LOCUS       sequence-348475-        3475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid expressing a pegRNA targeting the PEAR-GFP plasmid along 
            with an mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3475)
  AUTHORS   Simon DA, Talas A, Kulcsar PI, Biczok Z, Krausz SL, Varady G, Welker
            E
  TITLE     PEAR, a flexible fluorescent reporter for the identification and 
            enrichment of successfully prime edited cells.
  JOURNAL   Elife. 2022 Feb 23;11. pii: 69504. doi: 10.7554/eLife.69504.
  PUBMED    35196219
REFERENCE   2  (bases 1 to 3475)
  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: "Elife."; 
            date: "2022-02-23"; volume: "11. pii: 69504. doi"; pages: " 
            10.7554/eLife.69504"
COMMENT     Sequence Label: pDAS12230_pegRNA-PEAR-GFP(10PBS-24RT)-mCherry
FEATURES             Location/Qualifiers
     source          1..3475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        53..301
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     53..73
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     224..243
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        322..397
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        494..705
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      725..734
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             731..1441
                     /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(876..894)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1471..1488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1477..1684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(1749..2337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1829..1848)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2508..3299,3300..3368))
                     /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     3131..3150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3369..3473)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catttccccg aaaagtgcca cctgacgtcc agcagagatc cactttggcg ccgagggcct
       61 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
      121 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      181 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      241 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      301 cgaatgtcaa acccgcagtc tgttttagag ctagaaatag caagttaaaa taaggctagt
      361 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcggc tacgtgcagg taagtcctag
      421 actgcgggtt tttttttccg ttacataact tacggtaaat ggcccaattc gagtggctcc
      481 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      541 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      601 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
      661 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg
      721 atccgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
      781 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
      841 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      901 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
      961 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     1021 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     1081 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     1141 cggccccgta atgcagaaaa agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     1201 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     1261 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     1321 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     1381 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     1441 gatctacatg tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     1501 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1561 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1621 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg
     1681 gggagcggcc gctgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1741 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1801 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1861 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1921 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1981 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2041 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2101 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2161 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2221 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2281 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2341 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2401 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2461 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2521 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2581 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2641 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2701 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2761 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2821 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2881 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2941 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3001 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3061 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3121 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3181 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3241 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3301 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3361 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3421 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgca
//
