LOCUS       sequence-173709-        8859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ectopic mutated human MRP RNA contstruct from the 
            human RMRP promoter, also contains CMV-puro-t2A-mCherry expression 
            cassette for selection/transfection efficiency.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8859)
  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 8859)
  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-715MRPprom-MRPcucuc
FEATURES             Location/Qualifiers
     source          1..8859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     409..429
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             519..572
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      579..588
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             585..1295
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(730..748)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     998..1017
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1315..1348)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1404..1992
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1457..1477)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1875..1886)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1995..2011)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1996..2012)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2521..2701
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2763..3351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2843..2862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3522..4313,4314..4382))
                     /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     4145..4164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4383..4487)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4562..4581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4753..5132
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5134..5332
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             5350..5530
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5577..5702
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6199..6432
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6617..6661
                     /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    6959..7076
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        8231..8534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8535..8738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             join(8772..8859,1..509)
                     /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(8772..8791)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
       61 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
      121 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
      181 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
      241 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
      301 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
      361 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
      421 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
      481 cctggtgcat gacccgcaag cccggtgccg gatcgggaga gggcagagga agtctgctaa
      541 catgcggtga cgtcgaggag aatcctggcc caccggtcgc caccatggtg agcaagggcg
      601 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
      661 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
      721 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
      781 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
      841 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
      901 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
      961 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     1021 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1081 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1141 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     1201 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1261 actccaccgg cggcatggac gagctgtaca agtaagaatt cgtcgaggga cctaataact
     1321 tcgtatagca tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac
     1381 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     1441 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     1501 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     1561 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     1621 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     1681 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     1741 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     1801 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     1861 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     1921 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     1981 ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc
     2041 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
     2101 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
     2161 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     2221 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     2281 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
     2341 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
     2401 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
     2461 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
     2521 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     2581 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     2641 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     2701 agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2761 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2821 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2881 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2941 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3001 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3061 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3121 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3181 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3241 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3301 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3361 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3421 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3481 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3541 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3601 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3661 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3721 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3781 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3841 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3901 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3961 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4021 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4081 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4141 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4201 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4261 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4321 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4381 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4441 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4501 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     4561 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     4621 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     4681 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     4741 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     4801 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     4861 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4921 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4981 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     5041 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     5101 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     5161 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     5221 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     5281 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
     5341 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     5401 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     5461 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     5521 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     5581 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     5641 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     5701 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     5761 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     5821 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     5881 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     5941 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     6001 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag
     6061 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     6121 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     6181 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     6241 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     6301 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     6361 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     6421 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     6481 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     6541 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     6601 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     6661 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     6721 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     6781 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     6841 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     6901 gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt
     6961 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     7021 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     7081 tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgtac ctgcaaacac
     7141 ttgtacagca caaaagccgc gacggccgcg gtgtacagcg cccccagagg cagcgacccg
     7201 gcccgggtcc gctcgcgttg atccttgagc ggtggtcgcc gccggggttc cgtggctcca
     7261 gagccggggt gggctgcgtt ccctgcggag ggaggataca ggcgagtcag ccgagctggg
     7321 gcctgggacc cgtgcgcagt cccagccctc ctccagcagc cctagctggt acctgggtgt
     7381 gcccggaggt cgggattggc gcggtctcct aggaccccgg acagcgcaga cacaagcagc
     7441 agccccacca cacccaagag cgaaactgcg cccgccattg ccggcccacg ggtggaggga
     7501 tcgggcgggc ggtgccgaag cggtccggca ttggccggcc gccccaacgc gcacgcgcac
     7561 gcgagcaggc cggccggctc cggggaggcc acgcccactc cccgtagggc ggggccagac
     7621 catatttgca taagatagtg tcattctagc tttcctgtat ttgttcattt cgtgtctatt
     7681 agctattctg ctagccacaa tgcctctgaa agcctatagt cttagaaagt tatgcccgaa
     7741 aacggttttt ttaatctcac gccaccaact ttctcaccct aatcataaaa cacaatttct
     7801 ttagggctat aaaatactac tctgtgaagc tgaggacgtg gttcgtgctg aaggcctgta
     7861 tcctaggcta cacactgagg actctgttcc tcccctttcc gcctagggga aagtccccgg
     7921 acctcgggca ctctctgcca cgtgcatacg cacgtagaca ttccccgctt cccactccaa
     7981 agtccgccaa gaagcgtatc ccgctgagcg gcgtggcgcg ggggcgtcat ccgtcagctc
     8041 cctctagtta cgcaggcagt gcgtgtccgc gcaccaacca cacggggctc attctcagcg
     8101 cggctgtttt tttctcgagt actaggatcc attaggcggc cgcgtggata accgtattac
     8161 cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     8221 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     8281 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     8341 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     8401 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     8461 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     8521 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     8581 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     8641 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     8701 gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
     8761 gctagcccac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     8821 cccgggccgt acgcaccctc gccgccgcgt tcgccgact
//
