LOCUS       sequence-345398-        8107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes His-MBP-TEV-ybbR-H348E 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 (H348E)
FEATURES             Location/Qualifiers
     source          1..8107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     69..91
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(85..276)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1085..2167)
                     /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     2129..2148
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2168..2245)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2451..2470
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2554..2573
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2554..2572
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2573..2597
                     /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             2612..2634
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2653..2670
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2680..3780
                     /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     3754..3777
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3835..3855
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5524..5541
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5594..5612)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5608..5655
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5692..6147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5779..5798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5989..6010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6240..7055)
                     /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(6240..7055)
                     /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     6963..6982
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7047..7066
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7177..7765
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7666..7685
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7919..7936
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7951..8090
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8016..8035)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
       61 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
      121 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
      181 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
      241 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
      301 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
      361 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
      421 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
      481 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
      541 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
      601 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
      661 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
      721 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
      781 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
      841 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
      901 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
      961 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     1021 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     1081 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1141 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     1201 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     1261 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     1321 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     1381 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     1441 gcatcgcagt gggaacgata ccctcattca gcatttgcat ggtttgttga aaaccggaca
     1501 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     1561 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     1621 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     1681 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     1741 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     1801 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     1861 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     1921 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     1981 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     2041 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     2101 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     2161 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     2221 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     2281 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     2341 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     2401 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     2461 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     2521 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     2581 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
     2641 atgggttctt ctcaccatca ccatcaccat ggttcttcta tgaaaatcga agaaggtaaa
     2701 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
     2761 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
     2821 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
     2881 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
     2941 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
     3001 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
     3061 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
     3121 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
     3181 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
     3241 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
     3301 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
     3361 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
     3421 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     3481 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     3541 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     3601 tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa
     3661 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     3721 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     3781 aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac
     3841 ctgtacttcc aatccaatgc agattctctt gaatttattg ctagtaagct tgcggccgaa
     3901 tctgataaga aagaggaaga gaagtctgct gaaaccaaag aaccaactaa agagccaaca
     3961 aaggtcgaag aaccagttaa aaaggaggag aaaccagtcc agactgaaga aaagacggag
     4021 gaaaaatcgg aacttccaaa ggtagaagac cttaaaatct ctgaatcaac acataatacc
     4081 aacaatgcca atgttaccag tgctgatgcc ttgatcaagg aacaggaaga agaagtggat
     4141 gacgaagttg ttaacgatat gtttggtggt aaagatcacg tttctttaat tttcatgggt
     4201 catgttgatg ccggtaaatc tactatgggt ggtaatctac tatacttgac tggctctgtg
     4261 gataagagaa ctattgagaa atatgaaaga gaagccaagg atgcaggcag acaaggttgg
     4321 tacttgtcat gggtcatgga taccaacaaa gaagaaagaa atgatggtaa gactatcgaa
     4381 gttggtaagg cctactttga aactgaaaaa aggcgttata ccatattgga tgctcctggt
     4441 gaaaaaatgt acgtttccga gatgatcggt ggtgcttctc aagctgatgt tggtgttttg
     4501 gtcatttccg ccagaaaggg tgagtacgaa accggttttg agagaggtgg tcaaactcgt
     4561 gaacacgccc tattggccaa gacccaaggt gttaataaga tggttgtcgt cgtaaataag
     4621 atggatgacc caaccgttaa ctggtctaag gaacgttacg accaatgtgt gagtaatgtc
     4681 agcaatttct tgagagcaat tggttacaac attaagacag acgttgtatt tatgccagta
     4741 tccggctaca gtggtgcaaa tttgaaagat cacgtagatc caaaagaatg cccatggtac
     4801 accggcccaa ctctgttaga atatctggat acaatgaacc acgtcgaccg tcacatcaat
     4861 gctccattca tgttgcctat tgccgctaag atgaaggatc taggtaccat cgttgaaggt
     4921 aaaattgaat ccggtcatat caaaaagggt caatccaccc tactgatgcc taacaaaacc
     4981 gctgtggaaa ttcaaaatat ttacaacgaa actgaaaatg aagttgatat ggctatgtgt
     5041 ggtgagcaag ttaaactaag aatcaaaggt gttgaagaag aagacatttc accaggtttt
     5101 gtactaacat cgccaaagaa ccctatcaag agtgttacca agtttgtagc tcaaattgct
     5161 attgtagaat taaaatctat catagcagcc ggtttttcat gtgttatgca tgttcataca
     5221 gcaattgaag aggtacatat tgttaagtta ttgcacaaat tagaaaaggg taccaaccgt
     5281 aagtcaaaga aaccacctgc ttttgctaag aagggtatga aggtcatcgc tgttttagaa
     5341 actgaagctc cagtttgtgt ggaaacttac caagattacc ctcaattagg tagattcact
     5401 ttgagagatc aaggtaccac aatagcaatt ggtaaaattg ttaaaattgc cgagtaataa
     5461 cattggaagt ggataacgga tccgaattcg agcgccgtcg acaagcttgc ggccgcactc
     5521 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     5581 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     5641 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     5701 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5761 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5821 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5881 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5941 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     6001 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     6061 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     6121 ttaacaaact agtaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa
     6181 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     6241 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     6301 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     6361 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     6421 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     6481 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     6541 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     6601 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     6661 tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt tttcacctga atcaggatat
     6721 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     6781 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     6841 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     6901 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     6961 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     7021 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     7081 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7141 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7201 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7261 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7321 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7381 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7441 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7501 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7561 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7621 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7681 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7741 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7801 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7861 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7921 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     7981 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     8041 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     8101 cgccaac
//
