LOCUS       sequence-208591-        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   5..134
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     147..166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        147..165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        184..222
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     219..243
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      269..278
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             281..301
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             326..3481
                     /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             3482..3529
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3536..3616
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     promoter        3680..3928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3680..3700
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3851..3870
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3947..4022
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4042..4182
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4042..4171
                     /label=AAV2 ITR
     rep_origin      4257..4712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4344..4363)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4554..4575
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4729..4748)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4848..4870
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4908..4926)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4994..5098
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5099..5167,5168..5959)
                     /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(5317..5336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6130..6718
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6619..6638
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
       61 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      121 cactaggggt tcctgcggcc tctagataat acgactcact atagggggat ccacgtatgt
      181 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      241 atcgcctgga ggtaccgcca ccaccggtgc caccatggcc ccaaagaaga agcggaaggt
      301 cggtatccac ggagtcccag cagccaagcg gaactacatc ctgggcctgg acatcggcat
      361 caccagcgtg ggctacggca tcatcgacta cgagacacgg gacgtgatcg atgccggcgt
      421 gcggctgttc aaagaggcca acgtggaaaa caacgagggc aggcggagca agagaggcgc
      481 cagaaggctg aagcggcgga ggcggcatag aatccagaga gtgaagaagc tgctgttcga
      541 ctacaacctg ctgaccgacc acagcgagct gagcggcatc aacccctacg aggccagagt
      601 gaagggcctg agccagaagc tgagcgagga agagttctct gccgccctgc tgcacctggc
      661 caagagaaga ggcgtgcaca acgtgaacga ggtggaagag gacaccggca acgagctgtc
      721 caccaaagag cagatcagcc ggaacagcaa ggccctggaa gagaaatacg tggccgaact
      781 gcagctggaa cggctgaaga aagacggcga agtgcggggc agcatcaaca gattcaagac
      841 cagcgactac gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct accaccagct
      901 ggaccagagc ttcatcgaca cctacatcga cctgctggaa acccggcgga cctactatga
      961 gggacctggc gagggcagcc ccttcggctg gaaggacatc aaagaatggt acgagatgct
     1021 gatgggccac tgcacctact tccccgagga actgcggagc gtgaagtacg cctacaacgc
     1081 cgacctgtac aacgccctga acgacctgaa caatctcgtg atcaccaggg acgagaacga
     1141 gaagctggaa tattacgaga agttccagat catcgagaac gtgttcaagc agaagaagaa
     1201 gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac gaagaggata ttaagggcta
     1261 cagagtgacc agcaccggca agcccgagtt caccaacctg aaggtgtacc acgacatcaa
     1321 ggacattacc gcccggaaag agattattga gaacgccgag ctgctggatc agattgccaa
     1381 gatcctgacc atctaccaga gcagcgagga catccaggaa gaactgacca atctgaactc
     1441 cgagctgacc caggaagaga tcgagcagat ctctaatctg aagggctata ccggcaccca
     1501 caacctgagc ctgaaggcca tcaacctgat cctggacgag ctgtggcaca ccaacgacaa
     1561 ccagatcgct atcttcaacc ggctgaagct ggtgcccaag aaggtggacc tgtcccagca
     1621 gaaagagatc cccaccaccc tggtggacga cttcatcctg agccccgtcg tgaagagaag
     1681 cttcatccag agcatcaaag tgatcaacgc catcatcaag aagtacggcc tgcccaacga
     1741 catcattatc gagctggccc gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga
     1801 gatgcagaag cggaaccggc agaccaacga gcggatcgag gaaatcatcc ggaccaccgg
     1861 caaagagaac gccaagtacc tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa
     1921 gtgcctgtac agcctggaag ccatccctct ggaagatctg ctgaacaacc ccttcaacta
     1981 tgaggtggac cacatcatcc ccagaagcgt gtccttcgac aacagcttca acaacaaggt
     2041 gctcgtgaag caggaagaaa acagcaagaa gggcaaccgg accccattcc agtacctgag
     2101 cagcagcgac agcaagatca gctacgaaac cttcaagaag cacatcctga atctggccaa
     2161 gggcaagggc agaatcagca agaccaagaa agagtatctg ctggaagaac gggacatcaa
     2221 caggttctcc gtgcagaaag acttcatcaa ccggaacctg gtggatacca gatacgccac
     2281 cagaggcctg atgaacctgc tgcggagcta cttcagagtg aacaacctgg acgtgaaagt
     2341 gaagtccatc aatggcggct tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga
     2401 gcggaacaag gggtacaagc accacgccga ggacgccctg atcattgcca acgccgattt
     2461 catcttcaaa gagtggaaga aactggacaa ggccaaaaaa gtgatggaaa accagatgtt
     2521 cgaggaaaag caggccgaga gcatgcccga gatcgaaacc gagcaggagt acaaagagat
     2581 cttcatcacc ccccaccaga tcaagcacat taaggacttc aaggactaca agtacagcca
     2641 ccgggtggac aagaagccta atagagagct gattaacgac accctgtact ccacccggaa
     2701 ggacgacaag ggcaacaccc tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa
     2761 tgacaagctg aaaaagctga tcaacaagag ccccgaaaag ctgctgatgt accaccacga
     2821 cccccagacc taccagaaac tgaagctgat tatggaacag tacggcgacg agaagaatcc
     2881 cctgtacaag tactacgagg aaaccgggaa ctacctgacc aagtactcca aaaaggacaa
     2941 cggccccgtg atcaagaaga ttaagtatta cggcaacaaa ctgaacgccc atctggacat
     3001 caccgacgac taccccaaca gcagaaacaa ggtcgtgaag ctgtccctga agccctacag
     3061 attcgacgtg tacctggaca atggcgtgta caagttcgtg accgtgaaga atctggatgt
     3121 gatcaaaaaa gaaaactact acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct
     3181 gaagaagatc agcaaccagg ccgagtttat cgcctccttc tacaacaacg atctgatcaa
     3241 gatcaacggc gagctgtata gagtgatcgg cgtgaacaac gacctgctga accggatcga
     3301 agtgaacatg atcgacatca cctaccgcga gtacctggaa aacatgaacg acaagaggcc
     3361 ccccaggatc attaagacaa tcgcctccaa gacccagagc attaagaagt acagcacaga
     3421 cattctgggc aacctgtatg aagtgaaatc taagaagcac cctcagatca tcaaaaaggg
     3481 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc
     3541 atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt acccatacga
     3601 tgttccagat tacgcttaag aattcaataa aagatcttta ttttcattag atctgtgtgt
     3661 tggttttttg tgtacgcgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     3721 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     3781 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     3841 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     3901 atatatcttg tggaaaggac gaaacaccgg aagagcgagc tcttctgttt tagtactctg
     3961 gaaacagaat ctactaaaac aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga
     4021 gatttttggg cccgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4081 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4141 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4201 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4261 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4321 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4381 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4441 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4501 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4561 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4621 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4681 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4741 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4801 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4861 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4921 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4981 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5041 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5101 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5161 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5221 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5281 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5341 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5401 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5461 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5521 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5581 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5641 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5701 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5761 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     5821 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5881 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5941 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6001 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6061 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6121 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6181 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6241 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6301 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6361 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6421 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6481 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6541 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6601 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6661 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6721 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcac
//
