LOCUS       sequence-462526-        9250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SeqCas9 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 9250)
  TITLE     Yongming Wang Lab Plasmids 2024
REFERENCE   2  (bases 1 to 9250)
  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-SeqCas9-puro
FEATURES             Location/Qualifiers
     source          1..9250
                     /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          1054..1281
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1293..1302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1302..1367
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1374..1394
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5547..5594
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5610..5663
                     /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             5664..6263
                     /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(5664..5683)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6160..6180
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6291..6308)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6297..6504
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6513..6653
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6513..6642
                     /label=AAV2 ITR
     rep_origin      6728..7183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6815..6834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7025..7046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7200..7219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7319..7341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7379..7397)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7465..7569
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7570..7638,7639..8430)
                     /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(7788..7807)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8601..9189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9090..9109
                     /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 ggggggggcg cgcgccaggc gggggggggg gggggggggg gggggggggg gggggggcgc
      901 gcgccggggg gggggggggg gggggggggg cggggggggg ggagggggag gggggcggcg
      961 gcggcaaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1021 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     1081 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     1141 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
     1201 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
     1261 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg
     1321 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga
     1381 agaagcggaa ggtcggtatc cacggagtcc cagcagccga aaagacatac agtatcggcc
     1441 tggacatcgg caccaattct gtgggctggg ccgtgataac cgacgactac aaggtgcccg
     1501 ccaaaaagat gaaagtgctg ggaaatactg acaagaagta cattaagaag aacctgctgg
     1561 gcgccttgct gttcgatagc ggcgaaactg ccgaggccac cagactgaaa agagccgcca
     1621 ggagacggta tacaagaaga aagaacagac tgcggtacct gcaagagatc ttcgccaaag
     1681 agatggccaa ggtggatgag tccttctttc agcggctgga agaaagcttc ctgaccgatg
     1741 atgacaagac ttttgatagc catcctatct tcggcaacaa ggccgaagag gacacctacc
     1801 accaggagtt ccccaccatc taccacctga gaaagcacct ggctgatagc cccgagaagg
     1861 tggacctgag actggtgtac ctggccctgg cccacatgat caagttccgg ggccacttcc
     1921 tcatcgaagg ccagctgaac gccgagaaca ccgatgtgca gaagattttc gccgacttcg
     1981 tgggagtgta tgacagaacc tttgacgata gccatctgag cgagatcacc gtggacgccg
     2041 cttctatcct gaccgagaaa atcagcaaat ccagaagatt ggaaaacctc atcaagcagt
     2101 acccaaccga gaaaaagaac accctttttg gtaatctggt cgccctggct ctgggcctgc
     2161 aacctaactt caagaccaac tttaagctgt ccgaggacgc caagctccag tttagcaagg
     2221 atacctacga tgaggacctc gaggaactgc tgggcaagat cggagatgac tacgccgacc
     2281 tgttcacagc cgccaagaac ctgtacgacg ccatcctgct gagcggcatc ctgactgttg
     2341 atgacaacag caccaaggcc cctctgagcg ccagcatgat caaaagatat gaagaacacc
     2401 acgaggacct ggagaagctg aaaaccttta tcaaggtcaa caacttcgac aagtaccacg
     2461 agatcttcaa agacaagtcc aaaaatggct acgccggcta cattgaaaat ggcgtgaagc
     2521 aggacatctt ctacaagcac ctgaagagca tcatcagcga aaagaacggc ggccagtact
     2581 tcctggataa gatcgagaga gaggactttc tgaggaagca gcggaccttc gataatggct
     2641 ctatccctta ccagatccac ctgcaagaga tgcggacgat cctgcggcgg cagggcgagt
     2701 actacccctt cctgaaagag aaccaggcca agatcgagaa gatcctgaca tttagaatcc
     2761 cctactacgt ggggccactg gccagaaaga acagcagatt cgcctgggcc aagtaccaca
     2821 gcgacgaacc tatcacccct tggaatttcg atgaggtggt cgacaaggag aaaagcgccg
     2881 agaagtttat cacacgtatg accctgaacg acctgtacct gcctgaagag aaggtactgc
     2941 ctaaacactc ttacgtgtac gagaccttcg ccgtgtacaa cgagctgacc aagattaagt
     3001 atgtgaatga acagggcaaa agcttcttct tcgacgccaa catgaagcag gagatctttg
     3061 accacgtgtt caaggaaaac agaaaagtga ccaaggccaa gctgctgtca tacctgaaca
     3121 acgagttcga ggaattcaga atcaacgacc tgatcggcct ggacaaggat tctaaaagct
     3181 tcaatgccag cctgggcacc taccacgacc tgaaaaagat tctggacaag agcttcctgg
     3241 acgataagac aaacgaacag atcattgaag atatcgtcct gacactgact ctgttcgagg
     3301 atagagatat gatccacgag agactgcaaa aatacagcga catcttcaca agccagcagc
     3361 taaagaagct ggaaagacgc cactacaccg gatggggcag actgtcttac aagctgatta
     3421 acggcatccg gaacaaagag aataacaaga ccatcctgga tttcctcatc gacgacggcg
     3481 acgcaaatcg gaatttcatg cagctcatca acgacgacag cctgagcttc aaaacaacaa
     3541 tccaggaggc acaggtggtg ggagatgtgg acgacattga ggccgtggtg cacgacctgc
     3601 ctggcagccc tgctatcaag aaaggcatcc tgcaatctgt gaagattgtg gacgagctag
     3661 tgaaggtgat gggccacaac cctcagaaca tcgtgatcga aatggccaga gaaaaccaga
     3721 tcaccggcta tggcagaaac cggagcaacc agaggctgaa gcggctgcag gattccctga
     3781 aggaattcgg ctctgacatc ctatcaaaaa agaagccctc ctacgtggat agcaaggtgg
     3841 agaatagcca cctgcaaaac gaccggctgt tcctgtacta catccagaac ggcaaggaca
     3901 tgtacaccgg agaggaatta gacatcgatc acctgagcga ttacgacatt gatcacatca
     3961 tcccccaagc cttcatcaag gataacagca tcgacaatag agtgctcaca tcttctgcca
     4021 agaaccgggg caagtctgac gacgttccat ctatcgaaat cgtgcggaac agaaagagct
     4081 actggtacaa actgtataag agcggcctga tcagcaagag aaagttcgat aacctgacca
     4141 aggccgagag aggcggactg accgagacag acaaggctgg ctttatcaag agacagctgg
     4201 tagaaacccg gcagatcacc aaacatgtgg cccagatcct ggacgctaga ttcaacacaa
     4261 aatgcgacga gaacgacaag gtgatcagag atgtcaaggt aatcacactg aagagcagcc
     4321 tggtgtctca gttcagaaag gaatttaagt tctacaaggt gcgggaaatc aacgactacc
     4381 atcacgccca cgacgcttat ctgaacgctg tggtgggcac cgccctcttg aaaaaatacc
     4441 ccaagctggc ccctgagttc gtgtacggcg aatacaagaa atacgacgtg cggaaactgg
     4501 tggctaagag cagcgacaac cactccgagc tggggaaggc taccgctaag tatttcttct
     4561 actccaatct gatgaacttc ttcaagacag aggtgaagta cgccgacggc agagtgttcg
     4621 agcgccccga catcgaaacc aacgccgacg gcgaggtggt gtggaacaag cagagagact
     4681 tcaacatcgt gagaaaggtg ctgagctacc ctcaggtgaa catcgtgaaa aaagtggaag
     4741 tgcagaccgg tggcttcagc aaggaaagca tcctccctaa gggcgattct gacaaactga
     4801 tcccgagaaa gaccaagaag ctgcagtggg agacacagaa atacggcggc ttcgacagtc
     4861 ctaccgtggc ctacagtgtt ctggtggtcg ccgatgtcga gaaaggcaag accagaaagc
     4921 tgaaaaccgt gaaggaactg gtgggaataa gcattatgga aagaagcagc tttgaggaga
     4981 atcctgtatc ctttctggaa aagaaaggat accacaacgt gcaggaggac aagctgataa
     5041 agcttcctaa gtattcactg ttcgagttcg agggcggcag acggagactg ctggccagcg
     5101 ccaccgaact gcagaagggg aatgaggtgg tcctgcctca atacatggtg aacctgctgt
     5161 accacagcca gcatgtgaac aacagccaca agcctgagca ccttaactac gtgaagcagc
     5221 acaaggacga gttcaaggat atcttcaacc tgatcatctc tatcgcccgg atcaatatcc
     5281 tgaaacctaa ggtggttgac aacctgatca acgagttcac cgagtacgga caggaggaca
     5341 tcagcagcct gagcgagagc ttcatcaacc tgctgaaatt tatcagcttt ggcgccccag
     5401 gagcttttaa gttcctgaag ctggacgtga agcagtccaa cctgcggtat aagtccacca
     5461 cagaggccct gagtgctaca ctgatccacc agtctgtgac cggcctgtac gagacaagaa
     5521 tcgacctgtc taagttgggc gaagaaaaaa ggccggcggc cacgaaaaag gccggccagg
     5581 caaaaaagaa aaaggaattc ggcagtggag agggcagagg aagtctgcta acatgcggtg
     5641 acgtcgagga gaatcctggc ccaatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     5701 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     5761 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     5821 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     5881 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     5941 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     6001 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg
     6061 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     6121 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     6181 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     6241 tgacccgcaa gcccggtgcc tgagaattct aactagagct cgctgatcag cctcgactgt
     6301 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     6361 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     6421 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     6481 agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac
     6541 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     6601 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     6661 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     6721 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     6781 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     6841 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     6901 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     6961 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     7021 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     7081 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     7141 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     7201 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     7261 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     7321 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     7381 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     7441 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     7501 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     7561 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     7621 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     7681 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     7741 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     7801 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     7861 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     7921 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     7981 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     8041 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     8101 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     8161 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     8221 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     8281 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     8341 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     8401 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     8461 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     8521 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     8581 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     8641 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     8701 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     8761 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     8821 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     8881 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     8941 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     9001 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     9061 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     9121 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     9181 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     9241 gctcacatgt
//
