LOCUS       Exported                9201 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 9201)
  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 9201)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Dec 16, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9201
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
                     /note="color: #ffffff"
     promoter        727..1004
                     /label=chicken beta-actin promoter
                     /note="color: #ffffff; direction: RIGHT"
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /note="color: #------"
     regulatory      1244..1253
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /note="color: #cc99b2
                     Cleavage site after base 1318"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1325..1345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     misc_feature    1370..5497
                     /label=SeqCas9
                     /note="color: #800080"
     CDS             5498..5545
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5561..5614
                     /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."
                     /note="color: #cc99b2"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5615..6214
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /note="color: #ccffcc"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    6248..6455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   6464..6604
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     repeat_region   6464..6593
                     /label=AAV2 ITR
                     /note="color: #ffcc99"
     rep_origin      6679..7134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        7416..7520
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             7521..8381
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 7521 .. 7589 / #ccffcc / signal sequence
                        2: 7590 .. 8381 / #ccffcc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8552..9140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
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 aaaagacata
     1381 cagtatcggc ctggacatcg gcaccaattc tgtgggctgg gccgtgataa ccgacgacta
     1441 caaggtgccc gccaaaaaga tgaaagtgct gggaaatact gacaagaagt acattaagaa
     1501 gaacctgctg ggcgccttgc tgttcgatag cggcgaaact gccgaggcca ccagactgaa
     1561 aagagccgcc aggagacggt atacaagaag aaagaacaga ctgcggtacc tgcaagagat
     1621 cttcgccaaa gagatggcca aggtggatga gtccttcttt cagcggctgg aagaaagctt
     1681 cctgaccgat gatgacaaga cttttgatag ccatcctatc ttcggcaaca aggccgaaga
     1741 ggacacctac caccaggagt tccccaccat ctaccacctg agaaagcacc tggctgatag
     1801 ccccgagaag gtggacctga gactggtgta cctggccctg gcccacatga tcaagttccg
     1861 gggccacttc ctcatcgaag gccagctgaa cgccgagaac accgatgtgc agaagatttt
     1921 cgccgacttc gtgggagtgt atgacagaac ctttgacgat agccatctga gcgagatcac
     1981 cgtggacgcc gcttctatcc tgaccgagaa aatcagcaaa tccagaagat tggaaaacct
     2041 catcaagcag tacccaaccg agaaaaagaa cacccttttt ggtaatctgg tcgccctggc
     2101 tctgggcctg caacctaact tcaagaccaa ctttaagctg tccgaggacg ccaagctcca
     2161 gtttagcaag gatacctacg atgaggacct cgaggaactg ctgggcaaga tcggagatga
     2221 ctacgccgac ctgttcacag ccgccaagaa cctgtacgac gccatcctgc tgagcggcat
     2281 cctgactgtt gatgacaaca gcaccaaggc ccctctgagc gccagcatga tcaaaagata
     2341 tgaagaacac cacgaggacc tggagaagct gaaaaccttt atcaaggtca acaacttcga
     2401 caagtaccac gagatcttca aagacaagtc caaaaatggc tacgccggct acattgaaaa
     2461 tggcgtgaag caggacatct tctacaagca cctgaagagc atcatcagcg aaaagaacgg
     2521 cggccagtac ttcctggata agatcgagag agaggacttt ctgaggaagc agcggacctt
     2581 cgataatggc tctatccctt accagatcca cctgcaagag atgcggacga tcctgcggcg
     2641 gcagggcgag tactacccct tcctgaaaga gaaccaggcc aagatcgaga agatcctgac
     2701 atttagaatc ccctactacg tggggccact ggccagaaag aacagcagat tcgcctgggc
     2761 caagtaccac agcgacgaac ctatcacccc ttggaatttc gatgaggtgg tcgacaagga
     2821 gaaaagcgcc gagaagttta tcacacgtat gaccctgaac gacctgtacc tgcctgaaga
     2881 gaaggtactg cctaaacact cttacgtgta cgagaccttc gccgtgtaca acgagctgac
     2941 caagattaag tatgtgaatg aacagggcaa aagcttcttc ttcgacgcca acatgaagca
     3001 ggagatcttt gaccacgtgt tcaaggaaaa cagaaaagtg accaaggcca agctgctgtc
     3061 atacctgaac aacgagttcg aggaattcag aatcaacgac ctgatcggcc tggacaagga
     3121 ttctaaaagc ttcaatgcca gcctgggcac ctaccacgac ctgaaaaaga ttctggacaa
     3181 gagcttcctg gacgataaga caaacgaaca gatcattgaa gatatcgtcc tgacactgac
     3241 tctgttcgag gatagagata tgatccacga gagactgcaa aaatacagcg acatcttcac
     3301 aagccagcag ctaaagaagc tggaaagacg ccactacacc ggatggggca gactgtctta
     3361 caagctgatt aacggcatcc ggaacaaaga gaataacaag accatcctgg atttcctcat
     3421 cgacgacggc gacgcaaatc ggaatttcat gcagctcatc aacgacgaca gcctgagctt
     3481 caaaacaaca atccaggagg cacaggtggt gggagatgtg gacgacattg aggccgtggt
     3541 gcacgacctg cctggcagcc ctgctatcaa gaaaggcatc ctgcaatctg tgaagattgt
     3601 ggacgagcta gtgaaggtga tgggccacaa ccctcagaac atcgtgatcg aaatggccag
     3661 agaaaaccag atcaccggct atggcagaaa ccggagcaac cagaggctga agcggctgca
     3721 ggattccctg aaggaattcg gctctgacat cctatcaaaa aagaagccct cctacgtgga
     3781 tagcaaggtg gagaatagcc acctgcaaaa cgaccggctg ttcctgtact acatccagaa
     3841 cggcaaggac atgtacaccg gagaggaatt agacatcgat cacctgagcg attacgacat
     3901 tgatcacatc atcccccaag ccttcatcaa ggataacagc atcgacaata gagtgctcac
     3961 atcttctgcc aagaaccggg gcaagtctga cgacgttcca tctatcgaaa tcgtgcggaa
     4021 cagaaagagc tactggtaca aactgtataa gagcggcctg atcagcaaga gaaagttcga
     4081 taacctgacc aaggccgaga gaggcggact gaccgagaca gacaaggctg gctttatcaa
     4141 gagacagctg gtagaaaccc ggcagatcac caaacatgtg gcccagatcc tggacgctag
     4201 attcaacaca aaatgcgacg agaacgacaa ggtgatcaga gatgtcaagg taatcacact
     4261 gaagagcagc ctggtgtctc agttcagaaa ggaatttaag ttctacaagg tgcgggaaat
     4321 caacgactac catcacgccc acgacgctta tctgaacgct gtggtgggca ccgccctctt
     4381 gaaaaaatac cccaagctgg cccctgagtt cgtgtacggc gaatacaaga aatacgacgt
     4441 gcggaaactg gtggctaaga gcagcgacaa ccactccgag ctggggaagg ctaccgctaa
     4501 gtatttcttc tactccaatc tgatgaactt cttcaagaca gaggtgaagt acgccgacgg
     4561 cagagtgttc gagcgccccg acatcgaaac caacgccgac ggcgaggtgg tgtggaacaa
     4621 gcagagagac ttcaacatcg tgagaaaggt gctgagctac cctcaggtga acatcgtgaa
     4681 aaaagtggaa gtgcagaccg gtggcttcag caaggaaagc atcctcccta agggcgattc
     4741 tgacaaactg atcccgagaa agaccaagaa gctgcagtgg gagacacaga aatacggcgg
     4801 cttcgacagt cctaccgtgg cctacagtgt tctggtggtc gccgatgtcg agaaaggcaa
     4861 gaccagaaag ctgaaaaccg tgaaggaact ggtgggaata agcattatgg aaagaagcag
     4921 ctttgaggag aatcctgtat cctttctgga aaagaaagga taccacaacg tgcaggagga
     4981 caagctgata aagcttccta agtattcact gttcgagttc gagggcggca gacggagact
     5041 gctggccagc gccaccgaac tgcagaaggg gaatgaggtg gtcctgcctc aatacatggt
     5101 gaacctgctg taccacagcc agcatgtgaa caacagccac aagcctgagc accttaacta
     5161 cgtgaagcag cacaaggacg agttcaagga tatcttcaac ctgatcatct ctatcgcccg
     5221 gatcaatatc ctgaaaccta aggtggttga caacctgatc aacgagttca ccgagtacgg
     5281 acaggaggac atcagcagcc tgagcgagag cttcatcaac ctgctgaaat ttatcagctt
     5341 tggcgcccca ggagctttta agttcctgaa gctggacgtg aagcagtcca acctgcggta
     5401 taagtccacc acagaggccc tgagtgctac actgatccac cagtctgtga ccggcctgta
     5461 cgagacaaga atcgacctgt ctaagttggg cgaagaaaaa aggccggcgg ccacgaaaaa
     5521 ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag gaagtctgct
     5581 aacatgcggt gacgtcgagg agaatcctgg cccaatgacc gagtacaagc ccacggtgcg
     5641 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     5701 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     5761 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     5821 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     5881 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     5941 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     6001 agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     6061 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     6121 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     6181 cacctggtgc atgacccgca agcccggtgc ctgagaattc taactagagc tcgctgatca
     6241 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     6301 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     6361 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     6421 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     6481 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     6541 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     6601 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     6661 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6721 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     6781 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6841 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     6901 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6961 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     7021 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     7081 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     7141 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7201 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7261 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     7321 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     7381 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     7441 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     7501 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     7561 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     7621 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     7681 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     7741 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     7801 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     7861 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     7921 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     7981 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     8041 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     8101 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     8161 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     8221 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     8281 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     8341 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     8401 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     8461 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     8521 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     8581 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     8641 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     8701 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     8761 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     8821 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     8881 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     8941 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     9001 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     9061 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     9121 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     9181 gctggccttt tgctcacatg t
//
