LOCUS       sequence-208585-        6779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one vectors expressing both SaCas9 and LacZ sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6779)
  TITLE     Alex Hewitt Lab plasmids
REFERENCE   2  (bases 1 to 6779)
  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-U6-LacZvsSaCas9 sgRNA
FEATURES             Location/Qualifiers
     source          1..6779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     144..163
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        144..162
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        181..219
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     216..240
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      266..275
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             278..298
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             323..3478
                     /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             3479..3526
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3533..3613
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     promoter        3677..3925
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3677..3697
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3848..3867
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3948..4023
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4043..4183
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4043..4172
                     /label=AAV2 ITR
     rep_origin      4258..4713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4345..4364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4555..4576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4730..4749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4849..4871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4909..4927)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4995..5099
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5100..5168,5169..5960)
                     /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(5318..5337)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6131..6719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6620..6639
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggcctct agataatacg actcactata gggggatcca cgtatgtcga
      181 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      241 gcctggaggt accgccacca ccggtgccac catggcccca aagaagaagc ggaaggtcgg
      301 tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca tcggcatcac
      361 cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg ccggcgtgcg
      421 gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga gaggcgccag
      481 aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc tgttcgacta
      541 caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg ccagagtgaa
      601 gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc acctggccaa
      661 gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg agctgtccac
      721 caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg ccgaactgca
      781 gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat tcaagaccag
      841 cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc accagctgga
      901 ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct actatgaggg
      961 acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg agatgctgat
     1021 gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct acaacgccga
     1081 cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg agaacgagaa
     1141 gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga agaagaagcc
     1201 caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta agggctacag
     1261 agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg acatcaagga
     1321 cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga ttgccaagat
     1381 cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc tgaactccga
     1441 gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg gcacccacaa
     1501 cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca acgacaacca
     1561 gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt cccagcagaa
     1621 agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga agagaagctt
     1681 catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc ccaacgacat
     1741 cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga tcaacgagat
     1801 gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga ccaccggcaa
     1861 agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg aaggcaagtg
     1921 cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct tcaactatga
     1981 ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca acaaggtgct
     2041 cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt acctgagcag
     2101 cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc tggccaaggg
     2161 caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg acatcaacag
     2221 gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat acgccaccag
     2281 aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg tgaaagtgaa
     2341 gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta agaaagagcg
     2401 gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg ccgatttcat
     2461 cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc agatgttcga
     2521 ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca aagagatctt
     2581 catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt acagccaccg
     2641 ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca cccggaagga
     2701 cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca aggacaatga
     2761 caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc accacgaccc
     2821 ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga agaatcccct
     2881 gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa aggacaacgg
     2941 ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc tggacatcac
     3001 cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc cctacagatt
     3061 cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc tggatgtgat
     3121 caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta agaagctgaa
     3181 gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc tgatcaagat
     3241 caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc ggatcgaagt
     3301 gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca agaggccccc
     3361 caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca gcacagacat
     3421 tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca aaaagggcaa
     3481 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat cctacccata
     3541 cgatgttcca gattacgctt acccatacga tgttccagat tacgcttacc catacgatgt
     3601 tccagattac gcttaagaat tcaataaaag atctttattt tcattagatc tgtgtgttgg
     3661 ttttttgtgt acgcgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     3721 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     3781 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     3841 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     3901 tatcttgtgg aaaggacgaa acaccgcttt gcgaatacgc ccacgcggtt ttagtactct
     3961 ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg
     4021 agatttttgg gcccgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4081 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4141 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4201 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4261 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4321 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4381 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4441 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4501 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4561 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4621 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4681 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4741 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4801 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4861 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4921 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4981 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5041 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5101 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5161 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5221 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5281 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5341 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5401 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5461 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5521 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5581 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5641 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5701 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5761 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     5821 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5881 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5941 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6001 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6061 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6121 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6181 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6241 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6301 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6361 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6421 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6481 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6541 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6601 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6661 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6721 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatg
//
