LOCUS       sequence-239354-        8571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of PNRC1-W300A mutant C-MYC-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8571)
  AUTHORS   Gaviraghi M, Vivori C, Pareja Sanchez Y, Invernizzi F, Cattaneo A, 
            Santoliquido BM, Frenquelli M, Segalla S, Bachi A, Doglioni C, 
            Pelechano V, Cittaro D, Tonon G
  TITLE     Tumor suppressor PNRC1 blocks rRNA maturation by recruiting the 
            decapping complex to the nucleolus.
  JOURNAL   EMBO J. 2018 Dec 3;37(23). pii: embj.201899179. doi: 
            10.15252/embj.201899179. Epub 2018 Oct 29.
  PUBMED    30373810
REFERENCE   2  (bases 1 to 8571)
  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"; journalName: "EMBO J."; 
            date: "2018-12-3"; volume: "37(23). pii: embj.201899179. doi"; 
            pages: " 10.15252/embj.201899179. Epub 2018 Oct 29"
COMMENT     Sequence Label: pLenti C-MYC-DDK Puro PNRC1-W300A
FEATURES             Location/Qualifiers
     source          1..8571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..146
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        127..145
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1232..1261
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1280..1303
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1337..1910
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1360..1912
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1504..1521)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1731..1750
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1912..2511
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1912..1931)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2408..2428
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2541..2574)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2630..3218
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2683..2703)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3101..3112)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3221..3237)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3222..3238)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3747..3927
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3989..4577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4069..4088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4654..5313)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5228..5247
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5314..5418)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5493..5512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5683..6062
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6064..6262
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6280..6460
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6507..6632
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7129..7362
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7547..7591
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7889..8006
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        8089..8468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8469..8571,1..101)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg
       61 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
      121 attttgtaat acgactcact atagggcggc cgggaattcg tcgactggat ccggtaccga
      181 ggagatctgc cgccgcgatc gccggcgcgc cagatctcaa gcttaactag ctagcggacc
      241 gacgcgtatg actgtcgtct ccgtcccgca gcgggagccg ctcgtcctgg gtggccgcct
      301 tgcgccgctt ggcttttcct cccgaggtta ctttggggcc ctcccgatgg tgaccacggc
      361 tccgcctcct ttaccccgga tcccggaccc ccgggcactg cccccgaccc tcttccttcc
      421 tcatttccta gggggagatg gcccgtgtct gaccccccag cctcgcgctc cagcagctct
      481 gcccaaccgc agcctcgccg tggcgggagg cactcctcgg gcagcgccga agaagcggcg
      541 aaagaagaag gtgcgggcca gccccgcagg gcagctgccc agccgcttcc accagtacca
      601 gcagcaccgg ccgagtctgg agggcggccg gagccccgcg accggcccga gcggagcgca
      661 ggaggtcccg ggcccggccg ccgccttggc cccgagtcct gcagccgcag ccggcacgga
      721 gggagccagc cccgaccttg ccccgctgcg gcccgcggct cccggccaaa cccccctcag
      781 gaaagaggtt ttaaaatcaa agatgggaaa atcggagaaa attgcccttc cccatggcca
      841 gcttgttcat ggtatacact tgtatgagca accaaagata aacagacaga aaagcaaata
      901 taacttgcca ctaaccaaga tcacctctgc aaaaagaaat gaaaacaact tttggcagga
      961 ttctgtttca tctgacagaa ttcagaagca ggaaaaaaag ccttttaaaa ataccgagaa
     1021 cattaaaaat tcgcatttga agaaatcagc atttctaact gaagtgagcc aaaaggaaaa
     1081 ttatgctggg gcaaagttta gtgatccacc ttctcctagt gttcttccaa agcctcctag
     1141 tcacgcgatg ggaagcactg ttgaaaattc caaccaaaac agggagctga tggcagtaca
     1201 cttaaaaacg ctcctcaaag ttcaaactct cgagcagaaa ctcatctcag aagaggatct
     1261 ggcagcaaat gatatcctgg attacaagga tgacgacgat aaggtttaaa cctaggcgta
     1321 gcggccgcaa attccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct
     1381 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     1441 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     1501 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     1561 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     1621 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     1681 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     1741 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     1801 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     1861 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatgaccgag
     1921 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggcagt acgcaccctc
     1981 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccaga ccgccacatc
     2041 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     2101 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     2161 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     2221 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     2281 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     2341 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     2401 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     2461 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg atgtacaagt
     2521 aggattcgtc gagggaccta ataacttcgt atagcataca ttatacgaag ttatacatgt
     2581 ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct
     2641 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2701 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2761 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2821 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2881 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2941 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3001 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     3061 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3121 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3181 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc
     3241 gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt
     3301 gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac
     3361 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     3421 ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct
     3481 accacacaca aggctacttc cctgattggc agaactacac accagggcca gggatcagat
     3541 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag
     3601 aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg
     3661 acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg
     3721 cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg
     3781 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     3841 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     3901 tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga
     3961 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4021 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4081 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4141 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4201 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4261 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4321 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4381 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4441 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4501 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4561 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4621 acgaaaactc acgttaaggg attttggtca tgattacgcc ccgccctgcc actcatcgca
     4681 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
     4741 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt
     4801 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
     4861 cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc
     4921 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
     4981 gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
     5041 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaactc
     5101 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
     5161 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt
     5221 acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc
     5281 aacggtggta tatccagtga tttttttctc catactcttc ctttttcaat attattgaag
     5341 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5401 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     5461 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5521 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     5581 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     5641 cgttttgcgc tgcttcgcga tgtacgggcc agatatcgcg ttgacattga ttattgacta
     5701 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     5761 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     5821 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     5881 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     5941 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     6001 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     6061 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     6121 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     6181 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     6241 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
     6301 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     6361 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     6421 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     6481 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     6541 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     6601 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     6661 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     6721 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     6781 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     6841 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     6901 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     6961 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     7021 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     7081 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     7141 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     7201 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     7261 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     7321 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     7381 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     7441 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     7501 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     7561 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     7621 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     7681 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     7741 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     7801 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     7861 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     7921 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     7981 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     8041 ggttagtacc gggcccgctc tagacatgtc caatatgacc gccatgttga cattgattat
     8101 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     8161 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     8221 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     8281 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     8341 tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     8401 agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     8461 ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac
     8521 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg c
//
