LOCUS       sequence-462528-        9205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Slu1Cas9 and puromycin resistance genes, and cloning 
            backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9205)
  TITLE     Yongming Wang Lab Plasmids 2024
REFERENCE   2  (bases 1 to 9205)
  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: pAAV-CMV-Slu1Cas9-puro
FEATURES             Location/Qualifiers
     source          1..9205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1021..1248
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1260..1269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1269..1334
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1341..1361
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5502..5549
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5565..5618
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5619..6218
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5619..5638)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6115..6135
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6246..6263)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6252..6459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6468..6608
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6468..6597
                     /label=AAV2 ITR
     rep_origin      6683..7138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6770..6789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6980..7001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7155..7174)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7274..7296
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7334..7352)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7420..7524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7525..7593,7594..8385)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCIPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7743..7762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8556..9144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9045..9064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcc cggccctggg gggggggggg gggggggggg gggggggggg ggaggggggg
      901 gggggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      961 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     1021 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     1081 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     1141 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
     1201 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
     1261 ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag
     1321 acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     1381 cagccgaaaa aacctacagc atcggcctgg acataggcac aaatagcgtg ggctggagcg
     1441 tgatcacaga cgactacaag gttcctgcca aaaaaatgaa ggtgctcggc aacacagata
     1501 agaagtacat caaaaaaaac ctgctgggcg ccctgctgtt cgactctggc gaaaccgctg
     1561 aggtgaccag actgaaaaga acagctagga gaagatacac ccgtcggaag aaccggctga
     1621 gatacctgca ggagatcttc gccaaggaga tggtgaaggt ggatgaatct tttttccaga
     1681 gactggagga gagcttcctg acagatgacg acaagacctt cgactcccac cccatctttg
     1741 gcaacaaggc cgaggaggac gcctaccacc aggagttccc caccatctac cacctgagaa
     1801 agcacctggc cgacagcagc gaaaaggccg atctcagact ggtctacctg gccctggctc
     1861 acatgatcaa gttcagaggc cacttcctga ttgaaggcga gctgaacgcc gagaacaccg
     1921 acatccagaa actgttcaat gacttcgtgg agacctatga taagatcgcc gacgagagcc
     1981 acctgagcga agtggacgcc agcagcatcc tgacaaataa gatcagcaag agcagacggc
     2041 tggaaaacct gattaagcag taccctaccg agaagaaaaa caccctcttt ggcaatctgg
     2101 tggccctggt gctgggcctg caacctaact ttaaaaccaa cttcaaactc tccgaggatg
     2161 ccaagctgca gttcagcaag gacacctacg acgaggacct ggaagagctg ctgggcaaga
     2221 ttggcgacga ctacgccgac ctgttcaccg ccgccaagaa tctgtacgac gccatcctcc
     2281 tcagtggcat cctgacagtg gacgacaact ctaccaaagc ccctctgtct gctagcatga
     2341 tcaagagata cgaggaacac cacgaggacc tggagaagct gaagaagttc atcaaggtga
     2401 acaacttcga taagtaccat gagatcttca aggacaagtc caagaacggc tacgccggat
     2461 acatagagaa tggggtgaag caggacatct tttacaagca cctgaaaagc atcatcagcg
     2521 agaaaaacgg aggccagtac ttcctagaca agatcgagag agaggatttc ctgcggaagc
     2581 agcggacgtt cgacaacggc tctatccctc accagatcca cctgcaggag atgagagcca
     2641 tcctgcggca ccagggcgaa tactaccctt tcctgaagga aaaccagagc aagatcgaga
     2701 agatcctgac gtttcggatc ccttactacg tgggccccct ggccagaaag aacagcagat
     2761 tcgcctgggc caaatatcat tctgatgagc ctatcacccc ttggaacttt gacgaagtgg
     2821 tggacaaaga aaagtccgcc gaaaagttta tcaccagaat gacactgaat gacctgtacc
     2881 tacccgaaga gaaggtcctt ccgaagcata gccatgttta tgaaacatac gccgtgtaca
     2941 acgaactcac aaagatcaag tacgtgaacg agcagggaaa ggaaagcttt ttcgacagca
     3001 acatgaagca agaaattttc gaccacgtgt tcaaggagaa ccggaaagtc accaaggaga
     3061 agctgctgaa ctacctgaac aaggaatttc ctgagtacag aatcaaggac ctgatcggcc
     3121 tggacaagga gaacaagtcc ttcaacgcct ctctgggcac ctaccacgat ctgaagaaga
     3181 tcctggacaa gtccttcctg gacgacaagg tgaacgagga agtaatcgag gacatcatca
     3241 aaaccctgac cctgttcgag gacaaggaca tgatccacga gagactgcag aagtacagcg
     3301 acatcttcac tgccaatcaa ctgaagaagc tggaacggag acactacacc ggctggggca
     3361 gactatcgta caagctgatc aatggaataa gaaataaaga gaacaacaag accatcctgg
     3421 attatctgat cgacgacgga agcgccaacc ggaacttcat gcagcttatc aacgacgata
     3481 ccctgccatt caagcaaatc atccagaaaa gccaggtggt gggagatgtg gatgacatcg
     3541 aggccgtggt gcacgatctg cccggcagcc ctgccatcaa aaaaggcatc ctgcagtctg
     3601 tgaagatcgt agacgaactg gtgaaggtga tgggcggcaa ccctgataat atcgtgatcg
     3661 agatggccag agaaaaccag accaccaata gaggtagaag ccagagccag cagagactga
     3721 agaaactgca aagcagcctg aaggagctgg gctctaacat catcaacgag gagaaaccca
     3781 gctacatcga ggacaaagtg gagaacagcc acctgcaaaa tgatcagctg ttcctgtact
     3841 acatccagaa cggcaaggat atgtacaccg gcgacgagct ggatatcgat cacctgtctg
     3901 attacgatat tgatcacatc atcccccagg ccttcatcaa ggacgacagc attgacaatc
     3961 gggtgctgac aagttctgct aagaaccgcg gcaagtccga tgacgtgcct agcctggata
     4021 tcgttcgcgc tagaaaggca gaatgggtgc ggctgtacaa gagcggattg atcagcaagc
     4081 ggaaattcga taacctcacc aaggcagaaa gaggcggcct gacagaagcc gataaagcgg
     4141 gcttcatcaa gcggcagctg gtggaaacaa gacagatcac caagcacgtg gcccagatcc
     4201 ttgatgctcg gttcaatacc gaacacgatg aaaacgacaa ggtgatccgg gacgtgaagg
     4261 tgatcaccct gaagtctaac ctggtgagcc aatttagaaa ggacttcgag ttctacaagg
     4321 tcagagagat caacgattat catcacgccc acgacgcata cctgaacgcc gtggtcggca
     4381 cagccctgct gaagaagtac cccaagctgg cccctgagtt cgtgtacggc gaatacaaga
     4441 agtacgatgt gagaaaactc atcgctaagt ccagcgacga ccactccgag atgggcaagg
     4501 ccacagctaa gtacttcttt tacagcaacc tgatgaactt cttcaagacc aaggtgaaat
     4561 acgctgacgg cagcgtgtac gagagaccta tcatcgagac caatgccgac ggcgaagtgg
     4621 tgtggaacaa gcagaaggac ttcgagatca tccgcaaggt gctgagttat cctcaggtca
     4681 acatcgtcaa gaaggttgag gagcagaccg tgggccagaa cggcggtctg ttcgataata
     4741 acatcctggc caagccagag aaggatgata agaagaaact gtttcctatc aaaaagagcc
     4801 tgtcgacaac ctcctacggc ggatatgcca gggccacgat cgcctatagc atcctgatcg
     4861 tgtccgagtt cggcaaaaac aaggagctga aactgctggg cctgcctctg ctggacaagg
     4921 tgaagtacga gaagaatccc atcgcttacc tgaacagcat cggaatcatc aacgtgaaaa
     4981 gcatcttcaa gttaccaaag tactgcctgt tcgagttcca ggagaagaac accaactacc
     5041 ggagatacat ggtctccaac caggagctga agcgagccaa ccagcttttc ctgacctgta
     5101 aagaaacaga gctggtgtat aagcacatca agagcgacaa cgataacaca cacctggtgg
     5161 gcaatgaaaa gacaatcctg tctatttgga acaagctggt gatcttcatg gaacgtacca
     5221 agctgatcga taagaaactg tttgcctcac tgaaacagct gaaggacgag gtgaatctga
     5281 taaacattct ggatatcgcc agcaagagca tcgatttcac aaaaatcagt gctgggcaga
     5341 agaagatcgt gctgaacgac tctctgtctc tgcccatcaa acggtggcag ttcaagtccg
     5401 atgagaagat gctgaagcag ctgaacgacg ccaccctgat tcaccagagc attaccggac
     5461 tgtacgagac cagaatcgat ctgagcaagc tgggagaaga gaaaaggccg gcggccacga
     5521 aaaaggccgg ccaggcaaaa aagaaaaagg aattcggcag tggagagggc agaggaagtc
     5581 tgctaacatg cggtgacgtc gaggagaatc ctggcccaat gaccgagtac aagcccacgg
     5641 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     5701 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     5761 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     5821 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     5881 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     5941 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     6001 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     6061 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     6121 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     6181 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaga attctaacta gagctcgctg
     6241 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     6301 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     6361 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     6421 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     6481 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     6541 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     6601 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6661 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6721 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     6781 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6841 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     6901 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     6961 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     7021 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     7081 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     7141 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     7201 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     7261 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     7321 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     7381 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     7441 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     7501 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     7561 ttttttgcgg cattttgcat tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     7621 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     7681 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     7741 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     7801 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     7861 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     7921 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     7981 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     8041 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     8101 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     8161 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     8221 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     8281 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     8341 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     8401 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     8461 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     8521 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     8581 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     8641 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     8701 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     8761 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     8821 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     8881 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     8941 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     9001 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     9061 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     9121 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     9181 ttttgctggc cttttgctca catgt
//
