LOCUS       sequence-345392-        7825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes His-MBP-TEV-WT eRF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7825)
  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 7825)
  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 eRF1 (WT)
FEATURES             Location/Qualifiers
     source          1..7825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..66
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(60..251)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1060..2142)
                     /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     2104..2123
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2143..2220)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2426..2445
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2529..2548
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2529..2547
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2548..2572
                     /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             2587..2609
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2628..2645
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2655..3755
                     /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     3729..3752
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3810..3830
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5217..5234
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5287..5305)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5301..5348
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5385..5840
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5472..5491)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5682..5703
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5933..6748)
                     /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(5933..6748)
                     /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     6656..6675
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6740..6759
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6870..7458
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7359..7378
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7612..7629
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7644..7783
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7709..7728)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
       61 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
      121 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
      181 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
      241 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
      301 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
      361 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
      421 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
      481 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
      541 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
      601 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
      661 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
      721 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
      781 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
      841 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
      901 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
      961 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     1021 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1081 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1141 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     1201 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     1261 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     1321 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     1381 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgataccctc
     1441 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     1501 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     1561 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     1621 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     1681 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     1741 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     1801 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     1861 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     1921 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     1981 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     2041 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     2101 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     2161 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     2221 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     2281 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     2341 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     2401 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     2461 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     2521 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     2581 aataattttg tttaacttta agaaggagat ataccatggg ttcttctcac catcaccatc
     2641 accatggttc ttctatgaaa atcgaagaag gtaaactggt aatctggatt aacggcgata
     2701 aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag
     2761 tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg
     2821 atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc
     2881 tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg
     2941 atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc
     3001 tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc
     3061 tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt
     3121 acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca
     3181 agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc
     3241 tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag
     3301 ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca
     3361 tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat
     3421 ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc
     3481 tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata
     3541 aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc
     3601 cacgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc
     3661 agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc
     3721 agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac aacaacaaca
     3781 ataacaataa caacaacctc gggatcgagg aaaacctgta cttccaatcc aatgcaatgg
     3841 ataacgaggt tgaaaaaaat attgagatct ggaaggtcaa gaagttggtc caatctttag
     3901 aaaaagctag aggtaatggt acttctatga tttccttagt tattcctcct aagggtcaaa
     3961 ttccactgta ccaaaaaatg ttaacagatg aatatggtac tgcctcgaat attaaatcta
     4021 gggttaatcg tctttccgtt ttatctgcta tcacttccac ccaacaaaag ttgaagctat
     4081 ataatacttt gcccaagaac ggtttagttt tatattgtgg tgatatcatc actgaagatg
     4141 gtaaagaaaa aaaggtcact tttgacatcg aaccttacaa acctatcaac acatccttat
     4201 atttgtgtga taacaaattt catacagaag ttctttcgga attgcttcaa gctgacgaca
     4261 agttcggttt tatagtcatg gacggtcaag gtactttgtt tggttctgtg tccggtaata
     4321 cgagaactgt tttacataaa tttactgtcg atctgccaaa aaagcatggt agaggtggtc
     4381 aatctgcgct tcgttttgct cgtttaagag aagaaaaaag acataattat gtgagaaagg
     4441 tcgccgaagt tgctgttcaa aattttatta ctaatgacaa agtcaatgtt aagggtttaa
     4501 ttttagctgg ttctgctgac tttaagaccg atttggctaa atctgaatta ttcgatccaa
     4561 gactagcatg taaggttatt tccatcgtgg atgtttctta tggtggtgaa aacggtttca
     4621 accaggctat cgaactttct gccgaagcgt tggccaatgt caagtatgtt caagaaaaga
     4681 aattattgga ggcatatttt gacgaaattt cccaggacac tggtaaattc tgttatggta
     4741 tagatgatac tttaaaggca ttggatttag gtgcagtcga aaaattaatt gttttcgaaa
     4801 atttggaaac tatcagatat acatttaaag atgccgagga taatgaggtt ataaaattcg
     4861 ctgaaccaga agccaaggac aagtcgtttg ctattgacaa agctaccggc caagaaatgg
     4921 acgttgtctc cgaagaacct ttaattgaat ggctagcagc taactacaaa aacttcggtg
     4981 ctaccttgga attcatcaca gacaaatctt cagaaggtgc ccaatttgtc acaggttttg
     5041 gtggtattgg tgccatgctg cgttacaaag ttaattttga acaactagtt gatgaatctg
     5101 aggatgaata ttatgacgaa gatgaaggat ccgactatga tttcatttaa taacattgga
     5161 agtggataac ggatccgaat tcgagcgccg tcgacaagct tgcggccgca ctcgagcacc
     5221 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     5281 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     5341 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     5401 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5461 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5521 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5581 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5641 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5701 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5761 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5821 actagtaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5881 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     5941 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     6001 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     6061 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     6121 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     6181 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     6241 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     6301 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     6361 ggcgcaggaa cactgccagc gcatcaacaa tgttttcacc tgaatcagga tattcttcta
     6421 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     6481 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     6541 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     6601 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     6661 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     6721 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     6781 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6841 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6901 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6961 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     7021 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7081 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7141 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7201 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7261 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7321 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7381 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7441 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7501 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7561 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     7621 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     7681 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7741 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     7801 acccgctgac gcgccctgac gggct
//
