LOCUS       sequence-173911-        7135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ectopic 3xFLAG-Rpp25 construct from CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7135)
  AUTHORS   Goldfarb KC, Cech TR
  TITLE     Targeted CRISPR disruption reveals a role for RNase MRP RNA in human
            preribosomal RNA processing.
  JOURNAL   Genes Dev. 2017 Jan 1;31(1):59-71. doi: 10.1101/gad.286963.116. Epub
            2017 Jan 23.
  PUBMED    28115465
REFERENCE   2  (bases 1 to 7135)
  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.286963.116"; journalName: "Genes Dev"; date: 
            "2017-01-1- 1"; volume: "31"; issue: "1"; pages: "59-71"
COMMENT     Sequence Label: p_3xFLAG-Rpp25
FEATURES             Location/Qualifiers
     source          1..7135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        33..336
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        337..540
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             577..642
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(1268..1301)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1357..1945
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1410..1430)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1828..1839)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1948..1964)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1949..1965)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2474..2654
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2716..3304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2796..2815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3475..4266,4267..4335))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4098..4117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4336..4440)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4515..4534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4706..5085
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5087..5285
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             5303..5483
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5530..5655
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6152..6385
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6570..6614
                     /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    6912..7029
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
       61 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      121 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      181 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      241 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      301 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      361 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      421 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      481 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      541 ggtttagtga accgtcagat ccgctagccc accatggact acaaagacca tgacggtgat
      601 tataaagatc atgatatcga ttacaaggat gacgatgaca agatggagaa cttccgtaag
      661 gtgcgctccg aagaggcgcc agcggggtgc ggggccgagg gaggcggccc gggctccggc
      721 cccttcgcag acctggcgcc gggcgcggtg cacatgcggg tcaaggaagg cagcaagatc
      781 cggaacctga tggccttcgc caccgccagc atggcgcagc cagccacgcg cgccatcgtc
      841 ttcagcggct gcggccgggc caccaccaaa accgtcacgt gcgccgagat cctcaagcgc
      901 cgcctggcgg gcctgcacca ggtcacgcgg ctgcgctacc ggagcgtacg cgaggtgtgg
      961 cagagcctcc cgcctgggcc cacgcagggt cagacgcctg gcgagccggc cgctagtctc
     1021 agcgtactta agaacgtgcc cggcctcgcc atcctacttt ccaaggacgc gctggatccg
     1081 cgacagcccg gctaccagcc cccgaatccc catcctggtc cctcgtcccc gccagccgcg
     1141 ccagcgtcca agaggagcct aggggaaccc gcagctggag aaggctccgc gaagcgatcg
     1201 caacccgagc caggggttgc ggacgaggat cagacggcct aactcgagca attcgtcgag
     1261 ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta agggttccgg
     1321 ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     1381 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     1441 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     1501 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     1561 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     1621 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     1681 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     1741 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     1801 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     1861 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     1921 atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag acctagaaaa
     1981 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
     2041 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
     2101 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     2161 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg
     2221 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt
     2281 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg
     2341 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
     2401 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca
     2461 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     2521 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     2581 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     2641 gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2701 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2761 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2821 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2881 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2941 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3001 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3061 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3121 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3181 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3241 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3301 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3361 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3421 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3481 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3541 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3601 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3661 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3721 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3781 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3841 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3901 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3961 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4021 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4081 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4141 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4201 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4261 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4321 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4381 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4441 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     4501 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     4561 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     4621 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     4681 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     4741 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     4801 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     4861 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     4921 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     4981 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     5041 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     5101 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     5161 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     5221 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     5281 agcagcgcgt tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct
     5341 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     5401 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     5461 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
     5521 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
     5581 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
     5641 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
     5701 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
     5761 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
     5821 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
     5881 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
     5941 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccggc
     6001 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     6061 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     6121 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     6181 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     6241 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     6301 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     6361 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     6421 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     6481 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     6541 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     6601 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     6661 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     6721 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     6781 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat
     6841 ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca aatggcagta
     6901 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     6961 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     7021 caaaattttc gggtttatta cagggacagc agagatccag tttggttagt accgggccgc
     7081 gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta cgggg
//
