LOCUS       gRNA_GFP-T2.        3973 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for guide RNA (gRNA) targeting of an integrated GFP gene in 
            human cells using the GFP T2 target sequence.
ACCESSION   .
VERSION     .
KEYWORDS    gRNA_GFP-T2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE,
            Church GM.
  TITLE     RNA-guided human genome engineering via Cas9.
  JOURNAL   Science 2013;339:823-6.
  PUBMED    23287722
REFERENCE   2  (bases 1 to 3973)
  AUTHORS   Church Lab / Addgene #41820
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3973)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science"; 
            date: "2013"; volume: "339"; pages: "823-6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     promoter        406..646
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    655..674
                     /label=GFP T2
                     /note="GFP T2"
                     /note="GFP gRNA Target 2 sequence"
     misc_RNA        675..750
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      751..756
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     promoter        complement(862..880)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(887..903)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1041..1340
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     CDS             1692..2483
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     CDS             2692..3063
                     /label=BleoR
                     /note="antibiotic-binding protein"
     rep_origin      3204..3792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gccctttgta caaaaaagca ggctttaaag
      361 gaaccaattc agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc
      421 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
      481 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      541 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      601 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagcg
      661 caccatcttc ttcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
      721 caacttgaaa aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa
      781 agttggcatt aaagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg
      841 catctagagg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
      901 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
      961 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     1021 tgcgcagcct atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc
     1081 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc
     1141 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     1201 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     1261 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1321 acctgatgtt ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct
     1381 agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca
     1441 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca
     1501 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat
     1561 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     1621 tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag
     1681 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     1741 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     1801 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     1861 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     1921 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     1981 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     2041 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     2101 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     2161 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     2221 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2281 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     2341 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     2401 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     2461 atcgccttct tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt
     2521 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat
     2581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2641 agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag
     2701 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     2761 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     2821 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     2881 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     2941 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3001 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3061 gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3121 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3961 gaggaagcgg aag
//
