LOCUS       pCFD3-dU6-3gRNA.        6365 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Drosophila expression vector in which a guide RNA (gRNA) is 
            expressed from the U6-3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pCFD3-dU6-3gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6365)
  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 6365)
  AUTHORS   Bullock Lab / Addgene #49410
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6365)
  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..6365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..427
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3
                     snRNA (Port et al., 2014)"
     misc_RNA        446..521
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      522..527
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     promoter        complement(832..850)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(871..887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(895..911)
                     /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(919..949)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(964..985)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1273..1861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2035..2892)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2893..2997)
                     /label=AmpR promoter
     rep_origin      complement(3023..3478)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     3620..3636
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3643..3661
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3853..3970,4047..4140,4232..4838,4893..5026,
                     5087..5247,5321..5346))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=tryptophan oxygenase
                     /note="vermilion"
                     /note="selectable marker, required to synthesize the brown
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    5661..5730
                     /label=attB
                     /note="attB site for the phi-C31 integrase (Groth et al.,
                     2000)"
     protein_bind    5973..6358
                     /label=gypsy insulator
                     /note="chromatin insulator from Drosophila"
ORIGIN
        1 gaattctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
       61 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
      121 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
      181 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
      241 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
      301 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
      361 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
      421 aacgtcgggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt aaaataaggc
      481 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgcc tacctggagc
      541 ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt gctttcgcca gtatttatta
      601 tttttcatca atatgtattc aatttggtat gtatttagta attgtaatat atagacaatg
      661 gttttccgtt gacgtacata catctgacgt gtgtttattt agacataata gttatgtttt
      721 cacatctttt taatgttcgc ttaatgcgta tgcattctag acaattgtgc tcggcaacag
      781 tatatttgtg gtgtgccaac caacaacctg caggagctcc agcttttgtt ccctttagtg
      841 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      901 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
      961 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1021 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1081 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1681 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     1741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2161 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2341 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2881 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3001 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     3061 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3121 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3181 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3241 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3301 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3361 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3421 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     3481 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     3541 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     3601 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     3661 ggcgaattgg gtacaagctt ggatttattt tgttatgtta tatgtattat atgtcagaca
     3721 taaagaaaag gaacacatca aatgtgataa caaagactaa acaagtaatt ttattacacc
     3781 aaaacgacaa aacagtaggc agaacaaaca acgcatagcc aaacattgac gaattggata
     3841 ccctgccgat tgtcagacac ttttgttgat cagtttcttg cgaatggtct cgtccagcgg
     3901 tggaatcgcc tcgcggggaa tcagaaaagt ggacagattg aacagatcca gaaacacctt
     3961 gtaccgatca ctgaaaccaa aaaaaaacaa agggagaaca gtttgagttc attgatcccc
     4021 gatataatca catctgcgat gatcacctga gagtggagcg cagatattga tatccagacg
     4081 agccaccagt gcccaactgt tgggatccaa tcatgcgttg caccatgatc acgtgattgt
     4141 ctgcggcggg aatagaaagt atttggttag gaaaaccagt cttaaacata agatatattt
     4201 ataaaagagt atcaaagaat gcaatactta catctccact tggttattaa cgagtcgatg
     4261 tccatgagca gggtgagcaa ctggtgtggt tggctgaacc tgggttcatc cctatagaag
     4321 gtgatcatga tggctccctg aagggcacga tggctaaacc ggcgatcccc acgacgcacc
     4381 agtgcatcgt gcactgccgg atcaaagatg gagcgataca cctcgcgtcg cttctcaatg
     4441 tccatgaggc ggtagttttt cgccttctcc acgggctcct ccatggcgct ctgtacctgc
     4501 gcctccagga atcgatcgac gctctcctga aacttggccc agaagttgaa gccactctcc
     4561 tccagtccgg gcgtcctctc cagccatcgc tgcactagct ccagtagcga gggatctttc
     4621 tccgagttgc gaatcgagtt ccgcgcctcc tcgtcgctaa agacatccga gtacttctgg
     4681 ttgtatctca cccgctgctc tgtcagaact cccagcttgt tctcgatcaa acggaactgc
     4741 agcgactgaa aaccagatgc gggtgccagg tacttgcgga agtccatgaa gtctagcggg
     4801 gtcatggtct ccagaatggg cacttggtcc accaggagct gtacaaagga agttataaac
     4861 ggattttggt aagagattca gaaagcactc acttttagaa tcagaaccac tcggttcagt
     4921 cgcttgacaa tctccagcgt cttggtttca tcgatgacct ctgcatccaa catgtctcgt
     4981 atggagtcga actcaaagat gatctgcttg aaccaaagct cgtaggctgt ggcgaaggta
     5041 cttaaatgcc attgagtgtt gtcatcaaag ttgtaaacct actcaccctg gtgcgtgatg
     5101 atgaacagat gctcatcgtg cacgggtcgc ttgtcctcct cggacagcat acactgggca
     5161 tccagcagtt tgtccagcat cagatactct ccatagattt tgcccacttc cgtggttaat
     5221 ggcaccgccg aatcatcgtg atcgtttctg tatgggtttg aattgaatcg cagaactgaa
     5281 gatcgattgg cattcctgga cagcacgtgc tggtgctcac ccgtttcctg catagggaca
     5341 gctcatggtg cacagctcag atcagatcgt gactcctcga gcggcggatg ctggcgaact
     5401 gatctccgcc agcggaccgg agatgagacc ccagcgaacc gataacagag cgagagagct
     5461 ccagttccga ctgattgcac agtcggtgat ctgggcgatg ggcactgcca gataggctgg
     5521 gaattatcaa tcacttgagg tgaaagtgcg gcgcacacaa atccaagctt gatatcatcg
     5581 atctcgaggc tgcatccaac gcgttgggag ctctccggat caattcggct tcaggtaccg
     5641 tcgacgatgt aggtcacggt ctcgaagccg cggtgcgggt gccagggcgt gcccttgggc
     5701 tccccgggcg cgtactccac ctcacccatc tggtccatca tgatgaacgg gtcgaggtgg
     5761 cggtagttga tcccggcgaa cgcgcggcgc accgggaagc cctcgccctc gaaaccgctg
     5821 ggcgcggtgg tcacggtgag cacgggacgt gcgacggcgt cggctgggtg cggatacgcg
     5881 gggcagcgtc agcgggttct cgacggtcac ggcgggcatg tcgacaagcc gaattgatcc
     5941 actagaaggc ctaattcggt acactagttg gccacgtaat aagtgtgcgt tgaatttatt
     6001 cgcaaaaaca ttgcatattt tcggcaaagt aaaattttgt tgcatacctt atcaaaaaat
     6061 aagtgctgca tactttttag agaaaccaaa taatttttta ttgcataccc gtttttaata
     6121 aaatacattg cataccctct tttaataaaa aatattgcat actttgacga aacaaatttt
     6181 cgttgcatac ccaataaaag attattatat tgcatacccg tttttaataa aatacattgc
     6241 ataccctctt ttaataaaga atattgcata cgttgacgaa acaaattttc gttgcatacc
     6301 caataaaaga ttattatatt gcataccttt tcttgccata ccatttagcc gatcaattga
     6361 gatct
//
