LOCUS       .                       8232 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  .
ACCESSION   <unknown id>
VERSION     <unknown id>
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     CDS             5388..5408
                     /codon_start=1
                     /locus_tag=""
                     /note=""
                     /note="color: #993366"
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /protein_id=""
                     /transl_table=1
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             5361..5381
                     /codon_start=1
                     /locus_tag=""
                     /note=""
                     /note="color: #993366"
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /protein_id=""
                     /transl_table=1
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     CDS             5628..5651
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
                     /note="color: #cc99b2"
     CDS             5580..5609
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /transl_table=1
                     /translation="EQKLISEEDL"
                     /label="Myc"
                     /note="color: #cc99b2"
     CDS             5412..5561
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /transl_table=1
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
                     /label="VP64"
                     /note="color: #993366"
     promoter        951..1154
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /note="color: #ffffff"
                     /label="CMV promoter"
     enhancer        571..950
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
                     /label="CMV enhancer"
     rep_origin      complement(6441..7029)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /note="color: #ffff00"
                     /label="ori"
     polyA_signal    5698..6320
                     /note="human growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
                     /label="hGH poly(A) signal"
     CDS             complement(7193..8053)
                     /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             1257..5360
                     /codon_start=1
                     /locus_tag=""
                     /note="RNA-guided DNA-binding protein that lacks
                     endonuclease activity due to the D10A mutation in the RuvC
                     catalytic domain and the H840A mutation in the HNH
                     catalytic domain"
                     /note="color: #993366"
                     /product="catalytically dead mutant of the Cas9
                     endonuclease from the Streptococcus pyogenes Type II
                     CRISPR/Cas system"
                     /protein_id=""
                     /transl_table=1
                     /translation="MDKKYSIGL,A,IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI
                     KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM
                     IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR
                     RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL
                     AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL
                     VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED
                     LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA
                     RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
                     EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC
                     FDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE
                     ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN
                     FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
                     GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK
                     LYLYYLQNGRDMYVDQELDINRLSDYDVD,A,IVPQSFLKDDSIDNKVLTRSDKNRGKS
                     DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQ
                     ITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH
                     DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN
                     FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTG
                     GFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE
                     LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ
                     KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV
                     ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST
                     KEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /label="dCas9"
     misc_RNA        384..519
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system, modified to contain two MS2 RNA
                     aptamers"
                     /note="color: #00ccff"
                     /label="gRNA scaffold"
     terminator      520..525
                     /note="RNA polymerase III transcription terminator"
                     /note="color: #ffffff"
                     /label="polIII terminator"
     promoter        90..330
                     /note="Truncated RNA polymerase III promoter for human U6
                     snRNA used in pSIREN vectors such as
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site
                     is 8bp downstream of this feature."
                     /note="color: #ffffff"
                     /label="U6 promoter"
ORIGIN
        1 gaattcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc gtgaccgcgc
       61 gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt
      121 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
      181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      301 tcttgggttt atatatcttg tggaaaggac gcgggatcca ctggaccagg cagcagcgtc
      361 agaagacttt tttggaacgt ctcgttttag agctaggcca acatgaggat cacccatgtc
      421 tgcagggcct agcaagttaa aataaggcta gtccgttatc aacttggcca acatgaggat
      481 cacccatgtc tgcagggcca agtggcaccg agtcggtgct ttttttggtg tacatttata
      541 ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt
      601 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      661 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      721 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      781 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      841 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      901 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      961 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     1021 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1081 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1141 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc
     1201 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg
     1261 ataagaaata ctcaatagga ctggctattg gcacaaatag cgtcggatgg gctgtgatca
     1321 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     1381 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta
     1441 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc
     1501 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg
     1561 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata
     1621 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat
     1681 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga
     1741 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg
     1801 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta
     1861 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac
     1921 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca
     1981 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     2041 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa
     2101 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc
     2161 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga
     2221 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac
     2281 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     2341 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa
     2401 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc
     2461 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     2521 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa
     2581 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt
     2641 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     2701 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     2761 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata
     2821 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag
     2881 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta
     2941 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     3001 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta
     3061 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga
     3121 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc
     3181 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt
     3241 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt
     3301 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt
     3361 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg
     3421 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga
     3481 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg
     3541 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga
     3601 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg
     3661 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata
     3721 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatgccattg
     3781 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata
     3841 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact
     3901 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca
     3961 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     4021 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     4081 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac
     4141 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc
     4201 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc
     4261 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga
     4321 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata
     4381 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     4441 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca
     4501 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga
     4561 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta
     4621 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt
     4681 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag
     4741 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc
     4801 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata
     4861 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa
     4921 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt
     4981 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     5041 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc
     5101 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa
     5161 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg
     5221 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag
     5281 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     5341 tgagtcagct gggaggtgac cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
     5401 ggaaagtgga cgacgcattg gacgattttg atctggatat gctgggaagt gacgccctcg
     5461 atgattttga ccttgacatg cttggttcgg atgcccttga tgactttgac ctcgacatgc
     5521 tcggcagtga cgcccttgat gatttcgacc tggacatgct gacgcgtacg cggccgctcg
     5581 agcagaaact catctcagaa gaggatctgg cagcaaatga tatcctggat tacaaggatg
     5641 acgacgataa ggtttaaacg gccggccgcg gtcatagctg tttcctgaac agatcccggg
     5701 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     5761 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     5821 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     5881 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     5941 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     6001 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gaggcggggt
     6061 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     6121 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     6181 ttaaaataac tataccagca ggaggacgtc cagacacagc ataggctacc tggccatgcc
     6241 caaccggtgg gacatttgag ttgcttgctt ggcactgtcc tctcatgcgt tgggtccact
     6301 cagtagatgc ctgttgaatt gggtacgcgg ccagcggcga gcggtatcag ctcactcaaa
     6361 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtccgtaaa
     6421 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6481 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6541 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6601 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6661 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6721 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6781 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6841 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6901 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6961 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7021 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgacgcg
     7081 taactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7141 tttgcggccg caaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7201 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7261 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7321 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7381 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7441 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7501 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7561 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7621 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7681 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7741 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7801 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7861 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7921 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7981 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8041 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8101 tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcca
     8161 gagctgccag gaaacagcta tgaccatgta atacgactca ctatagggga tatcagctgg
     8221 atggcagtta ac
//
