LOCUS       sequence-339966-        5070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of transcript containing pegRNA and nick-sgRNA 
            targeting human RNF2 gene from the U6 promoter with pegRNA flanked 
            by Csy4 recognition site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5070)
  AUTHORS   Liu Y, Yang G, Huang S, Li X, Wang X, Li G, Chi T, Chen Y, Huang X, 
            Wang X
  TITLE     Enhancing prime editing by Csy4-mediated processing of pegRNA.
  JOURNAL   Cell Res. 2021 Jun 8. pii: 10.1038/s41422-021-00520-x. doi: 
            10.1038/s41422-021-00520-x.
  PUBMED    34103663
REFERENCE   2  (bases 1 to 5070)
  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: "Cell Res. 
            2021 Jun 8. pii: 10.1038/s41422-021-00520-x. doi: 
            10.1038/s41422-021-00520-x."
COMMENT     Sequence Label: PGL3-U6-RNF2_pegRNA-Csy4RS-nick_sgRNA-EGFP
FEATURES             Location/Qualifiers
     source          1..5070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     371..390
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(609..713)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      740..1195
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(827..846)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1037..1058
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1326..1374
                     /note="synthetic polyadenylation signal"
     misc_feature    1388..1479
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1428..1447
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1558..1806
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1558..1578
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1729..1748
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1827..1902
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1932..1951
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        1972..2047
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2099..2216
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2265..2775
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2694..2712
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(2784..2786,2787..2789,2790..3503)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2784..2786,2787..2789,2790..3500)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2829..2850)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3090..3109)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3437..3458
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3626..3747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3663..3682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3717..3736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3888..3905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4059..4647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4139..4158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4818..5070,1..539,540..608))
                     /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"
ORIGIN
        1 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
       61 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      121 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      181 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      241 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      301 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      361 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      421 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      481 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      541 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      601 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      661 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      721 ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
      781 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
      841 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
      901 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
      961 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1021 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1081 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1141 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttgccat
     1201 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     1261 cgccagccca agctaccatg ataagtaagt aatattaagg tacgggaggt acttggagcg
     1321 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     1381 tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg
     1441 ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgataggta ccgattagtg
     1501 aacggatctc gacggtatcg atcacgagac tagcctcgag cggccgcccc cttcaccgag
     1561 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     1621 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     1681 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1741 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1801 aacaccgtca tcttagtcat tacctggttt cagagctaga aatagcaagt tgaaataagg
     1861 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaacgaaca tctcaggtaa
     1921 tgactaagat ggttcactgc cgtataggca gtcaaccatt aagcaaaaca tgttttagag
     1981 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     2041 tcggtgcttt ttttaaagaa ttctcgacct cgagacaaat ggcagtattc atccacaatt
     2101 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2161 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2221 tttattacag ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc
     2281 cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg
     2341 aaacgcagcg gcgccgaccc tgggactcgc acattcttca cgtccgttcg cagcgtcacc
     2401 cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt
     2461 cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc
     2521 actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg
     2581 gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg
     2641 gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc
     2701 gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc
     2761 gacctctctc cccaggggga tccatggtga gcaagggcga ggagctgttc accggggtgg
     2821 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2881 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2941 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     3001 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     3061 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     3121 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3181 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3241 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     3301 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3361 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     3421 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3481 gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac
     3541 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccttga acctgaaaca
     3601 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     3661 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3721 tttgtccaaa ctcatcaatg tatcttagtc gaccgatgcc cttgagagcc ttcaacccag
     3781 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     3841 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
     3901 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3961 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4021 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4081 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4141 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4201 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4261 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4321 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4381 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4441 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4501 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4561 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4621 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4681 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4741 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4801 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4861 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4921 ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca
     4981 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5041 ttatccgcct ccatccagtc tattaattgt
//
