LOCUS       sequence-201953-        6775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone vector for cloning in target sgRNA for use with SaCas9 
            (SauCas9).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6775)
  TITLE     Alex Hewitt Lab plasmids
REFERENCE   2  (bases 1 to 6775)
  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"
COMMENT     Sequence Label: pX601 miniCMV-SaCas9-SpA-sgRNA scaffold
FEATURES             Location/Qualifiers
     source          1..6775
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     143..162
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        143..161
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        180..218
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     215..239
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      265..274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             277..297
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             322..3477
                     /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             3478..3525
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3532..3612
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     promoter        3676..3924
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3676..3696
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3847..3866
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3943..4018
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4038..4178
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4038..4167
                     /label=AAV2 ITR
     rep_origin      4253..4708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4340..4359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4550..4571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4725..4744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4844..4866
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4904..4922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4990..5094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5095..5163,5164..5955)
                     /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(5313..5332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6126..6714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6615..6634
                     /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 gataatacga ctcactatag ggggatccac gtatgtcgag
      181 gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg
      241 cctggaggta ccgccaccac cggtgccacc atggccccaa agaagaagcg gaaggtcggt
      301 atccacggag tcccagcagc caagcggaac tacatcctgg gcctggacat cggcatcacc
      361 agcgtgggct acggcatcat cgactacgag acacgggacg tgatcgatgc cggcgtgcgg
      421 ctgttcaaag aggccaacgt ggaaaacaac gagggcaggc ggagcaagag aggcgccaga
      481 aggctgaagc ggcggaggcg gcatagaatc cagagagtga agaagctgct gttcgactac
      541 aacctgctga ccgaccacag cgagctgagc ggcatcaacc cctacgaggc cagagtgaag
      601 ggcctgagcc agaagctgag cgaggaagag ttctctgccg ccctgctgca cctggccaag
      661 agaagaggcg tgcacaacgt gaacgaggtg gaagaggaca ccggcaacga gctgtccacc
      721 aaagagcaga tcagccggaa cagcaaggcc ctggaagaga aatacgtggc cgaactgcag
      781 ctggaacggc tgaagaaaga cggcgaagtg cggggcagca tcaacagatt caagaccagc
      841 gactacgtga aagaagccaa acagctgctg aaggtgcaga aggcctacca ccagctggac
      901 cagagcttca tcgacaccta catcgacctg ctggaaaccc ggcggaccta ctatgaggga
      961 cctggcgagg gcagcccctt cggctggaag gacatcaaag aatggtacga gatgctgatg
     1021 ggccactgca cctacttccc cgaggaactg cggagcgtga agtacgccta caacgccgac
     1081 ctgtacaacg ccctgaacga cctgaacaat ctcgtgatca ccagggacga gaacgagaag
     1141 ctggaatatt acgagaagtt ccagatcatc gagaacgtgt tcaagcagaa gaagaagccc
     1201 accctgaagc agatcgccaa agaaatcctc gtgaacgaag aggatattaa gggctacaga
     1261 gtgaccagca ccggcaagcc cgagttcacc aacctgaagg tgtaccacga catcaaggac
     1321 attaccgccc ggaaagagat tattgagaac gccgagctgc tggatcagat tgccaagatc
     1381 ctgaccatct accagagcag cgaggacatc caggaagaac tgaccaatct gaactccgag
     1441 ctgacccagg aagagatcga gcagatctct aatctgaagg gctataccgg cacccacaac
     1501 ctgagcctga aggccatcaa cctgatcctg gacgagctgt ggcacaccaa cgacaaccag
     1561 atcgctatct tcaaccggct gaagctggtg cccaagaagg tggacctgtc ccagcagaaa
     1621 gagatcccca ccaccctggt ggacgacttc atcctgagcc ccgtcgtgaa gagaagcttc
     1681 atccagagca tcaaagtgat caacgccatc atcaagaagt acggcctgcc caacgacatc
     1741 attatcgagc tggcccgcga gaagaactcc aaggacgccc agaaaatgat caacgagatg
     1801 cagaagcgga accggcagac caacgagcgg atcgaggaaa tcatccggac caccggcaaa
     1861 gagaacgcca agtacctgat cgagaagatc aagctgcacg acatgcagga aggcaagtgc
     1921 ctgtacagcc tggaagccat ccctctggaa gatctgctga acaacccctt caactatgag
     1981 gtggaccaca tcatccccag aagcgtgtcc ttcgacaaca gcttcaacaa caaggtgctc
     2041 gtgaagcagg aagaaaacag caagaagggc aaccggaccc cattccagta cctgagcagc
     2101 agcgacagca agatcagcta cgaaaccttc aagaagcaca tcctgaatct ggccaagggc
     2161 aagggcagaa tcagcaagac caagaaagag tatctgctgg aagaacggga catcaacagg
     2221 ttctccgtgc agaaagactt catcaaccgg aacctggtgg ataccagata cgccaccaga
     2281 ggcctgatga acctgctgcg gagctacttc agagtgaaca acctggacgt gaaagtgaag
     2341 tccatcaatg gcggcttcac cagctttctg cggcggaagt ggaagtttaa gaaagagcgg
     2401 aacaaggggt acaagcacca cgccgaggac gccctgatca ttgccaacgc cgatttcatc
     2461 ttcaaagagt ggaagaaact ggacaaggcc aaaaaagtga tggaaaacca gatgttcgag
     2521 gaaaagcagg ccgagagcat gcccgagatc gaaaccgagc aggagtacaa agagatcttc
     2581 atcacccccc accagatcaa gcacattaag gacttcaagg actacaagta cagccaccgg
     2641 gtggacaaga agcctaatag agagctgatt aacgacaccc tgtactccac ccggaaggac
     2701 gacaagggca acaccctgat cgtgaacaat ctgaacggcc tgtacgacaa ggacaatgac
     2761 aagctgaaaa agctgatcaa caagagcccc gaaaagctgc tgatgtacca ccacgacccc
     2821 cagacctacc agaaactgaa gctgattatg gaacagtacg gcgacgagaa gaatcccctg
     2881 tacaagtact acgaggaaac cgggaactac ctgaccaagt actccaaaaa ggacaacggc
     2941 cccgtgatca agaagattaa gtattacggc aacaaactga acgcccatct ggacatcacc
     3001 gacgactacc ccaacagcag aaacaaggtc gtgaagctgt ccctgaagcc ctacagattc
     3061 gacgtgtacc tggacaatgg cgtgtacaag ttcgtgaccg tgaagaatct ggatgtgatc
     3121 aaaaaagaaa actactacga agtgaatagc aagtgctatg aggaagctaa gaagctgaag
     3181 aagatcagca accaggccga gtttatcgcc tccttctaca acaacgatct gatcaagatc
     3241 aacggcgagc tgtatagagt gatcggcgtg aacaacgacc tgctgaaccg gatcgaagtg
     3301 aacatgatcg acatcaccta ccgcgagtac ctggaaaaca tgaacgacaa gaggcccccc
     3361 aggatcatta agacaatcgc ctccaagacc cagagcatta agaagtacag cacagacatt
     3421 ctgggcaacc tgtatgaagt gaaatctaag aagcaccctc agatcatcaa aaagggcaaa
     3481 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ctacccatac
     3541 gatgttccag attacgctta cccatacgat gttccagatt acgcttaccc atacgatgtt
     3601 ccagattacg cttaagaatt caataaaaga tctttatttt cattagatct gtgtgttggt
     3661 tttttgtgta cgcgtgaggg cctatttccc atgattcctt catatttgca tatacgatac
     3721 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     3781 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     3841 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     3901 atcttgtgga aaggacgaaa caccggaaga gcgagctctt ctgttttagt actctggaaa
     3961 cagaatctac taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt
     4021 tttgggcccg cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4081 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4141 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4201 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4261 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4321 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4381 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4441 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4501 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4561 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     4621 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4681 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4741 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4801 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4861 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4921 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4981 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5041 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5101 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5161 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5221 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5281 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5341 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5401 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5461 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5521 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5581 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5641 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5701 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5761 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     5821 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5881 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5941 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6001 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6061 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6121 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6181 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6241 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6301 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6361 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6421 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6481 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6541 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6601 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6661 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6721 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
