LOCUS       sequence-356612-        6160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of CeSmg7_1-395-G80E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6160)
  AUTHORS   Jonas S, Weichenrieder O, Izaurralde E
  TITLE     An unusual arrangement of two 14-3-3-like domains in the SMG5-SMG7 
            heterodimer is required for efficient nonsense-mediated mRNA decay.
  JOURNAL   Genes Dev. 2013 Jan 15;27(2):211-25. doi: 10.1101/gad.206672.112.
  PUBMED    23348841
REFERENCE   2  (bases 1 to 6160)
  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: 
            "10.1101/gad.206672"; journalName: "Genes Dev"; date: "2013-01-15- 
            15"; volume: "27"; issue: "2"; pages: "211-25"
COMMENT     Sequence Label: pRSFDuetMP-CeSmg7_1-395-G80E_S
FEATURES             Location/Qualifiers
     source          1..6160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             110..1210
                     /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
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGIDAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1184..1207
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1259..1282
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        2545..2563
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2564..2588
                     /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)."
     CDS             2697..2741
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2779..2797)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2793..2840
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3070..3885)
                     /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(3070..3885)
                     /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     3793..3812
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3886..3977)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3993..4742)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(4003..4020)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4243..4262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4938..6020)
                     /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     5982..6001
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6021..6098)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6144..6160,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcatcac aacactagta tgaaaatcga
      121 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
      181 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
      241 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
      301 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
      361 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
      421 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc
      481 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
      541 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
      601 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
      661 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
      721 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
      781 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
      841 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
      901 cgcaggtatt gacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
      961 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     1021 gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca caatggaaaa
     1081 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     1141 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     1201 cgcgcagact aattcgagct cgactagtgg atctggtggt ggtggcggca gcgcgggtct
     1261 ggaagttctg ttccaggggc cccaggatcc gatgtcggac gaatgggaac agcttactgt
     1321 agagttgaga aaaattccac gtggcaccga agctgcacca cagtatttga gacatttaat
     1381 gaaaatgttt gtcgctgact tcgaaactgc tgtatccaaa agattcgatg taaaattctg
     1441 gaataaactg aaatcaatga tggatgaaat aacaaaagca atggaaaatg atcgacttgt
     1501 taatcataat gttcaaaatc ttgcaattga atttttgact gatttatcac ttcttgttca
     1561 ttatcattat gaaattccaa attatggaaa cgatatctca aaacaattaa catggacacc
     1621 agatgtattt ctaaacagaa aacctatcaa gtcgaaaaag aatagtcgag tgttcatggc
     1681 ttatgttctg ttaagaatgg gagatttaat gagatataag gagaactacc caaaagctca
     1741 ggaatattac gagcaatcat gccgcattaa tccagctgat ggagctgtct ggaatcagct
     1801 aggattgata tcttcacttg gtgcaaaaaa cctggaaagt gtctattttc atacacgagc
     1861 tcttcatgcc actatggaat tcccaactgc ttctggtggc ttaacgaata ttttcaaaaa
     1921 ttttgcaaat cgagatattt caagaccaat gcctattaaa gatctatatt tatcctgcct
     1981 aggaagaatt catttccttc tagaaatcga agattcttct gtgcacctac aaaaaattgg
     2041 agaagaagct gcaacttcaa aagaaatgat tgttcctcta atgtctgtat ataaacatct
     2101 cgaggacgga actgaacttg aacaacgagc cgtagagtat gtcaaaacta tttggtgtac
     2161 ggcctaccga agtctcctga aaactttgga tgattataag gaggaatcaa aaaaactggc
     2221 agatgttcca catcttcttc atattctggc acttttactc tgtgcaccta aactccttcg
     2281 cggtattgaa gaccaaactg aagatgaagt aacatcaatt tgcgaatggc ttctatgcgc
     2341 aaattgtgat gagaaaatca aggattctga tgcttttgga tattttcatt gtcttcaaag
     2401 aattcaatat ccactcacta gaactcaatt ggctcagaaa ttagttgaaa tagaggatga
     2461 agatgaatct aaagattaag cggccgcata atgcttaagt cgaacagaaa gtaatcgtat
     2521 tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata
     2581 acaattcccc atcttagtat attagttaag tataagaagg agatatacat atggcagatc
     2641 tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag
     2701 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     2761 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2821 gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc
     2881 ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct
     2941 gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat
     3001 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3061 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3121 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3181 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3241 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     3301 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     3361 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     3421 tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac aattacaaac
     3481 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     3541 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     3601 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     3661 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     3721 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3781 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3841 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca
     3901 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3961 ttagaaaaat aaacaaatag gcatgcagcg ctcttccgct tcctcgctca ctgactcgct
     4021 acgctcggtc gttcgactgc ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt
     4081 atccacagaa tcaggggata aagccggaaa gaacatgtga gcaaaaagca aagcaccgga
     4141 agaagccaac gccgcaggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4201 atcgacgctc aagccagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4261 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4321 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt tggtatctca
     4381 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4441 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4501 cgccactggc agcagccatt ggtaactgat ttagaggact ttgtcttgaa gttatgcacc
     4561 tgttaaggct aaactgaaag aacagatttt ggtgagtgcg gtcctccaac ccacttacct
     4621 tggttcaaag agttggtagc tcagcgaacc ttgagaaaac caccgttggt agcggtggtt
     4681 tttctttatt tatgagatga tgaatcaatc ggtctatcaa gtcaacgaac agctattccg
     4741 ttactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     4801 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
     4861 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4921 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4981 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5041 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5101 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5161 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     5221 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5281 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5341 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5401 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5461 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5521 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5581 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5641 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5701 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5761 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5821 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5881 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5941 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6001 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6061 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6121 atgcgactcc tgcattagga aattaatacg actcactata
//
