic translation 
            termination.
  JOURNAL   Science. 2021 Aug 20;373(6557):876-882. doi: 
            10.1126/science.abi7801.
  PUBMED    34413231
REFERENCE   2  (bases 1 to 8107)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2021-08-20- 20"; volume: "373"; 
            issue: "6557"; pages: "876-882"
COMMENT     Sequence Label: Sc NybbR eRF3∆165N (WT)
FEATURES             Location/Qualifiers
     source          1..8107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     87..109
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(103..294)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1103..2185)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2147..2166
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2186..2263)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2469..2488
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2572..2591
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2572..2590
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2591..2615
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             2630..2652
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2671..2688
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2698..3798
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     3772..3795
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3853..3873
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5542..5559
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5612..5630)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5626..5673
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5710..6165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5797..5816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6007..6028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6258..7073)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6258..7073)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6981..7000
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7065..7084
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7195..7783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7684..7703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7937..7954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(7969..8107,1)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8034..8053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
       61 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
      121 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
      181 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
      241 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
      301 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
      361 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
      421 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
      481 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
      541 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
      601 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
      661 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
      721 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
      781 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
      841 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
      901 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
      961 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     1021 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     1081 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1141 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     1201 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     1261 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     1321 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     1381 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     1441 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatacc ctcattcagc atttgcatgg
     1501 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     1561 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     1621 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     1681 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     1741 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     1801 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     1861 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     1921 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     1981 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     2041 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     2101 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     2161 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     2221 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     2281 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     2341 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     2401 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     2461 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     2521 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     2581 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
     2641 ttaagaagga gatataccat gggttcttct caccatcacc atcaccatgg ttcttctatg
     2701 aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
     2761 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
     2821 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
     2881 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
     2941 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
     3001 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
     3061 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
     3121 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
     3181 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
     3241 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
     3301 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
     3361 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
     3421 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
     3481 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
     3541 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
     3601 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact
     3661 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
     3721 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
     3781 ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca acaataacaa taacaacaac
     3841 ctcgggatcg aggaaaacct gtacttccaa tccaatgcag attctcttga atttattgct
     3901 agtaagcttg cggccgaatc tgataagaaa gaggaagaga agtctgctga aaccaaagaa
     3961 ccaactaaag agccaacaaa ggtcgaagaa ccagttaaaa aggaggagaa accagtccag
     4021 actgaagaaa agacggagga aaaatcggaa cttccaaagg tagaagacct taaaatctct
     4081 gaatcaacac ataataccaa caatgccaat gttaccagtg ctgatgcctt gatcaaggaa
     4141 caggaagaag aagtggatga cgaagttgtt aacgatatgt ttggtggtaa agatcacgtt
     4201 tctttaattt tcatgggtca tgttgatgcc ggtaaatcta ctatgggtgg taatctacta
     4261 tacttgactg gctctgtgga taagagaact attgagaaat atgaaagaga agccaaggat
     4321 gcaggcagac aaggttggta cttgtcatgg gtcatggata ccaacaaaga agaaagaaat
     4381 gatggtaaga ctatcgaagt tggtaaggcc tactttgaaa ctgaaaaaag gcgttatacc
     4441 atattggatg ctcctggtca taaaatgtac gtttccgaga tgatcggtgg tgcttctcaa
     4501 gctgatgttg gtgttttggt catttccgcc agaaagggtg agtacgaaac cggttttgag
     4561 agaggtggtc aaactcgtga acacgcccta ttggccaaga cccaaggtgt taataagatg
     4621 gttgtcgtcg taaataagat ggatgaccca accgttaact ggtctaagga acgttacgac
     4681 caatgtgtga gtaatgtcag caatttcttg agagcaattg gttacaacat taagacagac
     4741 gttgtattta tgccagtatc cggctacagt ggtgcaaatt tgaaagatca cgtagatcca
     4801 aaagaatgcc catggtacac cggcccaact ctgttagaat atctggatac aatgaaccac
     4861 gtcgaccgtc acatcaatgc tccattcatg ttgcctattg ccgctaagat gaaggatcta
     4921 ggtaccatcg ttgaaggtaa aattgaatcc ggtcatatca aaaagggtca atccacccta
     4981 ctgatgccta acaaaaccgc tgtggaaatt caaaatattt acaacgaaac tgaaaatgaa
     5041 gttgatatgg ctatgtgtgg tgagcaagtt aaactaagaa tcaaaggtgt tgaagaagaa
     5101 gacatttcac caggttttgt actaacatcg ccaaagaacc ctatcaagag tgttaccaag
     5161 tttgtagctc aaattgctat tgtagaatta aaatctatca tagcagccgg tttttcatgt
     5221 gttatgcatg ttcatacagc aattgaagag gtacatattg ttaagttatt gcacaaatta
     5281 gaaaagggta ccaaccgtaa gtcaaagaaa ccacctgctt ttgctaagaa gggtatgaag
     5341 gtcatcgctg ttttagaaac tgaagctcca gtttgtgtgg aaacttacca agattaccct
     5401 caattaggta gattcacttt gagagatcaa ggtaccacaa tagcaattgg taaaattgtt
     5461 aaaattgccg agtaataaca ttggaagtgg ataacggatc cgaattcgag cgccgtcgac
     5521 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     5581 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     5641 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     5701 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5761 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5821 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5881 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5941 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     6001 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     6061 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     6121 aaaaatttaa cgcgaatttt aacaaactag taacgtttac aatttcaggt ggcacttttc
     6181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6241 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     6301 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     6361 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     6421 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     6481 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     6541 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     6601 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     6661 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatgttt
     6721 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     6781 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     6841 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     6901 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     6961 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     7021 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     7081 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     7141 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7201 tccttttttt ctgcacgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7261 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7321 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     7381 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7441 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7501 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7561 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7621 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7681 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7741 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7801 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7861 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7921 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     7981 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     8041 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     8101 tggctgc
//
