LOCUS       sequence-244574-        7977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expressing SaCas9 mRNA with one copy of PP7 aptamer and 2 copies
            of HBB 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7977)
  AUTHORS   Lu B, Javidi-Parsijani P, Makani V, Mehraein-Ghomi F, Sarhan WM, Sun
            D, Yoo KW, Atala ZP, Lyu P, Atala A
  TITLE     Delivering SaCas9 mRNA by lentivirus-like bionanoparticles for 
            transient expression and efficient genome editing.
  JOURNAL   Nucleic Acids Res. 2019 Feb 13. pii: 5316732. doi: 
            10.1093/nar/gkz093.
  PUBMED    30759231
REFERENCE   2  (bases 1 to 7977)
  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: "Nucleic 
            Acids Res. 2019 Feb 13. pii: 5316732. doi: 10.1093/nar/gkz093."
COMMENT     Sequence Label: pSaCas9-1xPP7-2x3'UTR
FEATURES             Location/Qualifiers
     source          1..7977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             813..3968
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3969..4016
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4359..4376)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4365..4572
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4582..4830
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4582..4602
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4753..4772
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4853..4928
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5240..5380
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5240..5369
                     /label=AAV2 ITR
     rep_origin      5455..5910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5542..5561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5752..5773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5927..5946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6046..6068
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6106..6124)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6192..6296
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6297..6365,6366..7157)
                     /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(6515..6534)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7328..7916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7817..7836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca
      841 tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg
      901 ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga
      961 gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc
     1021 tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg
     1081 ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc
     1141 acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg
     1201 agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg
     1261 ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat
     1321 tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc
     1381 accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct
     1441 actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg
     1501 agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct
     1561 acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg
     1621 agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga
     1681 agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta
     1741 agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg
     1801 acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga
     1861 ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc
     1921 tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg
     1981 gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca
     2041 acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt
     2101 cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga
     2161 agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc
     2221 ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga
     2281 tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga
     2341 ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg
     2401 aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct
     2461 tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca
     2521 acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt
     2581 acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc
     2641 tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg
     2701 acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat
     2761 acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg
     2821 tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta
     2881 agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg
     2941 ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc
     3001 agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca
     3061 aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt
     3121 acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca
     3181 cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca
     3241 aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc
     3301 accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga
     3361 agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa
     3421 aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc
     3481 tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc
     3541 cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc
     3601 tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta
     3661 agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc
     3721 tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc
     3781 ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca
     3841 agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca
     3901 gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca
     3961 aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat
     4021 cctaagctcg ctttcttgct gtccaatttc tattaaaggt tcctttgttc cctaagtcca
     4081 actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc ctaataaaaa
     4141 acatttattt tcattgctag ctcgctttct tgctgtccaa tttctattaa aggttccttt
     4201 gttccctaag tccaactact aaactggggg atattatgaa gggccttgag catctggatt
     4261 ctgcctaata aaaaacattt attttcattg cgaattcgga gcagacgata tggcgtcgct
     4321 ccgtcgactc tagaaaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     4381 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4441 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4501 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     4561 gcatgctggg gaggtacggc cgagggccta tttcccatga ttccttcata tttgcatata
     4621 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
     4681 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     4741 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     4801 ttatatatct tgtggaaagg acgaaacacc gagtaacggc agacttctcc acgttctagt
     4861 actctggaaa cagaatctac tagaacaagg caaaatgccg tgtttatctc gtcaacttgt
     4921 tggcgagata tcggagcaga cgatatggcg tcgctccagc gctcgctttc ttgctgtcca
     4981 atttctatta aaggttcctt tgttccctaa gtccaactac taaactgggg gatattatga
     5041 agggccttga gcatctggat tctgcctaat aaaaaacatt tattttcatt gcgctcgctt
     5101 tcttgctgtc caatttctat taaaggttcc tttgttccct aagtccaact actaaactgg
     5161 gggatattat gaagggcctt gagcatctgg attctgccta ataaaaaaca tttattttca
     5221 ttgctttttt tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     5281 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     5341 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     5401 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     5461 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5521 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5581 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5641 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     5701 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5761 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     5821 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5881 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5941 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6001 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6061 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     6121 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     6181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6241 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6301 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6361 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6421 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6481 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6541 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6601 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6661 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6721 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6781 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6841 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6901 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6961 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg
     7021 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7081 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7141 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7201 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7261 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7441 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7861 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7921 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
