LOCUS       sequence-343608-        8811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SaCas9 prime editor 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8811)
  AUTHORS   Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen
            C, Nelson JW, Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, 
            Liu DR
  TITLE     Enhanced prime editing systems by manipulating cellular determinants
            of editing outcomes.
  JOURNAL   Cell. 2021 Oct 28;184(22):5635-5652.e29. doi: 
            10.1016/j.cell.2021.09.018.
  PUBMED    34653350
REFERENCE   2  (bases 1 to 8811)
  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.2021.09"; journalName: "Cell"; date: "2021-10-28- 
            28"; volume: "184"; issue: "22"; pages: "5635-5652.e29"
COMMENT     Sequence Label: pCMV-SaPE2
FEATURES             Location/Qualifiers
     source          1..8811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        64..443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        444..647
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     598..618
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     689..708
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        689..707
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             757..777
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6109..6129
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6138..6155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6178..6195)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6184..6408
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6479..6495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6479..6495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6492..6514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6503..6519
                     /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(6527..6533,6534..6551,6552..6557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6572..6593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6710..6727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6881..7469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6961..6980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7640..8431,8432..8500))
                     /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     8263..8282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8501..8605)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8684..8703)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
       61 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      121 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      181 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      241 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      301 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      361 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      421 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      481 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      541 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      601 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
      661 gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag agccgccacc
      721 atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcggg
      781 aagcgaaatt acattctggg gctggacatt ggcattacat cagtgggcta tggcatcatt
      841 gactacgaga caagggacgt gatcgacgcc ggcgtgagac tgttcaagga ggccaacgtg
      901 gagaacaatg agggccggag atccaagagg ggagcaaggc gcctgaagcg gagaaggcgc
      961 cacagaatcc agagagtgaa gaagctgctg ttcgattaca acctgctgac cgaccactcc
     1021 gagctgtctg gcatcaatcc ttatgaggcc agagtgaagg gcctgtccca gaagctgtct
     1081 gaggaggagt ttagcgccgc cctgctgcac ctggcaaaga ggagaggcgt gcacaacgtg
     1141 aatgaggtgg aggaggacac cggcaacgag ctgtccacaa aggagcagat cagccgcaat
     1201 tccaaggccc tggaggagaa gtatgtggcc gagctgcagc tggagcggct gaagaaggat
     1261 ggcgaggtga ggggctccat caatcgcttc aagacctctg actacgtgaa ggaggccaag
     1321 cagctgctga aggtgcagaa ggcctaccac cagctggatc agtcctttat cgatacatat
     1381 atcgacctgc tggagacaag gcgcacatac tatgagggac caggagaggg ctctcccttc
     1441 ggctggaagg acatcaagga gtggtacgag atgctgatgg gccactgcac ctattttcca
     1501 gaggagctga gaagcgtgaa gtacgcctat aacgccgatc tgtacaacgc cctgaatgac
     1561 ctgaacaacc tggtcatcac cagggatgag aacgagaagc tggagtacta tgagaagttc
     1621 cagatcatcg agaacgtgtt caagcagaag aagaagccta cactgaagca gatcgccaag
     1681 gagatcctgg tgaacgagga ggacatcaag ggctaccgcg tgacctccac aggcaagcca
     1741 gagttcacca atctgaaggt gtatcacgat atcaaggaca tcacagcccg gaaggagatc
     1801 atcgagaacg ccgagctgct ggatcagatc gccaagatcc tgaccatcta tcagagctcc
     1861 gaggacatcc aggaggagct gaccaacctg aatagcgagc tgacacagga ggagatcgag
     1921 cagatcagca atctgaaggg ctacaccggc acacacaacc tgagcctgaa ggccatcaat
     1981 ctgatcctgg atgagctgtg gcacacaaac gacaatcaga tcgccatctt taaccggctg
     2041 aagctggtgc caaagaaggt ggacctgtcc cagcagaagg agatcccaac cacactggtg
     2101 gacgatttca tcctgtctcc cgtggtgaag cggagcttca tccagagcat caaagtgatc
     2161 aacgccatca tcaagaagta cggcctgccc aatgatatca tcatcgagct ggccagggag
     2221 aagaactcca aggacgccca gaagatgatc aatgagatgc agaagaggaa ccgccagacc
     2281 aatgagcgga tcgaggagat catcagaacc acaggcaagg agaacgccaa gtacctgatc
     2341 gagaagatca agctgcacga tatgcaggag ggcaagtgtc tgtattctct ggaggccatc
     2401 cctctggagg acctgctgaa caatccattc aactacgagg tggatcacat catcccccgg
     2461 agcgtgagct tcgacaattc ttttaacaat aaggtgctgg tgaagcagga ggaggccagc
     2521 aagaagggca ataggacccc tttccagtac ctgtctagct ccgattctaa gatcagctac
     2581 gagacattca agaagcacat cctgaatctg gccaagggca agggccgcat cagcaagacc
     2641 aagaaggagt acctgctgga ggagcgggac atcaacagat tctccgtgca gaaggacttc
     2701 atcaaccgga atctggtgga caccagatac gccacacgcg gcctgatgaa tctgctgcgg
     2761 tcttatttca gagtgaacaa tctggatgtg aaggtgaaga gcatcaacgg cggcttcacc
     2821 tcctttctgc ggagaaagtg gaagtttaag aaggagcgca acaagggcta taagcaccac
     2881 gccgaggatg ccctgatcat cgccaatgcc gacttcatct ttaaggagtg gaagaagctg
     2941 gacaaggcca agaaagtgat ggagaaccag atgttcgagg agaagcaggc cgagagcatg
     3001 cccgagatcg agacagagca ggagtacaag gagattttca tcacacctca ccagatcaag
     3061 cacatcaagg acttcaagga ctacaagtat tctcacaggg tggataagaa gcccaaccgc
     3121 gagctgatca atgacaccct gtatagcaca cggaaggacg ataagggcaa taccctgatc
     3181 gtgaacaatc tgaacggcct gtacgacaag gataatgaca agctgaagaa gctgatcaac
     3241 aagtctcccg agaagctgct gatgtaccac cacgatcctc agacatatca gaagctgaag
     3301 ctgatcatgg agcagtacgg cgacgagaag aacccactgt ataagtacta tgaggagaca
     3361 ggcaactacc tgacaaagta tagcaagaag gataatggcc ccgtgatcaa gaagatcaag
     3421 tactatggca acaagctgaa tgcccacctg gacatcaccg acgattaccc taactctcgc
     3481 aataaggtgg tgaagctgag cctgaagcca taccggttcg acgtgtacct ggacaacggc
     3541 gtgtataagt ttgtgacagt gaagaatctg gatgtgatca agaaggagaa ctactatgag
     3601 gtgaacagca agtgctacga ggaggccaag aagctgaaga agatcagcaa ccaggccgag
     3661 ttcatcgcct ctttttacaa caatgacctg atcaagatca atggcgagct gtatagagtg
     3721 atcggcgtga acaatgatct gctgaacaga atcgaagtga atatgatcga catcacctac
     3781 agggagtatc tggagaacat gaatgataag aggccccctc gcatcatcaa gaccatcgcc
     3841 tctaagacac agagcatcaa gaagtacagc acagacatcc tggggaacct gtatgaagtc
     3901 aagagcaaga aacatcctca gattatcaag aaaggctctg gaggatctag cggaggatcc
     3961 tctggcagcg agacaccagg aacaagcgag tcagcaacac cagagagcag tggcggcagc
     4021 agcggcggca gcagcaccct aaatatagaa gatgagtatc ggctacatga gacctcaaaa
     4081 gagccagatg tttctctagg gtccacatgg ctgtctgatt ttcctcaggc ctgggcggaa
     4141 accgggggca tgggactggc agttcgccaa gctcctctga tcatacctct gaaagcaacc
     4201 tctacccccg tgtccataaa acaatacccc atgtcacaag aagccagact ggggatcaag
     4261 ccccacatac agagactgtt ggaccaggga atactggtac cctgccagtc cccctggaac
     4321 acgcccctgc tacccgttaa gaaaccaggg actaatgatt ataggcctgt ccaggatctg
     4381 agagaagtca acaagcgggt ggaagacatc caccccaccg tgcccaaccc ttacaacctc
     4441 ttgagcgggc tcccaccgtc ccaccagtgg tacactgtgc ttgatttaaa ggatgccttt
     4501 ttctgcctga gactccaccc caccagtcag cctctcttcg cctttgagtg gagagatcca
     4561 gagatgggaa tctcaggaca attgacctgg accagactcc cacagggttt caaaaacagt
     4621 cccaccctgt ttaatgaggc actgcacaga gacctagcag acttccggat ccagcaccca
     4681 gacttgatcc tgctacagta cgtggatgac ttactgctgg ccgccacttc tgagctagac
     4741 tgccaacaag gtactcgggc cctgttacaa accctaggga acctcgggta tcgggcctcg
     4801 gccaagaaag cccaaatttg ccagaaacag gtcaagtatc tggggtatct tctaaaagag
     4861 ggtcagagat ggctgactga ggccagaaaa gagactgtga tggggcagcc tactccgaag
     4921 acccctcgac aactaaggga gttcctaggg aaggcaggct tctgtcgcct cttcatccct
     4981 gggtttgcag aaatggcagc ccccctgtac cctctcacca aaccggggac tctgtttaat
     5041 tggggcccag accaacaaaa ggcctatcaa gaaatcaagc aagctcttct aactgcccca
     5101 gccctggggt tgccagattt gactaagccc tttgaactct ttgtcgacga gaagcagggc
     5161 tacgccaaag gtgtcctaac gcaaaaactg ggaccttggc gtcggccggt ggcctacctg
     5221 tccaaaaagc tagacccagt agcagctggg tggccccctt gcctacggat ggtagcagcc
     5281 attgccgtac tgacaaagga tgcaggcaag ctaaccatgg gacagccact agtcattctg
     5341 gccccccatg cagtagaggc actagtcaaa caaccccccg accgctggct ttccaacgcc
     5401 cggatgactc actatcaggc cttgcttttg gacacggacc gggtccagtt cggaccggtg
     5461 gtagccctga acccggctac gctgctccca ctgcctgagg aagggctgca acacaactgc
     5521 cttgatatcc tggccgaagc ccacggaacc cgacccgacc taacggacca gccgctccca
     5581 gacgccgacc acacctggta cacggatgga agcagtctct tacaagaggg acagcgtaag
     5641 gcgggagctg cggtgaccac cgagaccgag gtaatctggg ctaaagccct gccagccggg
     5701 acatccgctc agcgggctga actgatagca ctcacccagg ccctaaagat ggcagaaggt
     5761 aagaagctaa atgtttatac tgatagccgt tatgcttttg ctactgccca tatccatgga
     5821 gaaatataca gaaggcgtgg gtggctcaca tcagaaggca aagagatcaa aaataaagac
     5881 gagatcttgg ccctactaaa agccctcttt ctgcccaaaa gacttagcat aatccattgt
     5941 ccaggacatc aaaagggaca cagcgccgag gctagaggca accggatggc tgaccaagcg
     6001 gcccgaaagg cagccatcac agagactcca gacacctcta ccctcctcat agaaaattca
     6061 tcaccctctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag
     6121 aggaaagtct aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct
     6181 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     6241 ccctggaagg tgccactccc actgtccttt cctaataaaa tgagaaaatt gcatcgcatt
     6301 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     6361 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     6421 aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt
     6481 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     6541 gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt
     6601 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6661 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6721 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6781 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6841 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6901 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6961 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     7021 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     7081 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     7141 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     7201 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     7261 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     7321 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     7381 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     7441 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7501 acactcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     7561 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7621 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7681 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7741 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7801 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7861 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7921 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7981 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     8041 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     8101 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     8161 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     8221 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     8281 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     8341 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     8401 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     8461 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     8521 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     8581 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     8641 gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg
     8701 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     8761 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg a
//
