LOCUS       sequence-347982-        5070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of transcript containing pegRNA and nick-sgRNA 
            targeting human ALDOB 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-ALDOB_pegRNA-Csy4RS-nick_sgRNA-EGFP
FEATURES             Location/Qualifiers
     source          1..5070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(82..873,874..942))
                     /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     705..724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(943..1047)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1074..1529
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1161..1180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1371..1392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1660..1708
                     /note="synthetic polyadenylation signal"
     misc_feature    1722..1813
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1762..1781
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1892..2140
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1892..1912
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2063..2082
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2162..2237
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2266..2285
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        2306..2381
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2433..2550
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2599..3109
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3028..3046
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(3118..3120,3121..3123,3124..3837)
                     /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(3118..3120,3121..3123,3124..3834)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3163..3184)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3424..3443)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3771..3792
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3960..4081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3997..4016)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4051..4070
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4222..4239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4393..4981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4473..4492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
       61 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      121 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      181 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac gctcaccggc
      241 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
      301 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
      361 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
      421 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
      481 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
      541 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
      601 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
      661 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
      721 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
      781 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
      841 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
      901 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
      961 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1021 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     1081 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1141 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1201 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1261 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1321 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1381 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1441 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1501 ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc gcaactgttg
     1561 ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac catgataagt
     1621 aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc tttattttca
     1681 ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa
     1741 aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc
     1801 agaacatttc tctatcgata ggtaccgatt agtgaacgga tctcgacggt atcgatcacg
     1861 agactagcct cgagcggccg cccccttcac cgagggccta tttcccatga ttccttcata
     1921 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     1981 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2041 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2101 tttcttggct ttatatatct tgtggaaagg acgaaacacc gccaccctgt gctgagtgta
     2161 tgtttcagag ctagaaatag caagttgaaa taaggctagt ccgttatcaa cttgaaaaag
     2221 tggcaccgag tcggtgctct ctccctccaa gacactcagc acagggttca ctgccgtata
     2281 ggcagtagct tcctatccaa tgccagtttt agagctagaa atagcaagtt aaaataaggc
     2341 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttaa agaattctcg
     2401 acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2461 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2521 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2581 ctttggccgc ggctcgaggg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc
     2641 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggac
     2701 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
     2761 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
     2821 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg
     2881 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
     2941 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
     3001 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
     3061 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatccatg
     3121 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3181 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     3241 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3301 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3361 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3421 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3481 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3541 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3601 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3661 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3721 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3781 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc
     3841 ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     3901 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     3961 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4021 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4081 agtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     4141 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     4201 ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4261 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4321 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4381 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4441 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4501 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4561 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4621 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4681 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4741 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4801 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4861 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4921 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4981 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5041 agggattttg gtcatgagat tatcaaaaag
//
