LOCUS       sequence-362045-        7448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting Grp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7448)
  TITLE     Various Crispr/Cas9 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7448)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 
            pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA-Grp
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      164..619
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(251..270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     461..482
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(636..655)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     755..777
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(815..833)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        901..1005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1006..1074,1075..1866)
                     /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(1224..1243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2037..2625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2526..2545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2687..2816
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2840..3219
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3220..3423
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3374..3394
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3442..3451
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3454..3474
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3499..6654
                     /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             6655..6702
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6709..6789
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6817..6834)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6823..7030
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        7037..7285
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7037..7057
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7208..7227
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7308..7383
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   join(7397..7448,1..89)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7397..7448,1..78)
                     /label=AAV2 ITR
ORIGIN
        1 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
       61 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
      121 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
      181 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      241 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      301 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      361 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      421 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
      481 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
      541 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
      601 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
      661 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
      721 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
      781 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
      841 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
      901 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
      961 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1021 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1081 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1141 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1201 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1261 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1321 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1381 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1441 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1501 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     1561 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1621 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1681 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     1741 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1801 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1861 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1921 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1981 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2041 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2101 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2161 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2221 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2281 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2341 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2401 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2461 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2521 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2581 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2641 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg
     2701 cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc
     2761 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg
     2821 cctctagact cgaggcgttg acattgatta ttgactagtt attaatagta atcaattacg
     2881 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     2941 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     3001 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     3061 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     3121 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     3181 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     3241 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     3301 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     3361 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     3421 gctctctggc taactaccgg tgccaccatg gccccaaaga agaagcggaa ggtcggtatc
     3481 cacggagtcc cagcagccaa gcggaactac atcctgggcc tggacatcgg catcaccagc
     3541 gtgggctacg gcatcatcga ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg
     3601 ttcaaagagg ccaacgtgga aaacaacgag ggcaggcgga gcaagagagg cgccagaagg
     3661 ctgaagcggc ggaggcggca tagaatccag agagtgaaga agctgctgtt cgactacaac
     3721 ctgctgaccg accacagcga gctgagcggc atcaacccct acgaggccag agtgaagggc
     3781 ctgagccaga agctgagcga ggaagagttc tctgccgccc tgctgcacct ggccaagaga
     3841 agaggcgtgc acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa
     3901 gagcagatca gccggaacag caaggccctg gaagagaaat acgtggccga actgcagctg
     3961 gaacggctga agaaagacgg cgaagtgcgg ggcagcatca acagattcaa gaccagcgac
     4021 tacgtgaaag aagccaaaca gctgctgaag gtgcagaagg cctaccacca gctggaccag
     4081 agcttcatcg acacctacat cgacctgctg gaaacccggc ggacctacta tgagggacct
     4141 ggcgagggca gccccttcgg ctggaaggac atcaaagaat ggtacgagat gctgatgggc
     4201 cactgcacct acttccccga ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg
     4261 tacaacgccc tgaacgacct gaacaatctc gtgatcacca gggacgagaa cgagaagctg
     4321 gaatattacg agaagttcca gatcatcgag aacgtgttca agcagaagaa gaagcccacc
     4381 ctgaagcaga tcgccaaaga aatcctcgtg aacgaagagg atattaaggg ctacagagtg
     4441 accagcaccg gcaagcccga gttcaccaac ctgaaggtgt accacgacat caaggacatt
     4501 accgcccgga aagagattat tgagaacgcc gagctgctgg atcagattgc caagatcctg
     4561 accatctacc agagcagcga ggacatccag gaagaactga ccaatctgaa ctccgagctg
     4621 acccaggaag agatcgagca gatctctaat ctgaagggct ataccggcac ccacaacctg
     4681 agcctgaagg ccatcaacct gatcctggac gagctgtggc acaccaacga caaccagatc
     4741 gctatcttca accggctgaa gctggtgccc aagaaggtgg acctgtccca gcagaaagag
     4801 atccccacca ccctggtgga cgacttcatc ctgagccccg tcgtgaagag aagcttcatc
     4861 cagagcatca aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa cgacatcatt
     4921 atcgagctgg cccgcgagaa gaactccaag gacgcccaga aaatgatcaa cgagatgcag
     4981 aagcggaacc ggcagaccaa cgagcggatc gaggaaatca tccggaccac cggcaaagag
     5041 aacgccaagt acctgatcga gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg
     5101 tacagcctgg aagccatccc tctggaagat ctgctgaaca accccttcaa ctatgaggtg
     5161 gaccacatca tccccagaag cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg
     5221 aagcaggaag aaaacagcaa gaagggcaac cggaccccat tccagtacct gagcagcagc
     5281 gacagcaaga tcagctacga aaccttcaag aagcacatcc tgaatctggc caagggcaag
     5341 ggcagaatca gcaagaccaa gaaagagtat ctgctggaag aacgggacat caacaggttc
     5401 tccgtgcaga aagacttcat caaccggaac ctggtggata ccagatacgc caccagaggc
     5461 ctgatgaacc tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc
     5521 atcaatggcg gcttcaccag ctttctgcgg cggaagtgga agtttaagaa agagcggaac
     5581 aaggggtaca agcaccacgc cgaggacgcc ctgatcattg ccaacgccga tttcatcttc
     5641 aaagagtgga agaaactgga caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa
     5701 aagcaggccg agagcatgcc cgagatcgaa accgagcagg agtacaaaga gatcttcatc
     5761 accccccacc agatcaagca cattaaggac ttcaaggact acaagtacag ccaccgggtg
     5821 gacaagaagc ctaatagaga gctgattaac gacaccctgt actccacccg gaaggacgac
     5881 aagggcaaca ccctgatcgt gaacaatctg aacggcctgt acgacaagga caatgacaag
     5941 ctgaaaaagc tgatcaacaa gagccccgaa aagctgctga tgtaccacca cgacccccag
     6001 acctaccaga aactgaagct gattatggaa cagtacggcg acgagaagaa tcccctgtac
     6061 aagtactacg aggaaaccgg gaactacctg accaagtact ccaaaaagga caacggcccc
     6121 gtgatcaaga agattaagta ttacggcaac aaactgaacg cccatctgga catcaccgac
     6181 gactacccca acagcagaaa caaggtcgtg aagctgtccc tgaagcccta cagattcgac
     6241 gtgtacctgg acaatggcgt gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa
     6301 aaagaaaact actacgaagt gaatagcaag tgctatgagg aagctaagaa gctgaagaag
     6361 atcagcaacc aggccgagtt tatcgcctcc ttctacaaca acgatctgat caagatcaac
     6421 ggcgagctgt atagagtgat cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac
     6481 atgatcgaca tcacctaccg cgagtacctg gaaaacatga acgacaagag gccccccagg
     6541 atcattaaga caatcgcctc caagacccag agcattaaga agtacagcac agacattctg
     6601 ggcaacctgt atgaagtgaa atctaagaag caccctcaga tcatcaaaaa gggcaaaagg
     6661 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccta cccatacgat
     6721 gttccagatt acgcttaccc atacgatgtt ccagattacg cttacccata cgatgttcca
     6781 gattacgctt aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     6841 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6901 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6961 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
     7021 atgctgggga ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
     7081 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     7141 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     7201 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     7261 tatcttgtgg aaaggacgaa acaccgccgc gcggcagtca ctgggctgtt ttagtactct
     7321 ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg
     7381 agatttttgc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     7441 cgctcgct
//
