LOCUS       sequence-408179-        7809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome encoding C-terminal PE6c 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 7809)
  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 7809)
  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-PE6c-dualU6-Rosa26-twinPE-attB
FEATURES             Location/Qualifiers
     source          1..7809
                     /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             4731..4751
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     4771..4790
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    4962..4999
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_RNA        complement(5000..5075)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5098..5411)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(5159..5179)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    5480..5517
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_RNA        complement(5518..5593)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5615..5863)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5673..5692)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5843..5863)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   5887..6016
                     /label=AAV2 ITR
     primer_bind     complement(6035..6052)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6035..6051)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6044..6066)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6193..6648
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6280..6299)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6490..6511
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6674..6778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6779..6847,6848..7639)
                     /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(6997..7016)
                     /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 ggggggggcg cgcgccaggs ggggsggggs ggggsgaggg 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 gaagcatcag cagctctaag cacaccctga
     3301 gccagatgaa caaggtgagc aacatcgtga aggaacccga gctgcccgac atctacaagg
     3361 aatttaagga catcaccgcc gacaccaata ccgagaagct gcctaagcct attaagggcc
     3421 tggaatttga agtggaactg acacaggaga actacagact gcctatccgg aactatcctc
     3481 tgactccagt caagatgcag gccatgaacg acgagatcaa tcaaggcctg aaaggcggca
     3541 tcatcagaga gagcaaggcc atcaacgcct gccctgttat attcgtgccc agaaaggaag
     3601 gcacactgcg catggtggtc gactacaggc ccctgaacaa gtacgtgaag cccaacgtct
     3661 accccctgcc actgattgaa caactgctgg ccaagatcca gggcagcacc attttcacca
     3721 agctggacct gaaaagcgcc taccaccaga tcagagtgcg aaagggagat gagcacaagc
     3781 tggccttccg gtgccccaga ggagtcttcg agtacctggt gatgccttac ggcatcaaga
     3841 cagcccctgc ccactttcag tacttcatca acacaatcct gggcgaggcc aaggaaagcc
     3901 acgtggtgtg ctacatggac gacatcctga tccattccaa gtccgagtcc gaacacgtga
     3961 aacatgtgaa ggacgtgctg caaaagctga agaacgctaa tctgatcatc aaccaggcca
     4021 aatgcgagtt tcaccagagc caagtgaagt tcctgggcta ccacatcagc gagaagggct
     4081 taaccccatg tcaggagaac atcgacaagg tgctgcagtg gaaacagcct aaaaaccaga
     4141 aggaactgag acagttcctg ggccaggtga actacctgag aaaattcatc cccaaaacca
     4201 gccagttgac ccaccctctg aacaaactcc tgaaaaagga tgtcagatgg aaatggaccc
     4261 ctacccagac ccaggctatc gagaatatca agcaatgtct ggtgtctcct cctgtgctga
     4321 ggcacttcga cttcagcaag aagatcctgc ttgagacaga cgtttctgat gtggccgtgg
     4381 gagccgtgct gagccagaag catgatgatg ataagtacta ccctgtgggc tattacagcg
     4441 ctaaaatgag caaagcccag ctgaattatt ctgtgtccga caaggagatg ctggctatca
     4501 tcaaaagcct ggagcactgg cggcactacc tggaatctac aatcgagccc ttcaagatcc
     4561 tgaccgacca cagaaacctg attggaagaa tcacaaacga gagcgaacca gagaacaagc
     4621 ggctggccag atggcagctg ttcctgcagg acttcaactt cgagatcaac tacagacctg
     4681 gctcttctgg cggctcaaaa agaaccgccg acggcagcga attcgagtct cccaagaaga
     4741 agaggaaagt ctaagtcgac ctttatttgt gaaatttgtg atgctattgc tttatttgta
     4801 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     4861 gttcaggggg agatgtggga ggttttttaa agcaagcttg caaaaaaatt ctagttggtt
     4921 taacgcgtaa ctagatagaa ccgcgctact gttcactcta aggcttgtcg acgacggcgg
     4981 tctccgtcgt caggatcatg caccgactcg gtgccacttt ttcaagttga taacggacta
     5041 gccttatttt aacttgctat ttctagctct aaaacctgtt agagtgaaca gtagacaaac
     5101 aaggcttttc tccaagggat atttatagtc tcaaaacaca caattacttt acagttaggg
     5161 tgagtttcct tttgtgctgt tttttaaaat aataatttag tatttgtatc tcttatagaa
     5221 atccaagcct atcatgtaaa atgtagctag tattaaaaag aacagattat ctgtctttta
     5281 tcgcacatta agcctctata gttactagga aatattatat gcaaattaac cggggcaggg
     5341 gagtagccga gcttctccca caagtctgtg cgagggggcc ggcgcgggcc tagagatggc
     5401 ggcgtcggat cgcgaattca aaaaaattct agttggttta acgcgtaact agatagaacc
     5461 gcgaatctgc tagtatatca tgatcctgac gacggagacc gccgtcgtcg acaagccgca
     5521 ccgactcggt gccacttttt caagttgata acggactagc cttattttaa cttgctattt
     5581 ctagctctaa aacacggata tactagcaga ttacggtgtt tcgtcctttc cacaagatat
     5641 ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa
     5701 aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt
     5761 gtactaatat ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt
     5821 atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcttcctgc ccgaccttgc
     5881 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     5941 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     6001 gcgagcgagc gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg
     6061 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     6121 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     6181 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6241 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     6301 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     6361 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     6421 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     6481 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     6541 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6601 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     6661 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6721 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     6781 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     6841 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6901 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6961 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     7021 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     7081 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     7141 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     7201 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     7261 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     7321 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     7381 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     7441 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     7501 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     7561 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     7621 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     7681 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     7741 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     7801 aggatcttc
//
