LOCUS       sequence-290546-        6431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of NcPAN2_1-321-P99R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6431)
  AUTHORS   Jonas S, Christie M, Peter D, Bhandari D, Loh B, Huntzinger E, 
            Weichenrieder O, Izaurralde E
  TITLE     An asymmetric PAN3 dimer recruits a single PAN2 exonuclease to 
            mediate mRNA deadenylation and decay.
  JOURNAL   Nat Struct Mol Biol. 2014 Jul;21(7):599-608. doi: 10.1038/nsmb.2837.
            Epub 2014 Jun 1.
  PUBMED    24880343
REFERENCE   2  (bases 1 to 6431)
  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.2837";
            journalName: "Nat Struct Mol Biol"; date: "2014-07"; volume: "21"; 
            issue: "7"; pages: "599-608"
COMMENT     Sequence Label: pnYC-NpM-NcPAN2_1-321-P99R_Y
FEATURES             Location/Qualifiers
     source          1..6431
                     /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             2489..3589
                     /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             3605..3628
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4663..4681)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4677..4724
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4899..4904,4905..4921,4922..4927))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      5039..5584
                     /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     5485..5504
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5701..5718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6110..6212
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(6213..6431,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6279..6298)
                     /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 ggaaactgaa gaaggtaaac tggtaatctg
     2521 gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga
     2581 taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt
     2641 tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta
     2701 cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta
     2761 tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt
     2821 tgaagcgtta tcgctgattt ataacaaaga cctgctgccg aacccgccaa aaacctggga
     2881 agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgccc tgatgttcaa
     2941 cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa
     3001 gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc
     3061 gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta
     3121 ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg
     3181 ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt
     3241 caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag
     3301 tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct
     3361 ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga
     3421 gttggcgaaa gatccacgta ttgccgccac gatggaaaac gcccagaaag gtgaaatcat
     3481 gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc
     3541 cgccagcggt cgtcagactg tcgatgaagc ccttaaagac gcgcagactg gtaccggcag
     3601 cggcctggaa gttctgttcc aggggcccca tatgatggat tctcgggatt ggacccagct
     3661 gggctgtgtt gcctacccgt caccaatcca tccagactac catgccggac ccgcgtcgac
     3721 aatagccttc gacaaccaag atgagctgct atggatcggc acccaaaagg gctttgccgg
     3781 ctctttcatc ggccgcgagc tcaagcgatt cactgccttc cgaatacacc ccgaaaccga
     3841 cggtcccctc cgccaattcc tcttcgtcga caagggcgtc atcttcctcg gaagccggag
     3901 cgtctacatg gcagcacgta gtggcgtgcg tatctggagt atccgccatg agtccatgca
     3961 ggatctgcgt gccatgtcct tcaccagcaa ggggacctcc gagatcctcg tggccggctg
     4021 gcagaacaag atgttggtga ttgacgtcaa caagggtgag gtcgtcaagg agctcccgac
     4081 acaagatcag tactccttcc tcaagatgag ccggtacatt tgcgcggcaa cgaacaaggg
     4141 caccgtcaac atactggacc ctatcacttt tactatcaag aaacaatggc aggcccacgg
     4201 ggctttcatc aacgatctgg acacgtccaa cgactttatt gtcacctgcg gtggctcaca
     4261 caggcagaca cacaacaccc cggccatact cgacccttac gtcaaggtgt ttgacctgaa
     4321 gaacatgagt gctatgaacc cagttccctt tgcgccgctg gccgctcacg tgaggatgca
     4381 cccgaggatg ttgacaacgg ccattgtcgt caaccaggcg ggccagatcc acgtgacgga
     4441 tctgttgaat ccaagcaaca gccaggtctg ctacacgcag cctcagggcg tggtgctaca
     4501 tttcgatgtg tcgagaaccg gagaaggaaa agcgcttgcg gataacaagc acaacaccta
     4561 cgtctgggga tcgcccaaca agatccagtt taccgagtga gctagcccta ggagatccgg
     4621 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     4681 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     4741 tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     4801 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     4861 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     4921 ctgtcaaaca tgagaattac aacttatatc gtatggggct gacttcaggt gctacatttg
     4981 aagagataaa ttgcactgaa atcaagaaat attttatctg attaataaga tgatcttctt
     5041 gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg
     5101 gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag
     5161 gagcgcagtc accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga
     5221 ctaactcctc taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc
     5281 gggttggact caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt
     5341 tcgtgcatac agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga
     5401 atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg
     5461 agagcgcacg agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5521 tcgccaccac tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg
     5581 gaaaaacggc tttgccgcgg ccctctcact tccctgttaa gtatcttcct ggcatcttcc
     5641 aggaaatctc cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt
     5701 agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg ctgacgcacc
     5761 ggtgcagcct tttttctcct gccacatgaa gcacttcact gacaccctca tcagtgccaa
     5821 catagtaagc cagtatacac tcccctagcg ctgatgtccg gcggtgcttt tgccgttacg
     5881 caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag ccactggagc
     5941 acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt
     6001 gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc
     6061 ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg
     6121 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt
     6181 tatcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     6241 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     6301 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     6361 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     6421 atccggaatt c
//
