LOCUS       .                       7032 bp    DNA     linear   UNK 01-JAN-1980
DEFINITION  .
ACCESSION   <unknown id>
VERSION     <unknown id>
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     terminator      77..81
                     /note="RNA polymerase III transcription terminator"
                     /note="color: #ffffff"
                     /label="polIII terminator"
     CDS             3314..3316
                     /codon_start=1
                     /product="start codon"
                     /transl_table=1
                     /translation="M"
                     /label="ATG"
                     /note="color: #993366"
     CDS             6515..6535
                     /codon_start=1
                     /locus_tag=""
                     /note=""
                     /note="color: #993366"
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /protein_id=""
                     /transl_table=1
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             3323..3343
                     /codon_start=1
                     /locus_tag=""
                     /note=""
                     /note="color: #993366"
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /protein_id=""
                     /transl_table=1
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             6536..6559
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
                     /note="color: #cc99b2"
     promoter        498..602
                     /gene="bla"
                     /label="AmpR promoter"
                     /note="color: #ffffff"
     promoter        3080..3283
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /note="color: #ffffff"
                     /label="CMV promoter"
     polyA_signal    6569..6776
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
                     /label="bGH poly(A) signal"
     enhancer        2776..3079
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
                     /label="CMV enhancer"
     rep_origin      1634..2222
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /note="color: #ffff00"
                     /label="ori"
     CDS             603..1463
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="color: #ccffcc"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     CDS             3344..6499
                     /codon_start=1
                     /note="generates RNA-guided double strand breaks in DNA"
                     /note="color: #993366"
                     /product="Cas9 (Csn1) endonuclease from the Staphylococcus
                     aureus Type II CRISPR/Cas system"
                     /protein_id=""
                     /standard_name=""
                     /transl_table=1
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
                     /label="SaCas9"
     regulatory      3308..3317
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     misc_RNA        1..76
                     /note="guide RNA scaffold for the Staphylococcus aureus
                     CRISPR/Cas9 system"
                     /note="color: #00ccff"
                     /label="Sa gRNA scaffold"
     repeat_region   2567..2707
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /note="color: #ffe4c4"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     repeat_region   96..236
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /note="color: #ffe4c4"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     promoter        6783..7031
                     /note="RNA polymerase III promoter for human U6 snRNA
                     (Domitrovich & Kunkel, 2003)"
                     /note="color: #ffffff"
                     /label="U6 promoter"
ORIGIN
        1 gttttagtac tctggaaaca gaatctacta aaacaaggca aaatgccgtg tttatctcgt
       61 caacttgttg gcgagatttt tgcggccgca gatctaggaa cccctagtga tggagttggc
      121 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
      181 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggcagc
      241 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
      301 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      361 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgccaaag ggcctcgtga
      421 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
      481 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      601 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      661 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      721 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
      781 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
      841 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
      901 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
      961 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1021 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1081 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1141 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1201 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1261 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1321 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1381 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1441 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1501 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1741 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     1801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2281 tgttctttcc tgcgttatcc cctgattctg tggataaccg taagcggaag agcgcccaat
     2341 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2401 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     2461 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     2521 ataacaattt cacacaggaa acagctatga ccatgattac gaattgcctg caggcagctg
     2581 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt
     2641 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     2701 ggttcctatc gatatcaagc tttaatagta atcaattacg gggtcattag ttcatagccc
     2761 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     2821 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2881 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2941 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     3001 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     3061 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     3121 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     3181 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     3241 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tggatatcct
     3301 taaggccgcc accatggcct cccccaaaaa gaagaggaaa gtgaagagga actacattct
     3361 gggcctggac attggcatca caagcgtggg ctacggcatc atcgattatg agaccagaga
     3421 tgtgattgac gctggagtga ggctgttcaa agaggctaac gtggagaaca acgagggcag
     3481 aaggagcaag aggggcgcta ggaggctcaa aaggaggagg agacatagga tccagagggt
     3541 gaagaagctg ctgttcgact acaatctgct cacagaccac agcgaactgt ccggcattaa
     3601 cccttacgaa gctagggtca aaggactgag ccagaagctc tccgaggaag agtttagcgc
     3661 cgccctgctc cacctcgcta agaggagggg agtgcacaat gtgaatgagg tggaggagga
     3721 caccggaaat gagctgagca ccaaggagca gatttccagg aactccaaag ccctggaaga
     3781 aaagtacgtg gctgaactgc agctcgagag actgaagaaa gacggagaag tgaggggaag
     3841 catcaacaga tttaaaacct ccgattatgt gaaggaagcc aagcaactcc tgaaggtgca
     3901 aaaggcctac catcagctgg atcagagctt cattgacacc tacatcgacc tgctcgagac
     3961 caggaggacc tattatgagg gccctggcga aggctccccc tttggatgga aggacatcaa
     4021 ggagtggtac gagatgctga tgggccactg cacctacttt cccgaggagc tgaggtccgt
     4081 gaaatatgcc tacaacgctg acctctacaa tgccctgaac gacctgaaca acctggtcat
     4141 caccagagat gagaacgaaa aactcgaata ttacgagaag ttccaaatca ttgaaaacgt
     4201 gttcaagcag aagaagaagc ccaccctcaa gcaaatcgct aaggagatcc tcgtcaacga
     4261 ggaggatatt aagggctaca gggtcaccag caccggcaag cccgaattta ccaacctgaa
     4321 ggtgtaccat gacatcaaag acatcaccgc tagaaaggag atcattgaga acgccgagct
     4381 gctggatcag atcgctaaga tcctgacaat ctaccagagc agcgaggata ttcaggagga
     4441 gctcacaaac ctcaactccg agctgaccca ggaggaaatc gaacaaattt ccaatctgaa
     4501 gggctataca ggcacccaca atctcagcct gaaggccatc aacctgatcc tggatgagct
     4561 gtggcacacc aatgacaacc agatcgctat ttttaatagg ctgaagctcg tgcccaagaa
     4621 ggtcgacctc agccaacaga aggaaatccc caccaccctg gtggatgatt ttatcctctc
     4681 ccccgtcgtg aagaggtcct tcattcaaag catcaaggtg atcaacgcca tcatcaagaa
     4741 gtacggcctc cctaacgata tcatcatcga gctggccagg gagaagaata gcaaggatgc
     4801 ccagaagatg attaatgaga tgcaaaaaag gaataggcag acaaatgaaa ggatcgagga
     4861 gatcattaga acaaccggaa aagagaatgc caaatacctg atcgagaaga tcaagctgca
     4921 cgacatgcag gagggaaagt gcctgtactc cctggaagcc atccccctgg aggatctgct
     4981 caataaccct ttcaactatg aggtggacca catcatccct aggtccgtga gctttgataa
     5041 cagctttaac aataaggtgc tggtgaagca ggaagaaaac agcaagaaag gaaacaggac
     5101 cccctttcag tacctgagct cctccgacag caagattagc tacgaaacct tcaagaagca
     5161 tattctgaac ctggccaagg gcaaaggcag aatctccaaa accaagaagg aatacctgct
     5221 ggaggaaagg gacattaaca ggttctccgt gcagaaggat tttattaata ggaacctcgt
     5281 ggacacaagg tacgctacaa ggggcctcat gaacctgctg aggtcctatt tcagagtcaa
     5341 caacctggat gtcaaggtga agtccatcaa cggcggcttc accagcttcc tcaggaggaa
     5401 gtggaagttt aaaaaagaga ggaataaggg ctacaagcat cacgctgaag atgctctgat
     5461 catcgctaac gccgacttca tttttaagga gtggaagaaa ctggacaaag ccaaaaaagt
     5521 catggaaaac cagatgtttg aggagaaaca ggccgagtcc atgcctgaaa ttgagaccga
     5581 gcaggagtac aaggagattt ttatcacccc tcatcaaatc aagcacatca aagactttaa
     5641 agattacaag tacagccaca gagtggataa aaaacccaac agggagctga ttaatgacac
     5701 cctgtattcc acaaggaaag acgacaaggg caataccctc atcgtgaata acctgaacgg
     5761 cctgtacgac aaggataatg ataaactgaa aaagctgatt aacaaatccc ccgagaagct
     5821 gctgatgtac caccatgacc cccagacata tcagaagctc aagctcatca tggagcaata
     5881 cggcgatgag aaaaaccctc tgtacaaata ttatgaagaa accggaaact atctcacaaa
     5941 atactccaag aaggataacg gccctgtgat taaaaagatt aagtattatg gcaacaagct
     6001 gaacgctcac ctggatatta ccgacgacta tcctaattcc agaaacaagg tggtcaaact
     6061 gtccctgaag ccctataggt ttgacgtgta cctggacaac ggagtgtaca agttcgtcac
     6121 cgtcaagaat ctggacgtga ttaagaagga gaactactat gaagtcaata gcaagtgcta
     6181 cgaggaggcc aagaagctga agaagattag caatcaggcc gagtttatcg ccagctttta
     6241 taataacgac ctgattaaga ttaatggcga actgtacagg gtgatcggag tcaacaacga
     6301 tctgctcaac aggatcgaag tcaacatgat cgacatcaca tacagggaat acctggagaa
     6361 catgaacgac aagaggcccc ccaggattat caagacaatt gccagcaaga cccagagcat
     6421 caaaaagtac tccaccgata tcctgggaaa tctctacgaa gtgaaatcca agaagcatcc
     6481 ccaaatcatt aagaagggcg gcggctccgg accccccaag aaaaagagga aggtggacta
     6541 taaggatgat gacgacaagt gaggatccct gtgccttcta gttgccagcc atctgttgtt
     6601 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6661 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     6721 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagaat
     6781 tcgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     6841 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     6901 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     6961 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     7021 gacgaaacac cg
//
