LOCUS       40924_10491        7255 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Drosophila expression vector for simultaneously expressing two guide
            RNA (gRNA) sequences from the U6-1 and U6-3 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7255)
  AUTHORS   Port F, Chen HM, Lee T, Bullock SL.
  TITLE     Optimized CRISPR/Cas tools for efficient germline and somatic genome
            engineering in Drosophila.
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 2014;111:E2967-76.
  PUBMED    25002478
REFERENCE   2  (bases 1 to 7255)
  AUTHORS   Bullock Lab / Addgene #49411
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7255)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
            Acad. Sci. U.S.A."; date: "2014"; volume: "111"; pages: "E2967-76"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     212..228
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        235..253
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2253..2322
                     /label=attB
                     /note="attB site for the phi-C31 integrase (Groth et al.,
                     2000)"
     protein_bind    2564..2949
                     /label=gypsy insulator
                     /note="chromatin insulator from Drosophila"
     promoter        2963..3759
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1
                     snRNA (Port et al., 2014)"
     misc_RNA        3778..3853
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3854..4274
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3
                     snRNA (Port et al., 2014)"
     promoter        complement(4679..4697)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4718..4734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4742..4758)
                     /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)."
     promoter        complement(4766..4796)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4811..4832)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5120..5708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5882..6739)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6740..6844)
                     /label=AmpR promoter
     rep_origin      complement(join(6870..7255,1..70))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
       61 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
      121 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
      181 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
      241 gactcactat agggcgaatt gggtacaagc ttggatttat tttgttatgt tatatgtatt
      301 atatgtcaga cataaagaaa aggaacacat caaatgtgat aacaaagact aaacaagtaa
      361 ttttattaca ccaaaacgac aaaacagtag gcagaacaaa caacgcatag ccaaacattg
      421 acgaattgga taccctgccg attgtcagac acttttgttg atcagtttct tgcgaatggt
      481 ctcgtccagc ggtggaatcg cctcgcgggg aatcagaaaa gtggacagat tgaacagatc
      541 cagaaacacc ttgtaccgat cactgaaacc aaaaaaaaac aaagggagaa cagtttgagt
      601 tcattgatcc ccgatataat cacatctgcg atgatcacct gagagtggag cgcagatatt
      661 gatatccaga cgagccacca gtgcccaact gttgggatcc aatcatgcgt tgcaccatga
      721 tcacgtgatt gtctgcggcg ggaatagaaa gtatttggtt aggaaaacca gtcttaaaca
      781 taagatatat ttataaaaga gtatcaaaga atgcaatact tacatctcca cttggttatt
      841 aacgagtcga tgtccatgag cagggtgagc aactggtgtg gttggctgaa cctgggttca
      901 tccctataga aggtgatcat gatggctccc tgaagggcac gatggctaaa ccggcgatcc
      961 ccacgacgca ccagtgcatc gtgcactgcc ggatcaaaga tggagcgata cacctcgcgt
     1021 cgcttctcaa tgtccatgag gcggtagttt ttcgccttct ccacgggctc ctccatggcg
     1081 ctctgtacct gcgcctccag gaatcgatcg acgctctcct gaaacttggc ccagaagttg
     1141 aagccactct cctccagtcc gggcgtcctc tccagccatc gctgcactag ctccagtagc
     1201 gagggatctt tctccgagtt gcgaatcgag ttccgcgcct cctcgtcgct aaagacatcc
     1261 gagtacttct ggttgtatct cacccgctgc tctgtcagaa ctcccagctt gttctcgatc
     1321 aaacggaact gcagcgactg aaaaccagat gcgggtgcca ggtacttgcg gaagtccatg
     1381 aagtctagcg gggtcatggt ctccagaatg ggcacttggt ccaccaggag ctgtacaaag
     1441 gaagttataa acggattttg gtaagagatt cagaaagcac tcacttttag aatcagaacc
     1501 actcggttca gtcgcttgac aatctccagc gtcttggttt catcgatgac ctctgcatcc
     1561 aacatgtctc gtatggagtc gaactcaaag atgatctgct tgaaccaaag ctcgtaggct
     1621 gtggcgaagg tacttaaatg ccattgagtg ttgtcatcaa agttgtaaac ctactcaccc
     1681 tggtgcgtga tgatgaacag atgctcatcg tgcacgggtc gcttgtcctc ctcggacagc
     1741 atacactggg catccagcag tttgtccagc atcagatact ctccatagat tttgcccact
     1801 tccgtggtta atggcaccgc cgaatcatcg tgatcgtttc tgtatgggtt tgaattgaat
     1861 cgcagaactg aagatcgatt ggcattcctg gacagcacgt gctggtgctc acccgtttcc
     1921 tgcataggga cagctcatgg tgcacagctc agatcagatc gtgactcctc gagcggcgga
     1981 tgctggcgaa ctgatctccg ccagcggacc ggagatgaga ccccagcgaa ccgataacag
     2041 agcgagagag ctccagttcc gactgattgc acagtcggtg atctgggcga tgggcactgc
     2101 cagataggct gggaattatc aatcacttga ggtgaaagtg cggcgcacac aaatccaagc
     2161 ttgatatcat cgatctcgag gctgcatcca acgcgttggg agctctccgg atcaattcgg
     2221 cttcaggtac cgtcgacgat gtaggtcacg gtctcgaagc cgcggtgcgg gtgccagggc
     2281 gtgcccttgg gctccccggg cgcgtactcc acctcaccca tctggtccat catgatgaac
     2341 gggtcgaggt ggcggtagtt gatcccggcg aacgcgcggc gcaccgggaa gccctcgccc
     2401 tcgaaaccgc tgggcgcggt ggtcacggtg agcacgggac gtgcgacggc gtcggctggt
     2461 gcggatacgc ggggcagcgt cagcgggttc tcgacggtca cggcgggcat gtcgacaagc
     2521 cgaattgatc cactagaagg cctaattcgg tacactagtt ggccacgtaa taagtgtgcg
     2581 ttgaatttat tcgcaaaaac attgcatatt ttcggcaaag taaaattttg ttgcatacct
     2641 tatcaaaaaa taagtgctgc atacttttta gagaaaccaa ataatttttt attgcatacc
     2701 cgtttttaat aaaatacatt gcataccctc ttttaataaa aaatattgca tactttgacg
     2761 aaacaaattt tcgttgcata cccaataaaa gattattata ttgcataccc gtttttaata
     2821 aaatacattg cataccctct tttaataaag aatattgcat acgttgacga aacaaatttt
     2881 cgttgcatac ccaataaaag attattatat tgcatacctt ttcttgccat accatttagc
     2941 cgatcaattg agatctgaat tcattttcaa cgtcctcgat agtatagtgg ttagtatccc
     3001 cgcctgtcac gcgggagacc ggggttcaat tccccgtcgg ggagaatctg tgattctttt
     3061 tttttttctt ttactttgtt atataaacaa tttttgtttt aattgaatct aatttgccat
     3121 tgcttttagg aatctcaggc atccagcaag cgtttgtccg ccgaatcgcc catcagtgaa
     3181 gaagatcctg tggcggctac gaaaatctcc ccggccatgt cggcctccac ctccagcgaa
     3241 aaacccatca gcgagctggc cacctctgtg ctgacccacc gctttccaga ctccacctcc
     3301 tcacccggcg aacatggcct tggacgaatg cagttgtcga tccgctacag cgcccagcgt
     3361 caaaaactag acgtgaccat acacaaaatc cagaagatac cacttcgcga tcccagcaat
     3421 atccccgatc cgtatgttaa gctgtatctg ttgcctggac gcaccaagga gtcgaaacgc
     3481 aagacgagcg tgatcaagga caactgcaac cccgtctacg atgcatcctt tgagtacctg
     3541 atttccattg ccgaactcag gcagacggaa ctggaggtga cggtgtgcac ccaaaaggga
     3601 ttcctatccg gcggtagtcc catcattggc atggtaggta cccgaaagca accccttagt
     3661 tacagacaca gcgcgtacgt ccttcgcatc cttatgattc ccaagtacat attctgcaag
     3721 agtacagtat atataggaaa gatatccggg tgaacttcgg ggtcttcgag aagacctgtt
     3781 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3841 accgagtcgg tgcttttttg ctcacctgtg attgctccta ctcaaataca aaaacatcaa
     3901 attttctgtc aataaagcat atttatttat atttatttta caggaaagaa ttccttttaa
     3961 agtgtatttt aacctataat gaaaaacgat taaaaaaaat acataaaata attcgaaaat
     4021 ttttgaatag cccaggttga taaaaattca tttcatacgt tttataactt atgcccctaa
     4081 gtattttttg accatagtgt ttcaattcta cattaatttt acagagtaga atgaaacgcc
     4141 acctactcag ccaagaggcg aaaaggttag ctcgccaagc agagagggcg ccagtgctca
     4201 ctacttttta taattctcaa cttctttttc cagactcagt tcgtatatat agacctattt
     4261 tcaatttaac gtcggggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa
     4321 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgcctac
     4381 ctggagcctg agagttgttc aataaaataa aaatgtttcg tttttttgct ttcgccagta
     4441 tttattattt ttcatcaata tgtattcaat ttggtatgta tttagtaatt gtaatatata
     4501 gacaatggtt ttccgttgac gtacatacat ctgacgtgtg tttatttaga cataatagtt
     4561 atgttttcac atctttttaa tgttcgctta atgcgtatgc attctagaca attgtgctcg
     4621 gcaacagtat atttgtggtg tgccaaccaa caacctgcag gagctccagc ttttgttccc
     4681 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4741 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4801 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4861 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4921 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4981 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5041 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5101 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5161 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5221 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5281 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5341 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5401 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5461 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5521 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5581 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5641 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5701 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5761 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5821 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5881 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5941 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6001 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6061 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6121 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6181 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6241 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6301 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6361 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6421 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6481 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6541 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6601 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6661 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6721 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6781 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6841 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     6901 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     6961 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     7021 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     7081 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     7141 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     7201 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctg
//
