LOCUS       pCas-Guide-AAVS1        8009 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian guide RNA (gRNA) vector for transgene integration at the 
            AAVS1 locus in the human genome. Use together with pAAVS1-puro-DNR.
ACCESSION   .
VERSION     .
KEYWORDS    pCas-Guide-AAVS1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8009)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8009)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        90..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        340..359
                     /label=AAVS1 target sequence
                     /note="AAVS1 target sequence"
     misc_RNA        360..435
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      436..441
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     enhancer        487..866
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        867..1070
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        1096..1114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1173..5276
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             5277..5297
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5304..5324
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             5346..5375
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             5394..5417
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     primer_bind     complement(5437..5453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    5464..6086
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(6207..6795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6973..7830)
                     /label=AmpR
                     /note="beta-lactamase"
     primer_bind     7945..7961
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 gcgggatcgg ttaatgtggc tctggttctg
      361 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
      421 gcaccgagtc ggtgcttttt ttggtgtaca tttatattgg ctcatgtcca atatgaccgc
      481 catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      541 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      601 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      661 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      721 tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc
      781 ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct
      841 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg
      901 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      961 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga
     1021 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
     1081 accgtcagaa ttttgtaata cgactcacta tagggcggcc gggaattcgt cgactggaac
     1141 cggtaccgag gagatctgcc gccgcgatcg ccatggataa gaaatactca ataggactgg
     1201 atattggcac aaatagcgtc ggatgggctg tgatcactga tgaatataag gttccttcta
     1261 aaaagttcaa ggttctggga aatacagacc gccacagtat caaaaaaaat cttatagggg
     1321 ctcttctgtt tgacagtgga gagacagccg aagctactag actcaaacgg acagctagga
     1381 gaaggtatac aagacggaag aataggattt gttatctcca ggagattttt tcaaatgaga
     1441 tggccaaagt ggatgatagt ttctttcata gacttgaaga gtcttttttg gtggaagaag
     1501 acaagaagca tgaaagacat cctatttttg gaaatatagt ggatgaagtt gcttatcacg
     1561 agaaatatcc aactatctat catctgagaa aaaaattggt ggattctact gataaagccg
     1621 atttgcgcct gatctatttg gccctggccc acatgattaa gtttagaggt cattttttga
     1681 ttgagggcga tctgaatcct gataatagtg atgtggacaa actgtttatc cagttggtgc
     1741 aaacctacaa tcaactgttt gaagaaaacc ctattaacgc aagtggagtg gatgctaaag
     1801 ccattctttc tgcaagattg agtaaatcaa gaagactgga aaatctcatt gctcagctcc
     1861 ccggtgagaa gaaaaatggc ctgtttggga atctcattgc tttgtcattg ggtttgaccc
     1921 ctaattttaa atcaaatttt gatttggcag aagatgctaa actccagctt tcaaaagata
     1981 cttacgatga tgatctggat aatctgttgg ctcaaattgg agatcaatat gctgatttgt
     2041 ttttggcagc taagaatctg tcagatgcta ttctgctttc agacatcctg agagtgaata
     2101 ctgaaataac taaggctccc ctgtcagctt caatgattaa acgctacgat gaacatcatc
     2161 aagacttgac tcttctgaaa gccctggtta gacaacaact tccagaaaag tataaagaaa
     2221 tcttttttga tcaatcaaaa aacggatatg caggttatat tgatggcggc gcaagccaag
     2281 aagaatttta taaatttatc aaaccaattc tggaaaaaat ggatggtact gaggaactgt
     2341 tggtgaaact gaatagagaa gatttgctgc gcaagcaacg gacctttgac aacggctcta
     2401 ttccccatca aattcacttg ggtgagctgc atgctatttt gagaagacaa gaagactttt
     2461 atccatttct gaaagacaat agagagaaga ttgaaaaaat cttgactttt aggattcctt
     2521 attatgttgg tccattggcc agaggcaata gtaggtttgc atggatgact cggaagtctg
     2581 aagaaacaat taccccatgg aattttgaag aagttgtcga taaaggtgct tcagctcaat
     2641 catttattga acgcatgaca aactttgata aaaatcttcc aaatgaaaaa gtgctgccaa
     2701 aacatagttt gctttatgag tattttaccg tttataacga attgacaaag gtcaaatatg
     2761 ttactgaagg aatgagaaaa ccagcatttc tttcaggtga acagaagaaa gccattgttg
     2821 atctgctctt caaaacaaat aggaaagtga ccgttaagca actgaaagaa gattatttca
     2881 aaaaaataga atgttttgat agtgttgaaa tttcaggagt tgaagataga tttaatgctt
     2941 cactgggtac ataccatgat ttgctgaaaa ttattaaaga taaagatttt ttggataatg
     3001 aagaaaatga agacatcctg gaggatattg ttctgacatt gaccctgttt gaagataggg
     3061 agatgattga ggaaagactt aaaacatacg ctcacctctt tgatgataag gtgatgaaac
     3121 agcttaaaag acgcagatat actggttggg gaaggttgtc cagaaaattg attaatggta
     3181 ttagggataa gcaatctggc aaaacaatac tggatttttt gaaatcagat ggttttgcca
     3241 atcgcaattt tatgcagctc atccatgatg atagtttgac atttaaagaa gacatccaaa
     3301 aagcacaagt gtctggacaa ggcgatagtc tgcatgaaca tattgcaaat ctggctggta
     3361 gccctgctat taaaaaaggt attctccaga ctgtgaaagt tgttgatgaa ttggtcaaag
     3421 tgatggggcg gcataagcca gaaaatatcg ttattgaaat ggcaagagaa aatcagacaa
     3481 ctcaaaaggg ccagaaaaat tccagagaga ggatgaaaag aatcgaagaa ggtatcaaag
     3541 aactgggaag tcagattctt aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa
     3601 agctctatct ctattatctc caaaatggaa gagatatgta tgtggaccaa gaactggata
     3661 ttaataggct gagtgattat gatgtcgatc acattgttcc acaaagtttc cttaaagacg
     3721 attcaataga caataaggtc ctgaccaggt ctgataaaaa tagaggtaaa tccgataacg
     3781 ttccaagtga agaagtggtc aaaaagatga aaaactattg gagacaactt ctgaacgcca
     3841 agctgatcac tcaaaggaag tttgataatc tgaccaaagc tgaaagagga ggtttgagtg
     3901 aacttgataa agctggtttt atcaaacgcc aattggttga aactcgccaa atcactaagc
     3961 atgtggcaca aattttggat agtcgcatga atactaaata cgatgaaaat gataaactta
     4021 ttagagaggt taaagtgatt accctgaaat ctaaactggt ttctgacttc agaaaagatt
     4081 tccaattcta taaagtgaga gagattaaca attaccatca tgcccatgat gcctatctga
     4141 atgccgtcgt tggaactgct ttgattaaga aatatccaaa acttgaaagc gagtttgtct
     4201 atggtgatta taaagtttat gatgttagga aaatgattgc taagtctgag caagaaatag
     4261 gcaaagcaac cgcaaagtat ttcttttact ctaatatcat gaacttcttc aaaacagaaa
     4321 ttacacttgc aaatggagag attcgcaaac gccctctgat cgaaactaat ggggaaactg
     4381 gagaaattgt ctgggataaa gggagagatt ttgccacagt gcgcaaagtg ttgtccatgc
     4441 cccaagtcaa tatcgtcaag aaaacagaag tgcagacagg cggattctct aaggagtcaa
     4501 ttctgccaaa aagaaattcc gacaagctga ttgctaggaa aaaagactgg gacccaaaaa
     4561 aatatggtgg ttttgatagt ccaaccgtgg cttattcagt cctggtggtt gctaaggtgg
     4621 aaaaagggaa atccaagaag ctgaaatccg ttaaagagct gctggggatc acaattatgg
     4681 aaagaagttc ctttgaaaaa aatcccattg actttctgga agctaaagga tataaggaag
     4741 ttaaaaaaga cctgatcatt aaactgccta aatatagtct ttttgagctg gaaaacggta
     4801 ggaaacggat gctggctagt gccggagaac tgcaaaaagg aaatgagctg gctctgccaa
     4861 gcaaatatgt gaattttctg tatctggcta gtcattatga aaagttgaag ggtagtccag
     4921 aagataacga acaaaaacaa ttgtttgtgg agcagcataa gcattatctg gatgagatta
     4981 ttgagcaaat cagtgaattt tctaagagag ttattctggc agatgccaat ctggataaag
     5041 ttcttagtgc atataacaaa catagagaca aaccaataag agaacaagca gaaaatatca
     5101 ttcatctgtt taccttgacc aatcttggag cacccgctgc ttttaaatac tttgatacaa
     5161 caattgatag gaaaagatat acctctacaa aagaagttct ggatgccact cttatccatc
     5221 aatccatcac tggtctttat gaaacacgca ttgatttgag tcagctggga ggtgacccca
     5281 agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa agtggacacg cgtacgcggc
     5341 cgctcgagca gaaactcatc tcagaagagg atctggcagc aaatgatatc ctggattaca
     5401 aggatgacga cgataaggtt taaacggccg gccgcggtca tagctgtttc ctgaacagat
     5461 cccgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     5521 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     5581 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     5641 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     5701 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     5761 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagagg
     5821 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     5881 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     5941 ctgattttaa aataactata ccagcaggag gacgtccaga cacagcatag gctacctggc
     6001 catgcccaac cggtgggaca tttgagttgc ttgcttggca ctgtcctctc atgcgttggg
     6061 tccactcagt agatgcctgt tgaattgggt acgcggccag cggcgagcgg tatcagctca
     6121 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtc
     6181 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6241 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6301 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6361 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6421 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6481 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6541 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6601 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6661 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6721 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6781 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6841 gacgcgtaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6901 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6961 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7021 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7081 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7141 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7201 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7261 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7321 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7381 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7441 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7501 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7561 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7621 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7681 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7741 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7801 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7861 ttatcagggt tattgtctca tgatgatatt attttatctt gtgcaatgta acatcagaga
     7921 ttttgagaca cgggccagag ctgccaggaa acagctatga ccatgtaata cgactcacta
     7981 taggggatat cagctggatg gcagttaac
//
