LOCUS       sequence-348477-        3467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pegRNA cloning plasmid with BFP marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3467)
  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 3467)
  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: pDAS12222_U6-pegRNA-BFP
FEATURES             Location/Qualifiers
     source          1..3467
                     /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        330..405
                     /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"
     CDS             731..1432
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1463..1480)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1469..1676
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(1741..2329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1821..1840)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2500..3291,3292..3360))
                     /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     3123..3142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3361..3465)
                     /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 cgggtcttcg gatccagatc tgaagacctg ttttagagct agaaatagca agttaaaata
      361 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctgaga cgatcgaaga
      421 attccgtctc attttttccg 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 atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
      781 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
      841 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
      901 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
      961 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     1021 agatgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     1081 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     1141 actcggctgg gaggccttca ccgagacact gtaccccgct gacggcggcc tggaaggcag
     1201 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     1261 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     1321 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     1381 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt aagatctaca
     1441 tgtcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1501 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1561 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1621 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg
     1681 ccgctgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1741 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1801 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1861 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1921 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1981 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2041 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2101 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2161 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2221 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2281 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2341 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2401 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2461 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2521 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2581 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2641 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2701 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2761 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2821 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2881 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2941 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3001 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3061 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3121 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3181 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3241 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3301 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3361 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3421 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgca
//
