LOCUS       sequence-345394-        7858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes His-MBP-TEV-ybbR-G180A eRF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7858)
  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 7858)
  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 eRF1 (G180A)
FEATURES             Location/Qualifiers
     source          1..7858
                     /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             5268..5285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5338..5356)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5352..5399
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5436..5891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5523..5542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5733..5754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5984..6799)
                     /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(5984..6799)
                     /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     6707..6726
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6791..6810
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6921..7509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7410..7429
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7663..7680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7695..7834
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7760..7779)
                     /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 tgcagattct cttgaattta ttgctagtaa gcttgcgatg gataacgagg
     3901 ttgaaaaaaa tattgagatc tggaaggtca agaagttggt ccaatcttta gaaaaagcta
     3961 gaggtaatgg tacttctatg atttccttag ttattcctcc taagggtcaa attccactgt
     4021 accaaaaaat gttaacagat gaatatggta ctgcctcgaa tattaaatct agggttaatc
     4081 gtctttccgt tttatctgct atcacttcca cccaacaaaa gttgaagcta tataatactt
     4141 tgcccaagaa cggtttagtt ttatattgtg gtgatatcat cactgaagat ggtaaagaaa
     4201 aaaaggtcac ttttgacatc gaaccttaca aacctatcaa cacatcctta tatttgtgtg
     4261 ataacaaatt tcatacagaa gttctttcgg aattgcttca agctgacgac aagttcggtt
     4321 ttatagtcat ggacggtcaa ggtactttgt ttggttctgt gtccggtaat acgagaactg
     4381 ttttacataa atttactgtc gatctgccaa aaaagcatgg tagagcgggt caatctgcgc
     4441 ttcgttttgc tcgtttaaga gaagaaaaaa gacataatta tgtgagaaag gtcgccgaag
     4501 ttgctgttca aaattttatt actaatgaca aagtcaatgt taagggttta attttagctg
     4561 gttctgctga ctttaagacc gatttggcta aatctgaatt attcgatcca agactagcat
     4621 gtaaggttat ttccatcgtg gatgtttctt atggtggtga aaacggtttc aaccaggcta
     4681 tcgaactttc tgccgaagcg ttggccaatg tcaagtatgt tcaagaaaag aaattattgg
     4741 aggcatattt tgacgaaatt tcccaggaca ctggtaaatt ctgttatggt atagatgata
     4801 ctttaaaggc attggattta ggtgcagtcg aaaaattaat tgttttcgaa aatttggaaa
     4861 ctatcagata tacatttaaa gatgccgagg ataatgaggt tataaaattc gctgaaccag
     4921 aagccaagga caagtcgttt gctattgaca aagctaccgg ccaagaaatg gacgttgtct
     4981 ccgaagaacc tttaattgaa tggctagcag ctaactacaa aaacttcggt gctaccttgg
     5041 aattcatcac agacaaatct tcagaaggtg cccaatttgt cacaggtttt ggtggtattg
     5101 gtgccatgct gcgttacaaa gttaattttg aacaactagt tgatgaatct gaggatgaat
     5161 attatgacga agatgaagga tccgactatg atttcattta ataacattgg aagtggataa
     5221 cggatccgaa ttcgagcgcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     5281 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     5341 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     5401 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     5461 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5521 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5581 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5641 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5701 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5761 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5821 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aactagtaac
     5881 gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5941 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     6001 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     6061 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     6121 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     6181 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     6241 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     6301 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     6361 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     6421 acactgccag cgcatcaaca atgttttcac ctgaatcagg atattcttct aatacctgga
     6481 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     6541 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     6601 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     6661 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     6721 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     6781 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     6841 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6901 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6961 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7021 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     7081 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7141 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7201 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7261 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7321 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7381 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7441 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7501 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7561 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7621 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7681 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     7741 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     7801 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgct
//
