LOCUS       Exported                8137 bp ds-DNA    linear   UNA 19-NOV-2014
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    pAF-U6-sgRNA-EFS-Cas9-2A-Cre-gBR Cassette
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8137)
  AUTHORS   FSR
  TITLE     Direct Submission
  JOURNAL   Exported Nov 19, 2014 from SnapGene Viewer 2.5.0
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8137
                     /organism="unspecified"
                     /mol_type="genomic DNA"
     misc_feature    1..30
                     /note="pAF Gibson"
     promoter        31..271
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    276..2160
                     /note="Filler"
     misc_RNA        2161..2236
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the CRISPR/Cas9 system"
     misc_feature    2284..2401
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    2445..2474
                     /note="pBF Gibson"
     promoter        2500..2711
                     /note="EF-1-alpha core promoter"
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    2726..2755
                     /note="gBF Gibson"
     CDS             2759..2782
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /note="FLAG"
                     /translation="DYKDDDDK"
     CDS             2789..2809
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             2834..6934
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6935..6982
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6998..7054
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7058..7078
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             7079..8107
                     /codon_start=1
                     /product="site-specific recombinase"
                     /note="Cre"
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    8108..8137
                     /note="gBR Gibson"
ORIGIN
        1 gatcagtgtg agggagtgta aagctggttt gagggcctat ttcccatgat tccttcatat
       61 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
      121 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
      181 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
      241 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagacggttg taaatgagca
      301 cacaaaatac acatgctaaa atattatatt ctatgacctt tataaaatca accaaaatct
      361 tctttttaat aactttagta tcaataatta gaatttttat gttccttttt gcaaactttt
      421 aataaaaatg agcaaaataa aaaaacgcta gttttagtaa ctcgcgttgt tttcttcacc
      481 tttaataata gctactccac cacttgttcc taagcggtca gctcctgctt caatcatttt
      541 ttgagcatct tcaaatgttc taactccacc agctgcttta actaaagcat tgtctttaac
      601 aactgacttc attagtttaa catcttcaaa tgttgcacct gattttgaaa atcctgttga
      661 tgttttaaca aattctaatc cagcttcaac agctatttca caagctttca tgatttcttc
      721 ttttgttaat aaacaatttt ccataataca tttaacaaca tgtgatccag ctgctttttt
      781 tacagctttc atgtcttcta aaactaattc ataatttttg tcttttaatg caccaatatt
      841 taataccata tcaatttctg ttgcaccatc tttaattgct tcagaaactt cgaatgcttt
      901 tgtagctgtt gtgcatgcac ctagaggaaa acctacaaca tttgttattc ctacatttgt
      961 gccttttaat aattctttac aatagcttgt tcaatatgaa ttaacacaaa ctgttgcaaa
     1021 atcaaattca attgcttcat cacataattg tttaatttca gctttcgtag catcttgttt
     1081 taataatgtg tgatctatat atttgtttag tttcattttt tctcctatat attcattttt
     1141 aattttaatt ctttaataat ttcgtctact ttaactttag cgttttgaac agattcacca
     1201 acacctataa aataaatttt tagtttaggt tcagttccac ttgggcgaac agcaaatcat
     1261 gacttatctt ctaaataaaa ttttagtaag tcttgtcctg gcatattata cattccatcg
     1321 atgtagtctt caacattaac aactttaagt ccagcaattt gagttaaggg tgttgctctc
     1381 aatgatttca ttaatggttc aatttttaat ttcttttctt ctggtttaaa attcaagttt
     1441 aaagtgaaag tgtaatatgc acccatttct ttaaataaat cttctaaata gtctactaat
     1501 gttttatttt gttttttata aaatcaagca gcctctgcta ttaatataga agcttgtatt
     1561 ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc ataagcaaaa
     1621 acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca tttaaatcca
     1681 gttaaagttt ttacaatatt aactccatat ttttcatgag cgattctatc acccaaatca
     1741 cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa gcgttttaga
     1801 tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa tcttacaaaa
     1861 tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt cataataata
     1921 atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg atcaaattct
     1981 ggatttggat ttacaacatt tttaaatgtt tcatcttcaa atgcatgctc ttcaacctca
     2041 ataacgttat atcctgattc acgtaatatt tttggggtaa atttagttcc tgttccatta
     2101 actgcgctaa aaataatttt taaatctttt ttagcttctt gctctttttt gtacgtctct
     2161 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     2221 ggcaccgagt cggtgctttt ttgaattcta gatcttgaga caaatggcag tattcatcca
     2281 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2341 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2401 tcgggtttat tacagggaca gcagagatcc actttggcgc cggcaaaccg ctgttcctag
     2461 gaatcccgag gccttcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2521 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2581 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2641 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2701 ccagaacaca ggtgtcgtga cgcgggaccc gacattagcg ctacagctta agcggatgga
     2761 ttacaaagac gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg
     2821 agtcccagca gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     2881 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     2941 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     3001 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     3061 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     3121 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     3181 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     3241 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     3301 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     3361 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     3421 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     3481 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     3541 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     3601 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     3661 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     3721 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     3781 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     3841 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     3901 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     3961 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     4021 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     4081 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     4141 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     4201 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     4261 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     4321 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     4381 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     4441 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     4501 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     4561 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     4621 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     4681 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     4741 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     4801 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     4861 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     4921 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     4981 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     5041 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     5101 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     5161 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     5221 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     5281 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     5341 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     5401 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     5461 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     5521 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     5581 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     5641 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     5701 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     5761 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     5821 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     5881 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     5941 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     6001 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     6061 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     6121 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     6181 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     6241 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     6301 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     6361 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     6421 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     6481 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     6541 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     6601 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     6661 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     6721 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     6781 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     6841 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     6901 gacacggatc gacctgtctc agctgggagg cgacaagcgt cctgctgcta ctaagaaagc
     6961 tggtcaagct aagaaaaaga aagctagcgg cagcggcgcc accaacttca gcctgctgaa
     7021 gcaggccggc gacgtggagg agaaccccgg ccccatgccc aagaagaaga ggaaggtgtc
     7081 caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga
     7141 ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg agcatacctg
     7201 gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa
     7261 atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc aggcgcgcgg
     7321 tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc atcgtcggtc
     7381 cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc ggatccgaaa
     7441 agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac gcactgattt
     7501 cgaccaggtt cgttcactca tggaaaatag cgatcgctgc caggatatac gtaatctggc
     7561 atttctgggg attgcttata acaccctgtt acgtatagcc gaaattgcca ggatcagggt
     7621 taaagatatc tcacgtactg acggtgggag aatgttaatc catattggca gaacgaaaac
     7681 gctggttagc accgcaggtg tagagaaggc acttagcctg ggggtaacta aactggtcga
     7741 gcgatggatt tccgtctctg gtgtagctga tgatccgaat aactacctgt tttgccgggt
     7801 cagaaaaaat ggtgttgccg cgccatctgc caccagccag ctatcaactc gcgccctgga
     7861 agggattttt gaagcaactc atcgattgat ttacggcgct aaggatgact ctggtcagag
     7921 atacctggcc tggtctggac acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc
     7981 tggagtttca ataccggaga tcatgcaagc tggtggctgg accaatgtaa atattgtcat
     8041 gaactatatc cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg
     8101 cgattagaaa cgttgttgtt tggggttgaa ttactct
//
