LOCUS       sequence-470051-        7294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector AAV-Cas9 system containing Cas9 from Staphylococcus 
            aureus (SaCas9) and its sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7294)
  AUTHORS   Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B,
            Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F
  TITLE     In vivo genome editing using Staphylococcus aureus Cas9.
  JOURNAL   Nature. 2015 Apr 1. doi: 10.1038/nature14299.
  PUBMED    25830891
REFERENCE   2  (bases 1 to 7294)
  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: "Nature. 
            2015 Apr 1. doi: 10.1038/nature14299."
COMMENT     Sequence Label: 
            pX602-AAV-TBG::NLS-SaCas9-NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA
FEATURES             Location/Qualifiers
     source          1..7294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..410
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     regulatory      429..438
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             441..461
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3642..3689
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3696..3722
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3723..3764
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     primer_bind     complement(3792..3809)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3798..4005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4017..4092)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(4113..4361)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4171..4190)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4341..4361)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4371..4511
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4371..4500
                     /label=AAV2 ITR
     rep_origin      4586..5041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4883..4904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5177..5199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5237..5255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5323..5427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5428..5496,5497..6288)
                     /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(5646..5665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6459..7047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6948..6967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7109..7238
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tgcatgtata atttctacag aacctattag aaaggatcac ccagcctctg cttttgtaca
       61 actttccctt aaaaaactgc caattccact gctgtttggc ccaatagtga gaactttttc
      121 ctgctgcctc ttggtgcttt tgcctatggc ccctattctg cctgctgaag acactcttgc
      181 cagcatggac ttaaacccct ccagctctga caatcctctt tctcttttgt tttacatgaa
      241 gggtctggca gccaaagcaa tcactcaaag ttcaaacctt atcatttttt gctttgttcc
      301 tcttggcctt ggttttgtac atcagctttg aaaataccat cccagggtta atgctggggt
      361 taatttataa ctaagagtgc tctagttttg caatacagga catgctataa aaatggaaag
      421 ataccggtgc caccatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
      481 cagccaagcg gaactacatc ctgggcctgg acatcggcat caccagcgtg ggctacggca
      541 tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca
      601 acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga
      661 ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc
      721 acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc
      781 tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca
      841 acgtgaacga ggtggaagag gacaccggca acgagctgtc caccagagag cagatcagcc
      901 ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga
      961 aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag
     1021 ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca
     1081 cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc
     1141 ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact
     1201 tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga
     1261 acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga
     1321 agttccagat catcgagaac gtgttcaagc agaagaagaa gcccaccctg aagcagatcg
     1381 ccaaagaaat cctcgtgaac gaagaggata ttaagggcta cagagtgacc agcaccggca
     1441 agcccgagtt caccaacctg aaggtgtacc acgacatcaa ggacattacc gcccggaaag
     1501 agattattga gaacgccgag ctgctggatc agattgccaa gatcctgacc atctaccaga
     1561 gcagcgagga catccaggaa gaactgacca atctgaactc cgagctgacc caggaagaga
     1621 tcgagcagat ctctaatctg aagggctata ccggcaccca caacctgagc ctgaaggcca
     1681 tcaacctgat cctggacgag ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc
     1741 ggctgaagct ggtgcccaag aaggtggacc tgtcccagca gaaagagatc cccaccaccc
     1801 tggtggacga cttcatcctg agccccgtcg tgaagagaag cttcatccag agcatcaaag
     1861 tgatcaacgc catcatcaag aagtacggcc tgcccaacga catcattatc gagctggccc
     1921 gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga gatgcagaag cggaaccggc
     1981 agaccaacga gcggatcgag gaaatcatcc ggaccaccgg caaagagaac gccaagtacc
     2041 tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac agcctggaag
     2101 ccatccctct ggaagatctg ctgaacaacc ccttcaacta tgaggtggac cacatcatcc
     2161 ccagaagcgt gtccttcgac aacagcttca acaacaaggt gctcgtgaag caggaagaaa
     2221 acagcaagaa gggcaaccgg accccattcc agtacctgag cagcagcgac agcaagatca
     2281 gctacgaaac cttcaagaag cacatcctga atctggccaa gggcaagggc agaatcagca
     2341 agaccaagaa agagtatctg ctggaagaac gggacatcaa caggttctcc gtgcagaaag
     2401 acttcatcaa ccggaacctg gtggatacca gatacgccac cagaggcctg atgaacctgc
     2461 tgcggagcta cttcagagtg aacaacctgg acgtgaaagt gaagtccatc aatggcggct
     2521 tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag gggtacaagc
     2581 accacgccga ggacgccctg atcattgcca acgccgattt catcttcaaa gagtggaaga
     2641 aactggacaa ggccaaaaaa gtgatggaaa accagatgtt cgaggaaagg caggccgaga
     2701 gcatgcccga gatcgaaacc gagcaggagt acaaagagat cttcatcacc ccccaccaga
     2761 tcaagcacat taaggacttc aaggactaca agtacagcca ccgggtggac aagaagccta
     2821 atagagagct gattaacgac accctgtact ccacccggaa ggacgacaag ggcaacaccc
     2881 tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa tgacaagctg aaaaagctga
     2941 tcaacaagag ccccgaaaag ctgctgatgt accaccacga cccccagacc taccagaaac
     3001 tgaagctgat tatggaacag tacggcgacg agaagaatcc cctgtacaag tactacgagg
     3061 aaaccgggaa ctacctgacc aagtactcca aaaaggacaa cggccccgtg atcaagaaga
     3121 ttaagtatta cggcaacaaa ctgaacgccc atctggacat caccgacgac taccccaaca
     3181 gcagaaacaa ggtcgtgaag ctgtccctga agccctacag attcgacgtg tacctggaca
     3241 atggcgtgta caagttcgtg accgtgaaga atctggatgt gatcaaaaaa gaaaactact
     3301 acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct gaagaagatc agcaaccagg
     3361 ccgagtttat cgcctccttc tacaacaacg atctgatcaa gatcaacggc gagctgtata
     3421 gagtgatcgg cgtgaacaac gacctgctga accggatcga agtgaacatg atcgacatca
     3481 cctaccgcga gtacctggaa aacatgaacg acaagaggcc ccccaggatc attaagacaa
     3541 tcgcctccaa gacccagagc attaagaagt acagcacaga cattctgggc aacctgtatg
     3601 aagtgaaatc taagaagcac cctcagatca tcaaaaaggg caaaaggccg gcggccacga
     3661 aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc atacgatgtt ccagattacg
     3721 ctagcggctt cgccaacgag cttggaccca ggttgatggg aaagtaagaa ttcctagagc
     3781 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3841 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3901 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3961 cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac caaaaatctc
     4021 gccaacaagt tgacgagata aacacggcat tttgccttgt tttagtagat tctgtttcca
     4081 gagtactaaa actgagacct gccgtggtct ccggtgtttc gtcctttcca caagatatat
     4141 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     4201 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     4261 actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac agccttgtat
     4321 cgtatatgca aatatgaagg aatcatggga aataggccct cagcggccgc aggaacccct
     4381 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4441 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4501 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4801 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4921 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5041 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5101 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5161 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5221 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5281 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6121 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     6181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7081 gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact
     7141 gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     7201 cagagaggga gtggccaact ccatcactag gggttcctgc ggcctctaga ctcgaggggc
     7261 tggaagctac ctttgacatc atttcctctg cgaa
//
