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=Slu2Cas9
     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 aaaagaccta
     1381 cagcatcggc ctggatatcg gcacaaatag cgtgggatgg tccgtgatca ccgatgacta
     1441 taaggtccct gccaagaaga tgaaggtcct gggcaacaca gataaaaagt acatcaagaa
     1501 gaacctcctg ggggctctgc tgttcgactc cggcgagaca gccgaggtga cccggctgaa
     1561 gcggacggcc agacgtagat acacccgcag aaagaacaga ctgagatacc tgcaggagat
     1621 cttcgccaag gaaatggcca aggtcgacga aagcttcttc cagagactgg aagaaagctt
     1681 tctgaccgat gatgacaaga ccttcgacag ccaccctata tttggcaaca aggccgagga
     1741 ggacgcttac catcaggagt ttcctacaat ctaccatctg cggaagcacc tggctgactc
     1801 aagcgagaag gcagacctgc ggctggtgta tctggccctg gcccacatga tcaagtttcg
     1861 tggtcatttc ttaatcgaag gcgagctgaa cgccgagaac accgacatcc agaagctgtt
     1921 taatgatttc gtggaaacat acgacaagat cgccgacgaa agccatctgt ctgaggtgga
     1981 cgccagttct atcctgacca acaagatttc taagtctcgg agactggaga acctgatcaa
     2041 gcaatacccc accgagaaga agaacaccct gttcggcaat ctggtggccc tcgttctggg
     2101 cctgcagcct aactttaaga caaacttcaa gctttccgag gacgctaagc tgcagtttag
     2161 caaggacacc tacgatgaag atctggagga actgctgggc aagatcggcg acgattacgc
     2221 tgacctgttc accgccgcca agaacctgta cgacgcaatc ctactaagtg gcattctaac
     2281 cgtggatgat aactcgacaa aagctcctct gagcgccagc atgatcaagc ggtacgagga
     2341 gcaccacgag gacctggaaa aactgaagaa attcatcaag gtgaacaact ttgacaagta
     2401 tcacgagatc ttcaaggaca aaagcaaaaa cggctacgcc ggctacatcg agaatggcgt
     2461 gaagcaggac atcttttaca agcatctgaa atccatcatc agcgaaaaga acggtggaca
     2521 gtacttcctg gataaaatcg agcgggagga cttcctgagg aagcagagaa ccttcgacaa
     2581 cggaagcatc ccccaccaga tccacctgca ggagatgaga gccattctgc ggagacaggg
     2641 agaatactac cccttcctga aggagaacca ggccaaaatc gaaaaaatcc tgacctttag
     2701 aatcccttac tacgtgggcc ccctggcccg caaagacagc cggttcgctt gggctaacta
     2761 ccactccgac gagcctatca cgccttggaa cttcgacgag gtggtggata aggaaaagtc
     2821 tgccgaaaag ttcatcacca gaatgaccct gaacgacctg tacctgcctg aagagaaggt
     2881 tcttccaaag cacagccacg tgtacgagac tttcaccgtg tacaacgagc tgactaagat
     2941 caagtacgtg aacgagcagg gcgagtcatt cttcttcgac gcgaatatga agcaggagat
     3001 tttcgaccac gtgttcaagg agaacagaaa ggtgaccaaa gctaaactgc tgtcttacct
     3061 gaacaacgaa ttcgaggaat tcagaatcaa cgacctaatc ggcttggaca aagacagcaa
     3121 gagcttcaac gccagcctgg gtacatacca cgacctcaag aagatcctgg acaaaagctt
     3181 cctggacgac aagaccaatg agcagatcat cgaggacatc gttctgaccc tgaccctgtt
     3241 cgaggataga gatatgattc acgagcggct gcagaagtac agcgattttt tcacatctca
     3301 gcagctcaag aagctggagc ggagacacta caccgggtgg ggcagactgt cttacaagct
     3361 gatcaacgga atccggaaca aggaaaacaa caagaccatc ctggacttcc tgatcgacga
     3421 cggccacgcc aacagaaact tcatgcagct tatcaacgac gagagcctca gctttaaaac
     3481 aattatccag gaggcccagg tggtgggcga tgtggacgac attgaggccg tggtgcacga
     3541 cctgcccggc tcccctgcta tcaagaaggg catcctgcaa tccgtgaaaa ttgtggacga
     3601 gctggtcaag gtgatgggag ataaccccga caacatcgtg atcgaaatgg ccagggaaaa
     3661 ccagaccacc aaccgggaaa gaagccagag ccagcaaaga ctgaagaagc tgcagaacag
     3721 cctgaaggag ctaggcagca atatcctgaa cgaggaaaag cccagctaca tcgaggataa
     3781 ggtcgagaat agccacctgc aaaacgacca gctgttcctg tactacatcc aaaacggcaa
     3841 agacatgtac acaggagatg agctggacat agatcacctg tctgactacg acatcgacca
     3901 catcattccc caggccttca tcaaggacaa ttctatcgac aatcgggtgc tgaccagcag
     3961 cgccaagaac agaggcaagt ctgatgatgt gccttctctg gacatcgtga gagcccggaa
     4021 ggccgagtgg gtgagactgt ataagtcagg cctgataagc aagagaaagt tcgacaacct
     4081 gaccaaggcc gagcggggcg gcctgactga agccgacaag gctggcttca ttaaacgaca
     4141 gctggttgaa acacggcaga taacaaagca cgtggcccag atcctggacg ctagattcaa
     4201 caccaagaga gatgagaacg acaaggtgat cagagacgtg aaggtgatca cactgaagtc
     4261 caacctggtc agccagttca gaaaagaatt caagttctac aaggtgcggg aaatcaacga
     4321 ctaccaccac gcccacgacg cctaccttaa cgctgtggtg ggcaccgccc tgctgaaaaa
     4381 ataccctaag ctgacacctg aattcgtcta cggcgaatat aagaagtacg atgtgcggaa
     4441 gctgattgcc aagagtagcg atgactactc agagatgggc aaggccaccg ccaaatattt
     4501 cttctattcc aaccttatga acttcttcaa gaccgaggtg aaatacgccg atggaagagt
     4561 gttcgagaga cctgatatcg agacaaatgc cgacggcgag gtggtgtgga acaagcagaa
     4621 agacttcgat atcgtcagaa aggtgctgag ctaccctcag gtgaacatcg tgaagaaagt
     4681 ggaagaacaa acagtcggcc aaaacggcgg cctgttcgat aacaacatcc tggccaagcc
     4741 tgagaaagac gataaaaaga agctgttccc tatcaaaaag tctttaagca ccacctccta
     4801 cggcggatac gccagagcta caatcgccta cagcatcctg atcgtgagcg agttcggcaa
     4861 aaacaaggag ctgaaactgc tgggcctgcc actgctggac aaggtgaagt atgaaaaaaa
     4921 ccctatcgcc tacctgaata gcattggcat catcaatgtg aagagcatct tcaagttgcc
     4981 aaagtactgc ctgtttgagt ttcaagaaaa aaatacaaat taccggaggt acatggtgtc
     5041 taatcaggag ctgaagagag ccaaccagct gtttctgaca tgtaaagaaa ccgagctggt
     5101 gtacaagtac atcaagagcg ataatgataa cacccacctg gtcggcaacg aaaagaccat
     5161 cctgagtatc tggaataagc tcgtgatatt catggaaaga accaagctga tcgataagaa
     5221 gctgttcgca agcctgaagc agctgaagga cgaagtggat ctgatcaaca tccttgacat
     5281 gacagccaag tctctcgact ttacgaaaat cagcgccggc cagaagaaga tcaccatcag
     5341 cgactccctg agcctgccaa tcaaaagatg gcagttcagc tctgatgaga agatgctgaa
     5401 gcaactgaaa accgccacgc tgatccacca gagcgtgacc ggactgtacg agaccagaat
     5461 cgatctgtcc aaactgggcg 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
//
