LOCUS       sequence-408180-        7890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome encoding C-terminal PEmaxDRNaseH and U6 expression 
            cassettes for Rosa26 twinPE pegRNA pairs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7890)
  AUTHORS   Doman JL, Pandey S, Neugebauer ME, An M, Davis JR, Randolph PB, 
            McElroy A, Gao XD, Raguram A, Richter MF, Everette KA, Banskota S, 
            Tian K, Tao YA, Tolar J, Osborn MJ, Liu DR
  TITLE     Phage-assisted evolution and protein engineering yield compact, 
            efficient prime editors.
  JOURNAL   Cell. 2023 Aug 31;186(18):3983-4002.e26. doi: 
            10.1016/j.cell.2023.07.039.
  PUBMED    37657419
REFERENCE   2  (bases 1 to 7890)
  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"; doi: 
            "10.1016/j.cell.2023.07"; journalName: "Cell"; date: "2023-08-31- 
            31"; volume: "186"; issue: "18"; pages: "3983-4002.e26"
COMMENT     Sequence Label: 
            v3em-Cterm-PEmaxdeltaRNaseH-dualU6-Rosa26-twinPE-attB
FEATURES             Location/Qualifiers
     source          1..7890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1018..1147
                     /label=AAV2 ITR
     enhancer        1170..1455
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1457..1734
                     /label=chicken beta-actin promoter
     intron          1735..1962
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2016..2036
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3231..3251
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4812..4832
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     4852..4871
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    5043..5080
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_RNA        complement(5081..5156)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5179..5492)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(5240..5260)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    5561..5598
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_RNA        complement(5599..5674)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5696..5944)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5754..5773)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5924..5944)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   5968..6097
                     /label=AAV2 ITR
     primer_bind     complement(6116..6133)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6116..6132)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6125..6147)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6274..6729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6361..6380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6571..6592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6755..6859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6860..6928,6929..7720)
                     /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     complement(7078..7097)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccagattta attaaggctg
     1021 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt
     1081 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     1141 ggttcctgcg gcctctagat cagggtaccc gttacataac ttacggtaaa tggcccgcct
     1201 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
     1261 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     1321 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     1381 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     1441 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     1501 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     1561 gcgggggggg ggggggggsg cgcgcsaggs ggggsggggs ggggcgaggg gcggggcggg
     1621 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     1681 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     1741 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     1801 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     1861 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
     1921 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca
     1981 tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtcatca
     2041 agattgctac acggaaatac ctgggaaagc agaacgtgta cgacatcggc gtggagcggg
     2101 atcacaactt cgccctgaag aatggcttta tcgccagcaa ttgtttcaac gaaatcggca
     2161 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     2221 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     2281 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     2341 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     2401 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     2461 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     2521 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     2581 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     2641 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     2701 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     2761 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     2821 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     2881 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     2941 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     3001 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     3061 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     3121 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt gactccggcg
     3181 gaagctctgg tggcagcaag cggaccgccg acggctctga attcgagagc cctaagaaga
     3241 aaagaaaggt gagcggaggc tctagcggcg gaagcaccct gaacattgaa gacgagtata
     3301 gactgcatga aacaagcaag gaacccgacg tgtccctggg ctccacctgg ctgtccgact
     3361 ttccccaggc ctgggccgag acaggaggaa tgggcctggc cgtgcggcag gcacccctga
     3421 tcatccctct gaaggccacc tctacacccg tgagcatcaa gcagtaccct atgtctcagg
     3481 aggccagact gggcatcaag cctcacatcc agaggctgct ggaccagggc atcctggtgc
     3541 catgccagag cccctggaac acaccactgc tgcccgtgaa gaagccaggc accaatgact
     3601 atagacccgt gcaggatctg agagaggtga acaagagggt ggaggatatc caccccaccg
     3661 tgcccaaccc ttacaatctg ctgtccggcc tgcccccttc tcaccagtgg tatacagtgc
     3721 tggacctgaa ggatgccttc ttttgtctga gactgcaccc taccagccag ccactgttcg
     3781 cctttgagtg gagggaccct gagatgggca tctctggcca gctgacctgg acacgcctgc
     3841 ctcagggctt caagaatagc ccaacactgt ttaacgaggc cctgcaccgc gacctggcag
     3901 atttccggat ccagcaccca gatctgatcc tgctgcagta cgtggacgat ctgctgctgg
     3961 ccgccaccag cgagctggat tgccagcagg gaacacgcgc cctgctgcag accctgggaa
     4021 acctgggata tagggcatcc gccaagaagg cccagatctg tcagaagcag gtgaagtacc
     4081 tgggctatct gctgaaggag ggccagagat ggctgacaga ggccaggaag gagacagtga
     4141 tgggccagcc aacacccaag accccaagac agctgaggga gttcctgggc aaagcaggat
     4201 tttgcaggct gttcatccca ggattcgcag agatggcagc acctctgtac ccactgacca
     4261 agccgggcac cctgtttaat tggggccctg accagcagaa ggcctatcag gagatcaagc
     4321 aggccctgct gacagcacca gccctgggcc tgccagacct gaccaagcct ttcgagctgt
     4381 ttgtggatga gaagcagggc tacgccaagg gcgtgctgac ccagaagctg ggaccatgga
     4441 gacggcccgt ggcctatctg tccaagaagc tggacccagt ggcagcagga tggccaccat
     4501 gcctgaggat ggtggcagca atcgccgtgc tgacaaagga tgccggcaag ctgaccatgg
     4561 gacagccact ggtcatcctg gcaccacacg cagtggaggc cctggtgaag cagcctccag
     4621 atcgctggct gtctaacgcc cggatgacac actaccaggc cctgctgctg gacaccgatc
     4681 gcgtgcagtt tggccctgtg gtggccctga atccagccac cctgctgcct ctgccagagg
     4741 agggcctgca gcacaactgt ctggactctg gcggctcaaa aagaaccgcc gacggcagcg
     4801 aattcgagtc tcccaagaag aagaggaaag tctaagtcga cctttatttg tgaaatttgt
     4861 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4921 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagctt
     4981 gcaaaaaaat tctagttggt ttaacgcgta actagataga accgcgctac tgttcactct
     5041 aaggcttgtc gacgacggcg gtctccgtcg tcaggatcat gcaccgactc ggtgccactt
     5101 tttcaagttg ataacggact agccttattt taacttgcta tttctagctc taaaacctgt
     5161 tagagtgaac agtagacaaa caaggctttt ctccaaggga tatttatagt ctcaaaacac
     5221 acaattactt tacagttagg gtgagtttcc ttttgtgctg ttttttaaaa taataattta
     5281 gtatttgtat ctcttataga aatccaagcc tatcatgtaa aatgtagcta gtattaaaaa
     5341 gaacagatta tctgtctttt atcgcacatt aagcctctat agttactagg aaatattata
     5401 tgcaaattaa ccggggcagg ggagtagccg agcttctccc acaagtctgt gcgagggggc
     5461 cggcgcgggc ctagagatgg cggcgtcgga tcgcgaattc aaaaaaattc tagttggttt
     5521 aacgcgtaac tagatagaac cgcgaatctg ctagtatatc atgatcctga cgacggagac
     5581 cgccgtcgtc gacaagccgc accgactcgg tgccactttt tcaagttgat aacggactag
     5641 ccttatttta acttgctatt tctagctcta aaacacggat atactagcag attacggtgt
     5701 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     5761 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     5821 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa
     5881 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     5941 cctcttcctg cccgaccttg cggccgcagg aacccctagt gatggagttg gccactccct
     6001 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     6061 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg
     6121 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     6181 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     6241 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     6301 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     6361 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     6421 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     6481 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     6541 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     6601 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     6661 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     6721 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6781 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6841 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6901 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6961 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     7021 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     7081 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     7141 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     7201 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7261 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7321 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7381 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7441 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7501 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7561 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7621 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7681 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7741 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7801 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7861 agaccccgta gaaaagatca aaggatcttc
//
