LOCUS       sequence-348470-        3414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid expressing an sgRNA 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 3414)
  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 3414)
  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: pDAS12137_sgRNA-PEAR-GFP-nick(+17)-mCherry
FEATURES             Location/Qualifiers
     source          1..3414
                     /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        323..398
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        433..644
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      664..673
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             670..1380
                     /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(815..833)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1410..1427)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1416..1623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(1688..2276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1768..1787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2447..3238,3239..3307))
                     /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     3070..3089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3308..3412)
                     /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 cgttcaagtc cgccatgccc gagttttaga gctagaaata gcaagttaaa ataaggctag
      361 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggctcg agtggctccg
      421 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
      481 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
      541 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
      601 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga
      661 tccgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      721 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
      781 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
      841 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
      901 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
      961 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1021 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1081 ggccccgtaa tgcagaaaaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     1141 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1201 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1261 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     1321 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtag
     1381 atctacatgt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1441 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1501 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1561 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     1621 ggagcggccg ctgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1681 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1741 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1801 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1861 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1921 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1981 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2041 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2101 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2161 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2221 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2281 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2341 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2401 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2461 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2521 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2581 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2641 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2701 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2761 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2821 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2881 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2941 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3001 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3061 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3121 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3181 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3241 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3301 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3361 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgca
//
