LOCUS       sequence-491702-        5920 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag human RPA194 with PAmCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5920)
  AUTHORS   Ide S, Imai R, Ochi H, Maeshima K
  TITLE     Transcriptional suppression of ribosomal DNA with phase separation.
  JOURNAL   Sci Adv. 2020 Oct 14;6(42). pii: 6/42/eabb5953. doi: 
            10.1126/sciadv.abb5953. Print 2020 Oct.
  PUBMED    33055158
REFERENCE   2  (bases 1 to 5920)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-10-14"; volume: "6(42). pii: 6/42/eabb5953. doi"; pages:
            " 10.1126/sciadv.abb5953. Print 2020 Oct"
COMMENT     Sequence Label: pGEM-PAmCherry-RPA194
FEATURES             Location/Qualifiers
     source          1..5920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     308..328
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             418..474
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             475..1182
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein"
                     /label=PAmCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(620..638)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     888..907
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(2040..2058)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2041..2058)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2076..2092)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2076..2092)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2089..2111)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2100..2116
                     /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)."
     promoter        complement(join(2124..2130,2131..2148,2149..2154))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2169..2190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2307..2324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2478..3066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2558..2577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3237..4028,4029..4097))
                     /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     3860..3879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4098..4202)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4280..4735)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4417..4438)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4629..4648
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4861..4883
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4875..4892
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4876..4892
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4899..4918
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4899..4917
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4948..4957
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4952..4961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5732..5920,1..408)
                     /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(5732..5751)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
       61 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
      121 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
      181 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
      241 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
      301 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
      361 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atccggagcc
      421 acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg tcccatggtg
      481 agcaagggcg aggaggataa catggccatc attaaggagt tcatgcgctt caaggtgcac
      541 atggaggggt ccgtgaacgg ccacgtgttc gagatcgagg gcgagggcga gggccgcccc
      601 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcacc
      661 tgggacatcc tgtcccctca attcatgtac ggctccaatg cctacgtgaa gcaccccgcc
      721 gacatccccg actactttaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
      781 aaattcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggtgag
      841 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
      901 aagaagacca tgggctggga ggccctctcc gagcggatgt accccgagga cggcgccctg
      961 aagggcgagg tcaagccgag agtgaagctg aaggacggcg gccactacga cgctgaggtc
     1021 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaaccgc
     1081 aagttggaca tcacctcaca caacgaggac tacaccatcg tggaacagta cgaacgtgcc
     1141 gagggccgcc actccaccgg cggcatggac gagctgtaca agggtggagg tggatctggt
     1201 ggaggtggat ctggtggcgg cggttcaatg ttgatctcca agaacatgcc ctggcggcgg
     1261 ctgcagggca tttccttcgg gatgtattcg gctgaagagc tcaagtaagg agttggtcgg
     1321 gtagcgtgcc cagcctggat cttgaggtcg gcgccctaaa ctacgattcc cagaagcgct
     1381 tttgcccagt ctatcctctt gagtccaggc ttgtctggcg cttgtcctgc tgggagccgt
     1441 agttctggga acgttcctgg gccggcgtgg actgcctgga taagaggccg gtggtgctgg
     1501 gggaaagagt ttgcgctgag gtaggggaat ataagggtgg ggagggtcct ggtctgcagc
     1561 gttatcatct gccagtctcc tcgttaacta acaagcattt gtggaattaa ccatagtgta
     1621 cctgacatgg ttaattaggc acgtagtaag tttcctcttg tttgtaaaat gattgtcctt
     1681 caagtgtcag ttcaggcgtt aacttctctt tgctaggacc ctgctatatt ctcggtctgg
     1741 ttgttaaatc aagggaactc tctttagtct tcattttgat caaggtagca tttgacattc
     1801 tccaatgcac tctcttgaaa ctttcctttc ttccagaacc ttatttgtct tcaagttctc
     1861 ctatttttat gacaaatact tctacctccc tctcttcctc tatcgatcta taacctcaca
     1921 aacatttatt gagcatccag ttttttccag gcaccaagct aggcgttaat gacacattgg
     1981 tgagcaaggg tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatt
     2041 ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2101 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2161 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2221 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2281 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2341 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2401 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2461 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2521 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2581 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2641 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2701 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2761 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2821 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2881 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2941 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3001 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3061 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3121 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3181 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3241 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3301 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3361 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3421 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3481 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3541 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3601 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3661 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3721 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3781 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3841 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3901 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3961 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4021 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4081 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4141 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4201 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     4261 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     4321 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4381 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     4441 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     4501 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     4561 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     4621 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4681 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt
     4741 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     4801 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     4861 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg
     4921 aattgggccc gacgtcgcat gctcccggcc gccatggcgg ccgcgggaat tcaataaatg
     4981 cgttgcgaat cctatttcaa agggccacct ccgcgtacct ggtcttaagg gcacgcgacc
     5041 gttaagggcg cgcatgagca aaccatttca tcgtgcctac ttccacagcc agctgggaaa
     5101 cataggacta aggtgactcc gcggtcttct tcgcggatac cctaagcgtc tcttacctgc
     5161 agctgcgtgc ctgttcacac aaacccgccc gccgacccgt cagcggctgg ccaagcctgc
     5221 tgcggaggcc cagccagcga acagcagata caaccgccat ctttgatttc tctgcagagc
     5281 atgttgggaa agcgtgcctc gaccttcagg aaggtggggc gcagtgggcg gggagcaggg
     5341 aacagctccg ctattggctg aagcggaaga agacgaaagc aatcataaaa tgggaggttg
     5401 caagctcatg gtttgaaaga cttcgtcacg gaagctaaaa gctctataca cccgatttgc
     5461 ctcggaggaa ttttcctaaa tgattatttt gatgtcttat atatctttga ttgttttcaa
     5521 aacaaaagag cgagcagaga gtcgtacaac tatttgttcc ccccccatgt agaagtgatc
     5581 tcatccacgt aaatgtcgtt cctgcgaccg cttccgcgcg caagcgcacg ttgaatcgcg
     5641 tggtgactcc gggcttgagg ttgaattaag aatagtcagg tggtgagtgg aacgtctctt
     5701 ggggtgtcgg aattcaaaac ggacctggag gatgaccgag tacaagccca cggtgcgcct
     5761 cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta
     5821 ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca
     5881 agaactcttc ctcacgcgcg tcgggctcga catcggcaag
//
