LOCUS       sequence-208586-        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"
     primer_bind     136..155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        136..154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        173..211
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     208..232
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      258..267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             270..290
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             315..3470
                     /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             3471..3518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3525..3605
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     promoter        3669..3917
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3669..3689
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3840..3859
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3940..4015
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4035..4175
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4035..4164
                     /label=AAV2 ITR
     rep_origin      4250..4705
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4337..4356)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4547..4568
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4722..4741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4841..4863
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4901..4919)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4987..5091
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5092..5160,5161..5952)
                     /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(5310..5329)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6123..6711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6612..6631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6773..6779,1..123)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
       61 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
      121 cctgcggcct ctagataata cgactcacta tagggggatc cacgtatgtc gaggtaggcg
      181 tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
      241 gtaccgccac caccggtgcc accatggccc caaagaagaa gcggaaggtc ggtatccacg
      301 gagtcccagc agccaagcgg aactacatcc tgggcctgga catcggcatc accagcgtgg
      361 gctacggcat catcgactac gagacacggg acgtgatcga tgccggcgtg cggctgttca
      421 aagaggccaa cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc agaaggctga
      481 agcggcggag gcggcataga atccagagag tgaagaagct gctgttcgac tacaacctgc
      541 tgaccgacca cagcgagctg agcggcatca acccctacga ggccagagtg aagggcctga
      601 gccagaagct gagcgaggaa gagttctctg ccgccctgct gcacctggcc aagagaagag
      661 gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc accaaagagc
      721 agatcagccg gaacagcaag gccctggaag agaaatacgt ggccgaactg cagctggaac
      781 ggctgaagaa agacggcgaa gtgcggggca gcatcaacag attcaagacc agcgactacg
      841 tgaaagaagc caaacagctg ctgaaggtgc agaaggccta ccaccagctg gaccagagct
      901 tcatcgacac ctacatcgac ctgctggaaa cccggcggac ctactatgag ggacctggcg
      961 agggcagccc cttcggctgg aaggacatca aagaatggta cgagatgctg atgggccact
     1021 gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc gacctgtaca
     1081 acgccctgaa cgacctgaac aatctcgtga tcaccaggga cgagaacgag aagctggaat
     1141 attacgagaa gttccagatc atcgagaacg tgttcaagca gaagaagaag cccaccctga
     1201 agcagatcgc caaagaaatc ctcgtgaacg aagaggatat taagggctac agagtgacca
     1261 gcaccggcaa gcccgagttc accaacctga aggtgtacca cgacatcaag gacattaccg
     1321 cccggaaaga gattattgag aacgccgagc tgctggatca gattgccaag atcctgacca
     1381 tctaccagag cagcgaggac atccaggaag aactgaccaa tctgaactcc gagctgaccc
     1441 aggaagagat cgagcagatc tctaatctga agggctatac cggcacccac aacctgagcc
     1501 tgaaggccat caacctgatc ctggacgagc tgtggcacac caacgacaac cagatcgcta
     1561 tcttcaaccg gctgaagctg gtgcccaaga aggtggacct gtcccagcag aaagagatcc
     1621 ccaccaccct ggtggacgac ttcatcctga gccccgtcgt gaagagaagc ttcatccaga
     1681 gcatcaaagt gatcaacgcc atcatcaaga agtacggcct gcccaacgac atcattatcg
     1741 agctggcccg cgagaagaac tccaaggacg cccagaaaat gatcaacgag atgcagaagc
     1801 ggaaccggca gaccaacgag cggatcgagg aaatcatccg gaccaccggc aaagagaacg
     1861 ccaagtacct gatcgagaag atcaagctgc acgacatgca ggaaggcaag tgcctgtaca
     1921 gcctggaagc catccctctg gaagatctgc tgaacaaccc cttcaactat gaggtggacc
     1981 acatcatccc cagaagcgtg tccttcgaca acagcttcaa caacaaggtg ctcgtgaagc
     2041 aggaagaaaa cagcaagaag ggcaaccgga ccccattcca gtacctgagc agcagcgaca
     2101 gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa tctggccaag ggcaagggca
     2161 gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg ggacatcaac aggttctccg
     2221 tgcagaaaga cttcatcaac cggaacctgg tggataccag atacgccacc agaggcctga
     2281 tgaacctgct gcggagctac ttcagagtga acaacctgga cgtgaaagtg aagtccatca
     2341 atggcggctt caccagcttt ctgcggcgga agtggaagtt taagaaagag cggaacaagg
     2401 ggtacaagca ccacgccgag gacgccctga tcattgccaa cgccgatttc atcttcaaag
     2461 agtggaagaa actggacaag gccaaaaaag tgatggaaaa ccagatgttc gaggaaaagc
     2521 aggccgagag catgcccgag atcgaaaccg agcaggagta caaagagatc ttcatcaccc
     2581 cccaccagat caagcacatt aaggacttca aggactacaa gtacagccac cgggtggaca
     2641 agaagcctaa tagagagctg attaacgaca ccctgtactc cacccggaag gacgacaagg
     2701 gcaacaccct gatcgtgaac aatctgaacg gcctgtacga caaggacaat gacaagctga
     2761 aaaagctgat caacaagagc cccgaaaagc tgctgatgta ccaccacgac ccccagacct
     2821 accagaaact gaagctgatt atggaacagt acggcgacga gaagaatccc ctgtacaagt
     2881 actacgagga aaccgggaac tacctgacca agtactccaa aaaggacaac ggccccgtga
     2941 tcaagaagat taagtattac ggcaacaaac tgaacgccca tctggacatc accgacgact
     3001 accccaacag cagaaacaag gtcgtgaagc tgtccctgaa gccctacaga ttcgacgtgt
     3061 acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa tctggatgtg atcaaaaaag
     3121 aaaactacta cgaagtgaat agcaagtgct atgaggaagc taagaagctg aagaagatca
     3181 gcaaccaggc cgagtttatc gcctccttct acaacaacga tctgatcaag atcaacggcg
     3241 agctgtatag agtgatcggc gtgaacaacg acctgctgaa ccggatcgaa gtgaacatga
     3301 tcgacatcac ctaccgcgag tacctggaaa acatgaacga caagaggccc cccaggatca
     3361 ttaagacaat cgcctccaag acccagagca ttaagaagta cagcacagac attctgggca
     3421 acctgtatga agtgaaatct aagaagcacc ctcagatcat caaaaagggc aaaaggccgg
     3481 cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atcctaccca tacgatgttc
     3541 cagattacgc ttacccatac gatgttccag attacgctta cccatacgat gttccagatt
     3601 acgcttaaga attcaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt
     3661 gtacgcgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     3721 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     3781 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     3841 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     3901 ggaaaggacg aaacaccgta cgtcgccgtc cagctcgacg ttttagtact ctggaaacag
     3961 aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt
     4021 gggcccgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4081 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4141 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4201 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4261 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4321 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4381 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4441 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4501 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4561 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4621 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4681 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4741 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4801 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4861 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4921 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4981 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5041 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5101 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5161 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5221 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5281 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5341 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5401 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5461 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5521 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5581 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5641 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5701 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5761 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     5821 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5881 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5941 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6001 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6061 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6121 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6181 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6241 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6301 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6361 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6421 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6481 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6541 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6601 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6661 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6721 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcag
//
