LOCUS       sequence-337167-        8899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression, sauCas9-KKH based prime editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8899)
  AUTHORS   Liu P, Liang SQ, Zheng C, Mintzer E, Zhao YG, Ponnienselvan K, Mir 
            A, Sontheimer EJ, Gao G, Flotte TR, Wolfe SA, Xue W
  TITLE     Improved prime editors enable pathogenic allele correction and 
            cancer modelling in adult mice.
  JOURNAL   Nat Commun. 2021 Apr 9;12(1):2121. doi: 10.1038/s41467-021-22295-w.
  PUBMED    33837189
REFERENCE   2  (bases 1 to 8899)
  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: "Nat 
            Commun."; date: "2021-04-9"; volume: "12(1):2121. doi"; pages: " 
            10.1038/s41467-021-22295-w"
COMMENT     Sequence Label: SauKKHCas9 PE2*
FEATURES             Location/Qualifiers
     source          1..8899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             508..528
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             537..554
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(577..594)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    583..807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(878..894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(878..894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(891..913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    902..918
                     /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)."
     promoter        complement(join(926..932,933..950,951..956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    971..992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1109..1126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1280..1868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1360..1379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2039..2830,2831..2899))
                     /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     2662..2681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2900..3004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3083..3102)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3274..3653
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3654..3857
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3808..3828
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3899..3908
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3911..3937
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3977..3997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 accgagaccg aggtaatctg ggctaaagcc ctgccagccg ggacatccgc tcagcgggct
       61 gaactgatag cactcaccca ggccctaaag atggcagaag gtaagaagct aaatgtttat
      121 actgatagcc gttatgcttt tgctactgcc catatccatg gagaaatata cagaaggcgt
      181 gggtggctca catcagaagg caaagagatc aaaaataaag acgagatctt ggccctacta
      241 aaagccctct ttctgcccaa aagacttagc ataatccatt gtccaggaca tcaaaaggga
      301 cacagcgccg aggctagagg caaccggatg gctgaccaag cggcccgaaa ggcagccatc
      361 acagagactc cagacacctc taccctcctc atagaaaatt catcaccctc tggcggctca
      421 aaaagaaccg ccgacggcag cgagaagcgc accgccgaca gccagcacag cacccccccc
      481 aagaccaagc gcaaggtgga attcgagccc aagaagaaga ggaaagtcta accggtcatc
      541 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
      601 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
      661 ctgtcctttc ctaataaaat gagaaaattg catcgcattg tctgagtagg tgtcattcta
      721 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
      781 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
      841 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
      901 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
      961 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1861 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
     1921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     1981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2401 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2641 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2701 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2761 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2821 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2881 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     2941 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3001 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     3061 atcccctagg gtctactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     3121 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     3181 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     3241 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     3301 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     3361 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     3421 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     3481 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     3541 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     3601 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     3661 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     3721 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     3781 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     3841 tctatataag cagagctggt ttagtgaacc gtcagatccg ctagagatcc gcggccgcgc
     3901 caccatgggg cccgccgcca agcgcgtgaa gctggacggc ggcaaacgga cagccgacgg
     3961 aagcgagttc gagtcaccaa agaagaagcg gaaagtcggt atccacggag tcccagcagc
     4021 caaacggaac tacatcctgg ggcttgacat tgggataacc agcgttggct acggaattat
     4081 tgattatgag acacgcgatg tgattgacgc cggggttagg ctgttcaaag aggccaacgt
     4141 tgaaaacaac gagggaagac ggagtaagcg cggagcaaga agactcaagc gcagacggag
     4201 acatcggatt cagagggtga aaaagctgct cttcgattac aatctcctga ccgatcatag
     4261 tgagctgagc ggaatcaacc cctacgaggc gcgagtgaaa gggctttccc agaagctgtc
     4321 cgaagaggag ttctccgccg cgttgctgca cctggccaaa cggagggggg ttcacaatgt
     4381 aaacgaagtg gaggaggaca cgggcaatga acttagtacg aaagaacaga tcagtaggaa
     4441 ctctaaggct ctcgaagaga aatacgtcgc tgagttgcag cttgagagac tgaaaaaaga
     4501 cggcgaagta cgcggatcta ttaataggtt caagacttca gattacgtaa aggaagccaa
     4561 gcagctcctg aaagtacaga aagcgtacca tcagctcgat cagagcttca tcgataccta
     4621 catagatttg ctggagacac ggaggacata ctacgagggc ccaggggaag gatctccttt
     4681 tgggtggaag gacatcaagg aatggtacga gatgcttatg ggacattgta catattttcc
     4741 ggaggagctc aggagcgtca agtacgccta caatgccgac ctgtacaatg ccctcaatga
     4801 cctcaataac ctcgtgatta ccagggacga gaacgagaag ctggagtact atgaaaagtt
     4861 ccagattatc gagaatgtgt ttaagcagaa gaagaagccg acacttaagc agattgcaaa
     4921 ggaaatcctc gtgaatgagg aagatatcaa gggatacaga gtgacaagta caggcaagcc
     4981 cgagttcaca aatctgaagg tgtaccacga tattaaggac ataaccgcac gaaaggagat
     5041 aatcgaaaac gctgagctcc tcgatcagat cgcaaaaatt cttaccatct accagtctag
     5101 tgaggacatt caggaggaac tgactaatct gaacagtgag ctcacccaag aggaaattga
     5161 gcagatttca aacctgaaag gctacaccgg gacgcacaat ctgagcctca aagcaatcaa
     5221 cctcattctg gatgaacttt ggcacacaaa tgacaaccaa attgccatat tcaaccgcct
     5281 gaaactggtg ccaaaaaaag tggatctgtc acagcaaaag gaaatcccta caaccttggt
     5341 tgacgatttt attctgtccc ccgttgtcaa gcggagcttc atccagtcaa tcaaggtgat
     5401 caatgccatc attaaaaaat acggattgcc aaacgatata attatcgagc ttgcacgaga
     5461 gaagaactca aaggacgccc agaagatgat taacgaaatg cagaagcgca accgccagac
     5521 aaacgaacgc atagaggaaa ttataagaac aaccggcaaa gagaatgcca agtatctgat
     5581 cgagaaaatc aagctgcacg acatgcaaga aggcaagtgc ctgtactctc tggaagctat
     5641 cccactcgaa gatctgctga ataatccatt caattacgag gtggaccaca tcatccctag
     5701 atccgtaagc tttgacaatt ccttcaataa caaagttctg gttaaacagg aggaagcctc
     5761 taaaaaaggg aaccggaccc cgttccagta cctgagctcc agtgacagca agattagcta
     5821 cgagactttt aagaaacata ttctgaatct ggccaaaggc aaaggcagga tcagcaagac
     5881 caagaaggag tacctcctcg aagaacgcga cattaacaga tttagtgtgc agaaagattt
     5941 catcaaccga aaccttgtcg atactcggta cgccacgaga ggcctgatga atctcctcag
     6001 gagctacttc cgcgtcaata atctggacgt taaagtcaag agcataaatg ggggattcac
     6061 cagctttctg aggagaaagt ggaagtttaa gaaggaacga aacaaaggat acaagcacca
     6121 tgctgaggat gctttgatca tcgctaacgc ggactttatc tttaaggaat ggaaaaagct
     6181 ggataaggca aagaaagtga tggaaaacca gatgttcgag gagaagcagg cagagtcaat
     6241 gcctgagatc gagacagagc aggaatacaa ggaaattttc atcacccctc atcagattaa
     6301 acacataaag gacttcaaag actataaata ctctcatagg gtggacaaaa aacccaatcg
     6361 caagctcatt aatgacaccc tgtactcaac acggaaggat gataaaggta ataccttgat
     6421 tgtgaataat cttaatggat tgtatgacaa agataacgac aagctcaaga agctgatcaa
     6481 caagtctcca gagaagctcc ttatgtatca ccacgaccca cagacttatc agaaattgaa
     6541 actgatcatg gagcaatacg gggatgagaa gaacccactc tacaaatatt atgaggaaac
     6601 aggtaattac ctgaccaagt actccaagaa ggataacgga ccagtgatca aaaagataaa
     6661 gtactatggc aacaaactta atgcgcattt ggacataact gacgattacc ccaattctcg
     6721 aaacaaggtt gtgaagctct ccctgaagcc ttatagattt gacgtgtacc tggataatgg
     6781 ggtttataaa ttcgtcaccg tgaaaaatct ggacgtgatc aaaaaggaga actattatga
     6841 agtaaactca aagtgctatg aggaggcgaa gaagctgaag aagatctcca atcaggccga
     6901 gttcatcgct tccttctata agaacgatct catcaagatc aatggagagc tttatcgcgt
     6961 cattggtgtg aacaatgact tgctgaacag gatcgaagtc aatatgatag acattaccta
     7021 ccgggagtat ctcgaaaaca tgaatgataa acggccgcct cacatcatca agacaatcgc
     7081 atctaaaact cagtcaataa aaaagtactc taccgatatc ctggggaatc tctatgaagt
     7141 gaagtcaaag aagcacccac aaatcattaa aaaaggttct ggaggatcta gcggaggatc
     7201 ctctggcagc gagacaccag gaacaagcga gtcagcaaca ccagagagca gtggcggcag
     7261 cagcggcggc agcagcaccc taaatataga agatgagtat cggctacatg agacctcaaa
     7321 agagccagat gtttctctag ggtccacatg gctgtctgat tttcctcagg cctgggcgga
     7381 aaccgggggc atgggactgg cagttcgcca agctcctctg atcatacctc tgaaagcaac
     7441 ctctaccccc gtgtccataa aacaataccc catgtcacaa gaagccagac tggggatcaa
     7501 gccccacata cagagactgt tggaccaggg aatactggta ccctgccagt ccccctggaa
     7561 cacgcccctg ctacccgtta agaaaccagg gactaatgat tataggcctg tccaggatct
     7621 gagagaagtc aacaagcggg tggaagacat ccaccccacc gtgcccaacc cttacaacct
     7681 cttgagcggg ctcccaccgt cccaccagtg gtacactgtg cttgatttaa aggatgcctt
     7741 tttctgcctg agactccacc ccaccagtca gcctctcttc gcctttgagt ggagagatcc
     7801 agagatggga atctcaggac aattgacctg gaccagactc ccacagggtt tcaaaaacag
     7861 tcccaccctg tttaatgagg cactgcacag agacctagca gacttccgga tccagcaccc
     7921 agacttgatc ctgctacagt acgtggatga cttactgctg gccgccactt ctgagctaga
     7981 ctgccaacaa ggtactcggg ccctgttaca aaccctaggg aacctcgggt atcgggcctc
     8041 ggccaagaaa gcccaaattt gccagaaaca ggtcaagtat ctggggtatc ttctaaaaga
     8101 gggtcagaga tggctgactg aggccagaaa agagactgtg atggggcagc ctactccgaa
     8161 gacccctcga caactaaggg agttcctagg gaaggcaggc ttctgtcgcc tcttcatccc
     8221 tgggtttgca gaaatggcag cccccctgta ccctctcacc aaaccgggga ctctgtttaa
     8281 ttggggccca gaccaacaaa aggcctatca agaaatcaag caagctcttc taactgcccc
     8341 agccctgggg ttgccagatt tgactaagcc ctttgaactc tttgtcgacg agaagcaggg
     8401 ctacgccaaa ggtgtcctaa cgcaaaaact gggaccttgg cgtcggccgg tggcctacct
     8461 gtccaaaaag ctagacccag tagcagctgg gtggccccct tgcctacgga tggtagcagc
     8521 cattgccgta ctgacaaagg atgcaggcaa gctaaccatg ggacagccac tagtcattct
     8581 ggccccccat gcagtagagg cactagtcaa acaacccccc gaccgctggc tttccaacgc
     8641 ccggatgact cactatcagg ccttgctttt ggacacggac cgggtccagt tcggaccggt
     8701 ggtagccctg aacccggcta cgctgctccc actgcctgag gaagggctgc aacacaactg
     8761 ccttgatatc ctggccgaag cccacggaac ccgacccgac ctaacggacc agccgctccc
     8821 agacgccgac cacacctggt acacggatgg aagcagtctc ttacaagagg gacagcgtaa
     8881 ggcgggagct gcggtgacc
//
