LOCUS       Exported                9189 bp ds-DNA     circular SYN 16-DEC-2024
DEFINITION  Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA 
            (V2.0).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9189)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 9189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Dec 16, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9189
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     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"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1325..1345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    1370..5485
                     /label=Slu1Cas9
     CDS             5486..5533
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5549..5602
                     /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             5603..6202
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    6236..6443
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6452..6592
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6452..6581
                     /label=AAV2 ITR
     rep_origin      6667..7122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        7404..7508
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7509..8369
                     /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"
     rep_origin      8540..9128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
     1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg aaaaaaccta
     1381 cagcatcggc ctggacatag gcacaaatag cgtgggctgg agcgtgatca cagacgacta
     1441 caaggttcct gccaaaaaaa tgaaggtgct cggcaacaca gataagaagt acatcaaaaa
     1501 aaacctgctg ggcgccctgc tgttcgactc tggcgaaacc gctgaggtga ccagactgaa
     1561 aagaacagct aggagaagat acacccgtcg gaagaaccgg ctgagatacc tgcaggagat
     1621 cttcgccaag gagatggtga aggtggatga atcttttttc cagagactgg aggagagctt
     1681 cctgacagat gacgacaaga ccttcgactc ccaccccatc tttggcaaca aggccgagga
     1741 ggacgcctac caccaggagt tccccaccat ctaccacctg agaaagcacc tggccgacag
     1801 cagcgaaaag gccgatctca gactggtcta cctggccctg gctcacatga tcaagttcag
     1861 aggccacttc ctgattgaag gcgagctgaa cgccgagaac accgacatcc agaaactgtt
     1921 caatgacttc gtggagacct atgataagat cgccgacgag agccacctga gcgaagtgga
     1981 cgccagcagc atcctgacaa ataagatcag caagagcaga cggctggaaa acctgattaa
     2041 gcagtaccct accgagaaga aaaacaccct ctttggcaat ctggtggccc tggtgctggg
     2101 cctgcaacct aactttaaaa ccaacttcaa actctccgag gatgccaagc tgcagttcag
     2161 caaggacacc tacgacgagg acctggaaga gctgctgggc aagattggcg acgactacgc
     2221 cgacctgttc accgccgcca agaatctgta cgacgccatc ctcctcagtg gcatcctgac
     2281 agtggacgac aactctacca aagcccctct gtctgctagc atgatcaaga gatacgagga
     2341 acaccacgag gacctggaga agctgaagaa gttcatcaag gtgaacaact tcgataagta
     2401 ccatgagatc ttcaaggaca agtccaagaa cggctacgcc ggatacatag agaatggggt
     2461 gaagcaggac atcttttaca agcacctgaa aagcatcatc agcgagaaaa acggaggcca
     2521 gtacttccta gacaagatcg agagagagga tttcctgcgg aagcagcgga cgttcgacaa
     2581 cggctctatc cctcaccaga tccacctgca ggagatgaga gccatcctgc ggcaccaggg
     2641 cgaatactac cctttcctga aggaaaacca gagcaagatc gagaagatcc tgacgtttcg
     2701 gatcccttac tacgtgggcc ccctggccag aaagaacagc agattcgcct gggccaaata
     2761 tcattctgat gagcctatca ccccttggaa ctttgacgaa gtggtggaca aagaaaagtc
     2821 cgccgaaaag tttatcacca gaatgacact gaatgacctg tacctacccg aagagaaggt
     2881 ccttccgaag catagccatg tttatgaaac atacgccgtg tacaacgaac tcacaaagat
     2941 caagtacgtg aacgagcagg gaaaggaaag ctttttcgac agcaacatga agcaagaaat
     3001 tttcgaccac gtgttcaagg agaaccggaa agtcaccaag gagaagctgc tgaactacct
     3061 gaacaaggaa tttcctgagt acagaatcaa ggacctgatc ggcctggaca aggagaacaa
     3121 gtccttcaac gcctctctgg gcacctacca cgatctgaag aagatcctgg acaagtcctt
     3181 cctggacgac aaggtgaacg aggaagtaat cgaggacatc atcaaaaccc tgaccctgtt
     3241 cgaggacaag gacatgatcc acgagagact gcagaagtac agcgacatct tcactgccaa
     3301 tcaactgaag aagctggaac ggagacacta caccggctgg ggcagactat cgtacaagct
     3361 gatcaatgga ataagaaata aagagaacaa caagaccatc ctggattatc tgatcgacga
     3421 cggaagcgcc aaccggaact tcatgcagct tatcaacgac gataccctgc cattcaagca
     3481 aatcatccag aaaagccagg tggtgggaga tgtggatgac atcgaggccg tggtgcacga
     3541 tctgcccggc agccctgcca tcaaaaaagg catcctgcag tctgtgaaga tcgtagacga
     3601 actggtgaag gtgatgggcg gcaaccctga taatatcgtg atcgagatgg ccagagaaaa
     3661 ccagaccacc aatagaggta gaagccagag ccagcagaga ctgaagaaac tgcaaagcag
     3721 cctgaaggag ctgggctcta acatcatcaa cgaggagaaa cccagctaca tcgaggacaa
     3781 agtggagaac agccacctgc aaaatgatca gctgttcctg tactacatcc agaacggcaa
     3841 ggatatgtac accggcgacg agctggatat cgatcacctg tctgattacg atattgatca
     3901 catcatcccc caggccttca tcaaggacga cagcattgac aatcgggtgc tgacaagttc
     3961 tgctaagaac cgcggcaagt ccgatgacgt gcctagcctg gatatcgttc gcgctagaaa
     4021 ggcagaatgg gtgcggctgt acaagagcgg attgatcagc aagcggaaat tcgataacct
     4081 caccaaggca gaaagaggcg gcctgacaga agccgataaa gcgggcttca tcaagcggca
     4141 gctggtggaa acaagacaga tcaccaagca cgtggcccag atccttgatg ctcggttcaa
     4201 taccgaacac gatgaaaacg acaaggtgat ccgggacgtg aaggtgatca ccctgaagtc
     4261 taacctggtg agccaattta gaaaggactt cgagttctac aaggtcagag agatcaacga
     4321 ttatcatcac gcccacgacg catacctgaa cgccgtggtc ggcacagccc tgctgaagaa
     4381 gtaccccaag ctggcccctg agttcgtgta cggcgaatac aagaagtacg atgtgagaaa
     4441 actcatcgct aagtccagcg acgaccactc cgagatgggc aaggccacag ctaagtactt
     4501 cttttacagc aacctgatga acttcttcaa gaccaaggtg aaatacgctg acggcagcgt
     4561 gtacgagaga cctatcatcg agaccaatgc cgacggcgaa gtggtgtgga acaagcagaa
     4621 ggacttcgag atcatccgca aggtgctgag ttatcctcag gtcaacatcg tcaagaaggt
     4681 tgaggagcag accgtgggcc agaacggcgg tctgttcgat aataacatcc tggccaagcc
     4741 agagaaggat gataagaaga aactgtttcc tatcaaaaag agcctgtcga caacctccta
     4801 cggcggatat gccagggcca cgatcgccta tagcatcctg atcgtgtccg agttcggcaa
     4861 aaacaaggag ctgaaactgc tgggcctgcc tctgctggac aaggtgaagt acgagaagaa
     4921 tcccatcgct tacctgaaca gcatcggaat catcaacgtg aaaagcatct tcaagttacc
     4981 aaagtactgc ctgttcgagt tccaggagaa gaacaccaac taccggagat acatggtctc
     5041 caaccaggag ctgaagcgag ccaaccagct tttcctgacc tgtaaagaaa cagagctggt
     5101 gtataagcac atcaagagcg acaacgataa cacacacctg gtgggcaatg aaaagacaat
     5161 cctgtctatt tggaacaagc tggtgatctt catggaacgt accaagctga tcgataagaa
     5221 actgtttgcc tcactgaaac agctgaagga cgaggtgaat ctgataaaca ttctggatat
     5281 cgccagcaag agcatcgatt tcacaaaaat cagtgctggg cagaagaaga tcgtgctgaa
     5341 cgactctctg tctctgccca tcaaacggtg gcagttcaag tccgatgaga agatgctgaa
     5401 gcagctgaac gacgccaccc tgattcacca gagcattacc ggactgtacg agaccagaat
     5461 cgatctgagc aagctgggag aagagaaaag gccggcggcc acgaaaaagg ccggccaggc
     5521 aaaaaagaaa aaggaattcg gcagtggaga gggcagagga agtctgctaa catgcggtga
     5581 cgtcgaggag aatcctggcc caatgaccga gtacaagccc acggtgcgcc tcgccacccg
     5641 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     5701 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     5761 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     5821 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     5881 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     5941 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     6001 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     6061 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     6121 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     6181 gacccgcaag cccggtgcct gagaattcta actagagctc gctgatcagc ctcgactgtg
     6241 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     6301 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     6361 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     6421 gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact
     6481 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     6541 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     6601 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     6661 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     6721 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     6781 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     6841 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     6901 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     6961 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     7021 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     7081 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     7141 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     7201 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     7261 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     7321 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     7381 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     7441 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     7501 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     7561 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     7621 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     7681 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     7741 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     7801 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     7861 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     7921 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     7981 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     8041 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     8101 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     8161 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     8221 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     8281 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     8341 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     8401 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     8461 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     8521 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     8581 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     8641 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     8701 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     8761 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     8821 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     8881 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     8941 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     9001 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     9061 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     9121 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     9181 ctcacatgt
//
