LOCUS       sequence-289672-        5867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsNot2_429-540.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5867)
  AUTHORS   Braun JE, Truffault V, Boland A, Huntzinger E, Chang CT, Haas G, 
            Weichenrieder O, Coles M, Izaurralde E
  TITLE     A direct interaction between DCP1 and XRN1 couples mRNA decapping to
            5' exonucleolytic degradation.
  JOURNAL   Nat Struct Mol Biol. 2012 Dec;19(12):1324-31. doi: 
            10.1038/nsmb.2413. Epub 2012 Nov 11.
  PUBMED    23142987
REFERENCE   2  (bases 1 to 5867)
  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: "10.1038/nsmb.2413";
            journalName: "Nat Struct Mol Biol"; date: "2012-12"; volume: "19"; 
            issue: "12"; pages: "1324-31"
COMMENT     Sequence Label: pnYC-NpHM-HsNot2_429-540_P
FEATURES             Location/Qualifiers
     source          1..5867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2294..2313
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2402..2421
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2402..2420
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2421..2445
                     /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             2460..2482
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2501..2518
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2534..3634
                     /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="METEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     CDS             3650..3673
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4099..4117)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4113..4160
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4335..4340,4341..4357,4358..4363))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      4475..5020
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     4921..4940
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5137..5154
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5546..5648
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(5649..5867,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5715..5734)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 ggtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
       61 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
      121 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
      181 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
      241 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
      301 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
      361 gccgtctgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
      421 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
      481 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga
      541 cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac
      601 cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt
      661 tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca
      721 gagcctgata aaaacggtta gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      781 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgcttg tttcggcgtg
      841 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ctgccggcac ctgtcctacg
      901 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      961 cggaaggagc taccggacag cggtgcggac tgttgtaact cagaataaga aatgaggccg
     1021 ctcatggcgt tgactctcag tcatagtatc gtggtatcac cggttggttc cactctctgt
     1081 tgcgggcaac ttcagcagca cgtaggggac ttccgcgttt ccagacttta cgaaacacgg
     1141 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     1201 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     1261 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     1321 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
     1381 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
     1441 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
     1501 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     1561 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccagtga
     1621 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     1681 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     1741 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     1801 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     1861 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     1921 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     1981 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     2041 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac
     2101 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     2161 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     2221 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     2281 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     2341 tgatgccggc cacgatgcgt ccggcgtaga ggatcagact aagatctcga tcccgcgaaa
     2401 ttaatacgac tcactatagg ggaattgtga gcggataaca attccccact agtaataatt
     2461 ttgtttaact ttaagaagga gatataccat gagcggcagc catcatcatc atcatcacag
     2521 cggcagcggc tccatggaaa ctgaagaagg taaactggta atctggatta acggcgataa
     2581 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     2641 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     2701 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     2761 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     2821 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     2881 gatttataac aaagacctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     2941 ggataaagaa ctgaaagcga aaggtaagag cgccctgatg ttcaacctgc aagaaccgta
     3001 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     3061 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     3121 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     3181 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     3241 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     3301 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     3361 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     3421 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     3481 acgtattgcc gccacgatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     3541 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     3601 gactgtcgat gaagccctta aagacgcgca gactggtacc ggcagcggcc tggaagttct
     3661 gttccagggg ccccatatgg ataagctggc tgcaataaaa cttggccgat atggtgaaga
     3721 ccttctcttc tatctctatt acatgaatgg aggagacgta ttacaacttt tagctgcagt
     3781 ggagcttttt aaccgtgatt ggagatacca caaagaagaa cgagtatgga ttaccagggc
     3841 accaggcatg gagccaacaa tgaaaaccaa tacctatgag aggggaacat attacttctt
     3901 tgactgtctt aactggagga aagtagctaa ggagttccat ctggaatatg acaaattaga
     3961 agaacggcct cacctgccat ccaccttcaa ctacaaccct gctcagcaag ccttctaagg
     4021 atcctaatag tctagagcta gccctaggag atccggctgc taacaaagcc cgaaaggaag
     4081 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     4141 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     4201 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     4261 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     4321 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattacaact
     4381 tatatcgtat ggggctgact tcaggtgcta catttgaaga gataaattgc actgaaatca
     4441 agaaatattt tatctgatta ataagatgat cttcttgaga tcgttttggt ctgcgcgtaa
     4501 tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag gttctctgag
     4561 ctaccaactc tttgaaccga ggtaactggc ttggaggagc gcagtcacca aaacttgtcc
     4621 tttcagttta gccttaaccg gcgcatgact tcaagactaa ctcctctaaa tcaattacca
     4681 gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt tggactcaag acgatagtta
     4741 ccggataagg cgcagcggtc ggactgaacg gggggttcgt gcatacagtc cagcttggag
     4801 cgaactgcct acccggaact gagtgtcagg cgtggaatga gacaaacgcg gccataacag
     4861 cggaatgaca ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg agccgccagg
     4921 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc caccactgat ttgagcgtca
     4981 gatttcgtga tgcttgtcag gggggcggag cctatggaaa aacggctttg ccgcggccct
     5041 ctcacttccc tgttaagtat cttcctggca tcttccagga aatctccgcc ccgttcgtaa
     5101 gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg agtcagtgag cgaggaagcg
     5161 gaatatatcc tgtatcacat attctgctga cgcaccggtg cagccttttt tctcctgcca
     5221 catgaagcac ttcactgaca ccctcatcag tgccaacata gtaagccagt atacactccc
     5281 ctagcgctga tgtccggcgg tgcttttgcc gttacgcacc accccgtcag tagctgaaca
     5341 ggagggacag ctgatagaaa cagaagccac tggagcacct caaaaacacc atcatacact
     5401 aaatcagtaa gttggcagca tcacccgacg cactttgcgc cgaataaata cctgtgacgg
     5461 aagatcactt cgcagaataa ataaatcctg gtgtccctgt tgataccggg aagccctggg
     5521 ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata
     5581 atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg
     5641 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     5701 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     5761 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     5821 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattc
//
