LOCUS       sequence-345395-        8074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes His-MBP-TEV-WT eRF3∆165N.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8074)
  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 8074)
  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 eRF3∆165N (WT)
FEATURES             Location/Qualifiers
     source          1..8074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..84
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(78..269)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1078..2160)
                     /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     2122..2141
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2161..2238)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2444..2463
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2547..2566
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2547..2565
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2566..2590
                     /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             2605..2627
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2646..2663
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2673..3773
                     /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     3747..3770
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3828..3848
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5484..5501
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5554..5572)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5568..5615
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5652..6107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5739..5758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5949..5970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6200..7015)
                     /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(6200..7015)
                     /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     6923..6942
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7007..7026
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7137..7725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7626..7645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7879..7896
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7911..8050
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7976..7995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
       61 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
      121 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
      181 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
      241 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
      301 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
      361 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      421 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      481 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      541 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      601 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      661 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      721 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      781 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      841 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      901 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      961 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     1021 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1081 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1141 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1201 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1261 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1321 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1381 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1441 agtgggaacg ataccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1501 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1561 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1621 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1681 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1741 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1801 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1861 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1921 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1981 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     2041 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2101 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2161 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2221 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     2281 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2341 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2401 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2461 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2521 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2581 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggtt
     2641 cttctcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt aaactggtaa
     2701 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
     2761 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
     2821 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
     2881 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
     2941 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
     3001 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
     3061 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
     3121 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
     3181 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
     3241 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
     3301 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
     3361 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
     3421 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg
     3481 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
     3541 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     3601 aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
     3661 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     3721 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
     3781 gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa aacctgtact
     3841 tccaatccaa tgcagccgaa tctgataaga aagaggaaga gaagtctgct gaaaccaaag
     3901 aaccaactaa agagccaaca aaggtcgaag aaccagttaa aaaggaggag aaaccagtcc
     3961 agactgaaga aaagacggag gaaaaatcgg aacttccaaa ggtagaagac cttaaaatct
     4021 ctgaatcaac acataatacc aacaatgcca atgttaccag tgctgatgcc ttgatcaagg
     4081 aacaggaaga agaagtggat gacgaagttg ttaacgatat gtttggtggt aaagatcacg
     4141 tttctttaat tttcatgggt catgttgatg ccggtaaatc tactatgggt ggtaatctac
     4201 tatacttgac tggctctgtg gataagagaa ctattgagaa atatgaaaga gaagccaagg
     4261 atgcaggcag acaaggttgg tacttgtcat gggtcatgga taccaacaaa gaagaaagaa
     4321 atgatggtaa gactatcgaa gttggtaagg cctactttga aactgaaaaa aggcgttata
     4381 ccatattgga tgctcctggt cataaaatgt acgtttccga gatgatcggt ggtgcttctc
     4441 aagctgatgt tggtgttttg gtcatttccg ccagaaaggg tgagtacgaa accggttttg
     4501 agagaggtgg tcaaactcgt gaacacgccc tattggccaa gacccaaggt gttaataaga
     4561 tggttgtcgt cgtaaataag atggatgacc caaccgttaa ctggtctaag gaacgttacg
     4621 accaatgtgt gagtaatgtc agcaatttct tgagagcaat tggttacaac attaagacag
     4681 acgttgtatt tatgccagta tccggctaca gtggtgcaaa tttgaaagat cacgtagatc
     4741 caaaagaatg cccatggtac accggcccaa ctctgttaga atatctggat acaatgaacc
     4801 acgtcgaccg tcacatcaat gctccattca tgttgcctat tgccgctaag atgaaggatc
     4861 taggtaccat cgttgaaggt aaaattgaat ccggtcatat caaaaagggt caatccaccc
     4921 tactgatgcc taacaaaacc gctgtggaaa ttcaaaatat ttacaacgaa actgaaaatg
     4981 aagttgatat ggctatgtgt ggtgagcaag ttaaactaag aatcaaaggt gttgaagaag
     5041 aagacatttc accaggtttt gtactaacat cgccaaagaa ccctatcaag agtgttacca
     5101 agtttgtagc tcaaattgct attgtagaat taaaatctat catagcagcc ggtttttcat
     5161 gtgttatgca tgttcataca gcaattgaag aggtacatat tgttaagtta ttgcacaaat
     5221 tagaaaaggg taccaaccgt aagtcaaaga aaccacctgc ttttgctaag aagggtatga
     5281 aggtcatcgc tgttttagaa actgaagctc cagtttgtgt ggaaacttac caagattacc
     5341 ctcaattagg tagattcact ttgagagatc aaggtaccac aatagcaatt ggtaaaattg
     5401 ttaaaattgc cgagtaataa cattggaagt ggataacgga tccgaattcg agcgccgtcg
     5461 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     5521 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     5581 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     5641 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5701 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5761 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5821 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5881 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5941 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     6001 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     6061 acaaaaattt aacgcgaatt ttaacaaact agtaacgttt acaatttcag gtggcacttt
     6121 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6181 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     6241 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     6301 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     6361 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     6421 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     6481 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     6541 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     6601 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt
     6661 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     6721 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     6781 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     6841 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     6901 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     6961 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     7021 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     7081 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7141 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7201 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7261 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     7321 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7381 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7441 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7501 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7561 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7621 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7681 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7741 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     7801 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     7861 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     7921 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     7981 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     8041 catggctgcg ccccgacacc cgccaacacc cgct
//
