LOCUS       sequence-339967-        5069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of transcript containing pegRNA and nick-sgRNA 
            targeting human SDHB 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 5069)
  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 5069)
  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-SDHB_pegRNA-Csy4RS-nick_sgRNA-EGFP
FEATURES             Location/Qualifiers
     source          1..5069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     239..258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(477..581)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      608..1063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(695..714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     905..926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1194..1242
                     /note="synthetic polyadenylation signal"
     misc_feature    1256..1347
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1296..1315
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1426..1674
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1426..1446
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1597..1616
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1696..1771
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1799..1818
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        1839..1914
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1966..2083
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2132..2642
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2561..2579
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(2651..2653,2654..2656,2657..3370)
                     /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(2651..2653,2654..2656,2657..3367)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2696..2717)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2957..2976)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3304..3325
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3493..3614
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3530..3549)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3584..3603
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3755..3772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3926..4514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4006..4025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4685..5069,1..407,408..476))
                     /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 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
       61 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      121 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      181 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      241 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      301 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      361 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      421 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      481 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      541 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      601 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      661 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      721 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      781 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      841 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      901 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      961 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1021 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg
     1081 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag
     1141 ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa
     1201 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat
     1261 acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg
     1321 caagtgcagg tgccagaaca tttctctatc gataggtacc gattagtgaa cggatctcga
     1381 cggtatcgat cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc
     1441 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     1501 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1561 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1621 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgctcag
     1681 gtaatccacc tgcctgtttc agagctagaa atagcaagtt gaaataaggc tagtccgtta
     1741 tcaacttgaa aaagtggcac cgagtcggtg cttgggaggc cgagtcaggt ggattaccgt
     1801 tcactgccgt ataggcagat tttataggcc aggcgtgggt tttagagcta gaaatagcaa
     1861 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     1921 taaagaattc tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1981 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2041 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2101 cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca
     2161 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     2221 ccgaccctgg gactcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     2281 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     2341 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
     2401 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     2461 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     2521 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     2581 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     2641 agggggatcc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2701 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2761 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2821 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2881 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2941 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3001 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3061 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3121 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3181 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3241 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3301 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3361 gtacaagtaa agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     3421 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3481 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3541 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3601 atcaatgtat cttagtcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     3661 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     3721 cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3781 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3841 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3901 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3961 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4021 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4081 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4141 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4201 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4261 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4321 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4381 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4441 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4501 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4561 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4621 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4681 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4741 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4801 gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa
     4861 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4921 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4981 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5041 cattcagctc cggttcccaa cgatcaagg
//
