LOCUS       sequence-345393-        7858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes His-MBP-TEV-ybbR-WT 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 (WT)
FEATURES             Location/Qualifiers
     source          1..7858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..38
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(32..223)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1032..2114)
                     /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     2076..2095
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2115..2192)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2398..2417
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2501..2520
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2501..2519
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2520..2544
                     /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             2559..2581
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2600..2617
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2627..3727
                     /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     3701..3724
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3782..3802
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5222..5239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5292..5310)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5306..5353
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5390..5845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5477..5496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5687..5708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5938..6753)
                     /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(5938..6753)
                     /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     6661..6680
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6745..6764
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6875..7463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7364..7383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7617..7634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7649..7788
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7714..7733)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
       61 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
      121 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
      181 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
      241 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
      301 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
      361 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
      421 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
      481 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
      541 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
      601 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
      661 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
      721 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
      781 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
      841 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
      901 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
      961 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     1021 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     1081 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     1141 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     1201 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     1261 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     1321 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     1381 gcaaccagca tcgcagtggg aacgataccc tcattcagca tttgcatggt ttgttgaaaa
     1441 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     1501 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     1561 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     1621 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     1681 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     1741 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     1801 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     1861 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     1921 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     1981 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     2041 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     2101 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     2161 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     2221 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     2281 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     2341 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     2401 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     2461 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     2521 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
     2581 atataccatg ggttcttctc accatcacca tcaccatggt tcttctatga aaatcgaaga
     2641 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
     2701 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
     2761 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
     2821 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
     2881 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
     2941 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
     3001 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
     3061 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
     3121 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
     3181 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
     3241 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
     3301 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
     3361 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     3421 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
     3481 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     3541 gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc
     3601 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     3661 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     3721 gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga
     3781 ggaaaacctg tacttccaat ccaatgcaga ttctcttgaa tttattgcta gtaagcttgc
     3841 gatggataac gaggttgaaa aaaatattga gatctggaag gtcaagaagt tggtccaatc
     3901 tttagaaaaa gctagaggta atggtacttc tatgatttcc ttagttattc ctcctaaggg
     3961 tcaaattcca ctgtaccaaa aaatgttaac agatgaatat ggtactgcct cgaatattaa
     4021 atctagggtt aatcgtcttt ccgttttatc tgctatcact tccacccaac aaaagttgaa
     4081 gctatataat actttgccca agaacggttt agttttatat tgtggtgata tcatcactga
     4141 agatggtaaa gaaaaaaagg tcacttttga catcgaacct tacaaaccta tcaacacatc
     4201 cttatatttg tgtgataaca aatttcatac agaagttctt tcggaattgc ttcaagctga
     4261 cgacaagttc ggttttatag tcatggacgg tcaaggtact ttgtttggtt ctgtgtccgg
     4321 taatacgaga actgttttac ataaatttac tgtcgatctg ccaaaaaagc atggtagagg
     4381 tggtcaatct gcgcttcgtt ttgctcgttt aagagaagaa aaaagacata attatgtgag
     4441 aaaggtcgcc gaagttgctg ttcaaaattt tattactaat gacaaagtca atgttaaggg
     4501 tttaatttta gctggttctg ctgactttaa gaccgatttg gctaaatctg aattattcga
     4561 tccaagacta gcatgtaagg ttatttccat cgtggatgtt tcttatggtg gtgaaaacgg
     4621 tttcaaccag gctatcgaac tttctgccga agcgttggcc aatgtcaagt atgttcaaga
     4681 aaagaaatta ttggaggcat attttgacga aatttcccag gacactggta aattctgtta
     4741 tggtatagat gatactttaa aggcattgga tttaggtgca gtcgaaaaat taattgtttt
     4801 cgaaaatttg gaaactatca gatatacatt taaagatgcc gaggataatg aggttataaa
     4861 attcgctgaa ccagaagcca aggacaagtc gtttgctatt gacaaagcta ccggccaaga
     4921 aatggacgtt gtctccgaag aacctttaat tgaatggcta gcagctaact acaaaaactt
     4981 cggtgctacc ttggaattca tcacagacaa atcttcagaa ggtgcccaat ttgtcacagg
     5041 ttttggtggt attggtgcca tgctgcgtta caaagttaat tttgaacaac tagttgatga
     5101 atctgaggat gaatattatg acgaagatga aggatccgac tatgatttca tttaataaca
     5161 ttggaagtgg ataacggatc cgaattcgag cgccgtcgac aagcttgcgg ccgcactcga
     5221 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     5281 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     5341 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     5401 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5461 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5521 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5581 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5641 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5701 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5761 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5821 aacaaactag taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5881 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     5941 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     6001 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     6061 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     6121 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     6181 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     6241 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     6301 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     6361 caaccggcgc aggaacactg ccagcgcatc aacaatgttt tcacctgaat caggatattc
     6421 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     6481 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     6541 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     6601 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     6661 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     6721 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     6781 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6841 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6901 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6961 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7021 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7081 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7141 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7201 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7261 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7321 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7381 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7441 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7501 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     7561 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     7621 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     7681 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7741 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     7801 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagac
//
