LOCUS       sequence-462529-        9123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Dpi2Cas9 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 9123)
  TITLE     Yongming Wang Lab Plasmids 2024
REFERENCE   2  (bases 1 to 9123)
  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-Dpi2Cas9-puro
FEATURES             Location/Qualifiers
     source          1..9123
                     /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          1062..1289
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1301..1310
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1310..1375
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1382..1402
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5420..5467
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5483..5536
                     /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             5537..6136
                     /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(5537..5556)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6033..6053
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6164..6181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6170..6377
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6386..6526
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6386..6515
                     /label=AAV2 ITR
     rep_origin      6601..7056
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6688..6707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6898..6919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7073..7092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7192..7214
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7252..7270)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7338..7442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7443..7511,7512..8303)
                     /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(7661..7680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8474..9062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8963..8982
                     /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 gggggggggg gcccccgggg gggggggggg gggggggggg gggggggggg gggggggggg
      901 gggggggggc gcgggggggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
      961 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
     1021 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
     1081 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
     1141 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga
     1201 gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg
     1261 agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga
     1321 ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc
     1381 cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagcccaga aaaactacac
     1441 catcggcctg gacatcggca ccaattctgt tggctgggcc gtgatgaagg acgactacac
     1501 cctgatcaga aagcggatca aagtgctggg caacaccgac atcaaaaaga tcaagaaaaa
     1561 cttctggggc gtgagactat tcgataaggg cgagacagcc aaggaaacca gactgaagag
     1621 aggcacacgg agaagatacc agcggcggag aaacagactt atctacctgc aggatttttt
     1681 tcagcagccc atgctggcca ttgacgagaa ctttttccac agactggacg actctttctt
     1741 cgtgcctgac gacaagtcct atgaccggca ccccattttt ggctcactgg aagaagaggt
     1801 ggcctaccac aacacatacc ccaccatcta ccatctgcgc aaacacctgg ccgataaccc
     1861 cgagaaggcc gacctgcgtc tggtgtacac agccctcgcc cacatcgtga agtacagagg
     1921 ccacttcctg atcgagggcg agctgaacac cgaaaatatc agcatcggcg aaacctttga
     1981 gcaatttctc gatacataca acgagatctt caagaccgag ctcacaggcg aggtggacaa
     2041 ggtggaggat atcctgaaaa gtaaggagag cagaacccag aagtacgaac aaattatcaa
     2101 gctgtttcca agcgagaaca agaccggcat gtttggccag ttcatcaagt tcatcgtggg
     2161 caataaggcc aagtttaaga ccatcttcga cctggaagag gaccaggact tccagctgcc
     2221 tgatgaaaat tacgaggaga acctgaacga gctgctggcc atgacagacg atgcgctgct
     2281 ggatgtgttc atggccgcta agaacgtgta cgacgccgtg gaaatgagcg ccatcatcag
     2341 cacagatacc ggcaacagca aggccgtgct cagcaaccag atgattaact tctacgacga
     2401 gcataaggtg gatctggccc agctgaagca gttctttaag acttacctgc ctgataagta
     2461 ctacgagtgc ttcagcgact ctagcaaaaa tggctacgct ggctatatcg acggcaaaac
     2521 aaaccaggag aatttctaca agtacatcga aaaagtgatg aaaaccgtga aaagcgatga
     2581 gaaggactat ttcctggaca agatcgaccg cgaggtgttc ctgagaaagc agagaagctt
     2641 ctacaacagc gtgatccccc accagatcca cctgcaggag atgcaggcca tcctggacca
     2701 ccagtcccaa tactacagct ttctggctga gaacagagac aagatcgaaa gcctggtgac
     2761 cttcaggatt ccatactacg tcggacctct gaccgctagc gaccaatccg agttcgcctg
     2821 gatggaaaga caaagcgacg agcctatcag accttggaac ttcgatgaaa tcgtgaacaa
     2881 ggagagatct gccgagaaat tcattgagag aatgaccaat atggatacct acctgctgga
     2941 ggaaaaggtt ctgcctaagc ggtcactgct gtaccaaaca ttcgaggtgt acaacgaact
     3001 cacaaaggtg cggtacacaa atgaacaggg caagaccgaa aaactgaacc ggcagcagaa
     3061 aaccgagatc atcgagacac tgttcaagca gaagaacaga gtgcgggaaa aggatatcgc
     3121 gaattacctg gaacagtacg gctacgttga tggcaccgac atcaagggca tcgaggacaa
     3181 gttcaacgcc tctctgagca catacaacga cctggccaag atcgacggcg ccaaggccta
     3241 cctggacgac ccagagtacg ccgacatgtg ggaggacatc atcaagatcc tgacggtgtt
     3301 cgaggacaag gccatgagaa agaaacagct gcagacctac agcgacaccc tgagccccga
     3361 gatcctgaaa aagctggagc ggaagcacta cacaggatgg ggaagattct ccaagaaact
     3421 gatcaatggc ctgagagatg aaggaagcaa caagaccatc ctggactacc tcaagtctga
     3481 tgagggctct tccgggccta caaacagaaa cttcatccag ctgatccggg acaataccct
     3541 gagcttcaag aagaagattg aggacgccag aaccattgag gacaccaccc acatctatga
     3601 taccgtggcc gatctgcccg gcagccctgc catcaaaaag gggatcagac aagccctcaa
     3661 aatcgtggaa gaaatcatcg atatcattgg atacgagccc gagaacatcg tggtcgagat
     3721 ggccagagaa agccagacca caaaaaaggg aaaagacctg agcaaggaaa gactggaaaa
     3781 gctgaccgaa gccatcaagg aattcgacgg tcctaccgat atcaaagtta aggacctgaa
     3841 gaatgaaaac ctgagaaacg acagactgta cctgtactac ctgcagaatg gcagagacat
     3901 gtacaccaac gagccactgg acatcaacaa cctgagcaag tacgacatcg accacatcat
     3961 ccctcagtcc ttcacaacag acaactctat agataacaag gtcctggtca gccggaccaa
     4021 gaaccagggc aacaagagcg atgacgtgcc cagcatcaac atcgtgcaca agatgaagcc
     4081 cttctggcgg caactgcaca aggccggatt gatcagcgag agaaagctca agaacctgac
     4141 caaggctgaa cacggcggcc tgaccgaggc cgacaaagcc catttcctga accggcaact
     4201 tgtggaaacc cggcagatca ccaaacacgt ggctaatctg ctggacagcc agtataacac
     4261 ggccgaggaa cagcggatta acatcgtcct gctgaagagc tccatgacca gcagattccg
     4321 gagagagttt gaaatgtaca aggtgagaga gatcaacgac taccaccacg cccatgatgc
     4381 ttatttgaac gctgtggtgg ccatcaccat catgaaagtc tatcctaacc tgaagcagca
     4441 gttcgtgtat ggccagtaca agaaaactag catgtttaag gaggaaaagg ccaccgcaag
     4501 aaagtacttc tacagcaata tcaccaaatt cttcaagaag gaaaaggtgg tgaacgagga
     4561 aaccggcgaa attctgtggg acacagaaaa gcacatttct atcatcaaaa gagtgctgtc
     4621 ctggaagcag atgaacatcg tgaagaaggt ggagaagcag aagggccagc tgtggaaaga
     4681 gaccatccac cctaaggggg atagcgacac cctgatccca atcaaaaagg gcatggaccc
     4741 tcagaaatac ggcggcacaa acaacatctc tgagtcttat gccgtggcca taaaacagat
     4801 cgtgggcaag aagaagaaga ttaagagcga actggtgagc atccctatcg tggatagaga
     4861 aaagtacgag cagaacccta tcaattacct ggagaagaac gggtacaacg agtctatcat
     4921 catcggtgaa gtgttcaaat accagctgtt cgaactggag gatggaacaa aaagaatgat
     4981 cgcaaccgcc aaggaatttc agaaggccaa ccagctgatc ctgcctaatt acttagtgcg
     5041 gctgctgtac aacgccaagc ggtacgacag agtgaaccac ctggaatcag tggaatacat
     5101 ccacagccac atcagcgagt tcgacgacct cttcgagcac gtgatcaact tcgccaaaaa
     5161 atatatcaaa cctgagaaga acatcgagaa ggccatcgag ctgtacaaca agtacggcct
     5221 gaaggacccc gccctggtga gcgagagttt catcaacctg atgaagttca ccagcatcgg
     5281 agcccctgct gaaatcgagt tcttcggcga aaagatccct cgcagacagc tgaagagcat
     5341 caccgagttc aggggctcta cactgatctt tcaaagcatc acaggcctgt acgaaaccag
     5401 atacaagctg gaagataata aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     5461 gaaaaaggaa ttcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga
     5521 ggagaatcct ggcccaatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     5581 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     5641 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     5701 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     5761 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     5821 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     5881 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca
     5941 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     6001 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     6061 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     6121 caagcccggt gcctgagaat tctaactaga gctcgctgat cagcctcgac tgtgccttct
     6181 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6241 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     6301 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     6361 agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     6421 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     6481 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     6541 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     6601 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6661 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6721 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6781 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     6841 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6901 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     6961 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     7021 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     7081 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     7141 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     7201 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     7261 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     7321 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     7381 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7441 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7501 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7561 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7621 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7681 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7741 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7801 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7861 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7921 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7981 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     8041 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     8101 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     8161 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     8221 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     8281 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     8341 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     8401 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8461 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     8521 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     8581 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     8641 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     8701 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     8761 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     8821 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8881 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     8941 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     9001 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     9061 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     9121 tgt
//
