LOCUS       sequence-201948-        6779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one vectors expressing both SaCas9 and YFP 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-YFPvsSaCas9 sgRNA
FEATURES             Location/Qualifiers
     source          1..6779
                     /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        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 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 caccgtacgt cgccgtccag ctcgacgttt 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 gccctagcgc 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 cactcaaccc tatctcgggc tattcttttg atttataagg
     4621 gattttgccg atttcggcct 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 tactgtcctt 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 ctcacatgt
//
