LOCUS       Exported                6617 bp ds-DNA     circular SYN 10-JAN-2018
DEFINITION  multiplex gRNA plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    pCFD5
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6617)
  AUTHORS   Port F, Bullock SL
  TITLE     Augmenting CRISPR applications in Drosophila with tRNA-flanked 
            sgRNAs.
  JOURNAL   Nat Methods. 2016 Oct;13(10):852-4. doi: 10.1038/nmeth.3972. Epub 
            2016 Sep 5.
  PUBMED    27595403
REFERENCE   2  (bases 1 to 6617)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6815
                     /dnas_title="pCFD5-frame0,1,2"
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(25..42)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(196..784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     complement(276..295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(955..1815)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     primer_bind     1578..1597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1816..1920)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1947..2403
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2034..2053)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2244..2265
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2529..2551
                     /label="M13/pUC Forward"
                     /note="In lacZ gene"
     primer_bind     2543..2560
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     2544..2560
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     2570..2589
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2570..2588
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(2780..2897,2974..3067,3159..3765,
                     3820..3953,4014..4174,4248..4273))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=vermilion
                     /note="selectable marker, required to synthesize the
                     brown eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLS
                     EEDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRV
                     VLILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYN
                     QKYSDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFL
                     EAQVQSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGA
                     IMITFYRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQ
                     YLRSTLSDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    4588..4657
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al.,
                     2000)"
     protein_bind    4899..5284
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, and Mod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        5298..5718
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3
                     snRNA (Port et al., 2014)"
     misc_RNA        6184..6259
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     primer_bind     complement(6570..6590)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6570..6588)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6609..6625)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     complement(6609..6625)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6622..6644)
                     /label="M13/pUC Reverse"
                     /note="In lacZ gene"
     protein_bind    6633..6649
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(6657..6687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6702..6723
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the
                     presence of cAMP."
     misc_feature    5786..5814
                     /note="overlap with fragment"
     misc_feature    6184..6213
                     /note="overlap with fragment"
     misc_RNA        5836..5911
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     misc_feature    5816..5835
                     /note="frame 0"
     misc_RNA        6010..6085
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     misc_feature    6164..6183
                     /note="frame 2"
     misc_feature    5990..6009
                     /note="frame 1"
     misc_feature    5786..5858
                     /note="frag1F"
     misc_feature    5967..6009
                     /note="frag1R"
     misc_feature    5990..6032
                     /note="frag2F"
     misc_feature    6141..6213
                     /note="frag2R"
     misc_feature    5585..5603
                     /note=""
ORIGIN      
        1 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
       61 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      121 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      661 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      901 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1081 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1261 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1801 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1921 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg
     1981 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2041 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2101 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2161 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2221 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2281 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2341 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2401 tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     2461 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     2521 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     2581 actatagggc gaattgggta caagcttgga tttattttgt tatgttatat gtattatatg
     2641 tcagacataa agaaaaggaa cacatcaaat gtgataacaa agactaaaca agtaatttta
     2701 ttacaccaaa acgacaaaac agtaggcaga acaaacaacg catagccaaa cattgacgaa
     2761 ttggataccc tgccgattgt cagacacttt tgttgatcag tttcttgcga atggtctcgt
     2821 ccagcggtgg aatcgcctcg cggggaatca gaaaagtgga cagattgaac agatccagaa
     2881 acaccttgta ccgatcactg aaaccaaaaa aaaacaaagg gagaacagtt tgagttcatt
     2941 gatccccgat ataatcacat ctgcgatgat cacctgagag tggagcgcag atattgatat
     3001 ccagacgagc caccagtgcc caactgttgg gatccaatca tgcgttgcac catgatcacg
     3061 tgattgtctg cggcgggaat agaaagtatt tggttaggaa aaccagtctt aaacataaga
     3121 tatatttata aaagagtatc aaagaatgca atacttacat ctccacttgg ttattaacga
     3181 gtcgatgtcc atgagcaggg tgagcaactg gtgtggttgg ctgaacctgg gttcatccct
     3241 atagaaggtg atcatgatgg ctccctgaag ggcacgatgg ctaaaccggc gatccccacg
     3301 acgcaccagt gcatcgtgca ctgccggatc aaagatggag cgatacacct cgcgtcgctt
     3361 ctcaatgtcc atgaggcggt agtttttcgc cttctccacg ggctcctcca tggcgctctg
     3421 tacctgcgcc tccaggaatc gatcgacgct ctcctgaaac ttggcccaga agttgaagcc
     3481 actctcctcc agtccgggcg tcctctccag ccatcgctgc actagctcca gtagcgaggg
     3541 atctttctcc gagttgcgaa tcgagttccg cgcctcctcg tcgctaaaga catccgagta
     3601 cttctggttg tatctcaccc gctgctctgt cagaactccc agcttgttct cgatcaaacg
     3661 gaactgcagc gactgaaaac cagatgcggg tgccaggtac ttgcggaagt ccatgaagtc
     3721 tagcggggtc atggtctcca gaatgggcac ttggtccacc aggagctgta caaaggaagt
     3781 tataaacgga ttttggtaag agattcagaa agcactcact tttagaatca gaaccactcg
     3841 gttcagtcgc ttgacaatct ccagcgtctt ggtttcatcg atgacctctg catccaacat
     3901 gtctcgtatg gagtcgaact caaagatgat ctgcttgaac caaagctcgt aggctgtggc
     3961 gaaggtactt aaatgccatt gagtgttgtc atcaaagttg taaacctact caccctggtg
     4021 cgtgatgatg aacagatgct catcgtgcac gggtcgcttg tcctcctcgg acagcataca
     4081 ctgggcatcc agcagtttgt ccagcatcag atactctcca tagattttgc ccacttccgt
     4141 ggttaatggc accgccgaat catcgtgatc gtttctgtat gggtttgaat tgaatcgcag
     4201 aactgaagat cgattggcat tcctggacag cacgtgctgg tgctcacccg tttcctgcat
     4261 agggacagct catggtgcac agctcagatc agatcgtgac tcctcgagcg gcggatgctg
     4321 gcgaactgat ctccgccagc ggaccggaga tgagacccca gcgaaccgat aacagagcga
     4381 gagagctcca gttccgactg attgcacagt cggtgatctg ggcgatgggc actgccagat
     4441 aggctgggaa ttatcaatca cttgaggtga aagtgcggcg cacacaaatc caagcttgat
     4501 atcatcgatc tcgaggctgc atccaacgcg ttgggagctc tccggatcaa ttcggcttca
     4561 ggtaccgtcg acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc
     4621 cttgggctcc ccgggcgcgt actccacctc acccatctgg tccatcatga tgaacgggtc
     4681 gaggtggcgg tagttgatcc cggcgaacgc gcggcgcacc gggaagccct cgccctcgaa
     4741 accgctgggc gcggtggtca cggtgagcac gggacgtgcg acggcgtcgg ctggtgcgga
     4801 tacgcggggc agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga caagccgaat
     4861 tgatccacta gaaggcctaa ttcggtacac tagttggcca cgtaataagt gtgcgttgaa
     4921 tttattcgca aaaacattgc atattttcgg caaagtaaaa ttttgttgca taccttatca
     4981 aaaaataagt gctgcatact ttttagagaa accaaataat tttttattgc atacccgttt
     5041 ttaataaaat acattgcata ccctctttta ataaaaaata ttgcatactt tgacgaaaca
     5101 aattttcgtt gcatacccaa taaaagatta ttatattgca tacccgtttt taataaaata
     5161 cattgcatac cctcttttaa taaagaatat tgcatacgtt gacgaaacaa attttcgttg
     5221 catacccaat aaaagattat tatattgcat accttttctt gccataccat ttagccgatc
     5281 aattgagatc tgaattcttt tttgctcacc tgtgattgct cctactcaaa tacaaaaaca
     5341 tcaaattttc tgtcaataaa gcatatttat ttatatttat tttacaggaa agaattcctt
     5401 ttaaagtgta ttttaaccta taatgaaaaa cgattaaaaa aaatacataa aataattcga
     5461 aaatttttga atagcccagg ttgataaaaa ttcatttcat acgttttata acttatgccc
     5521 ctaagtattt tttgaccata gtgtttcaat tctacattaa ttttacagag tagaatgaaa
     5581 cgccacctac tcagccaaga ggcgaaaagg ttagctcgcc aagcagagag ggcgccagtg
     5641 ctcactactt tttataattc tcaacttctt tttccagact cagttcgtat atatagacct
     5701 attttcaatt taacgtcggg gctttgagtg tgtgtagaca tcaagcatcg gtggttcagt
     5761 ggtagaatgc tcgcctgcca cgcgggcggc ccgggttcga ttcccggccg atgcagccag
     5821 tacccaaaaa gcggggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     5881 tcaacttgaa aaagtggcac cgagtcggtg ctaacaaagc accagtggtc tagtggtaga
     5941 atagtaccct gccacggtac agacccgggt tcgattcccg gctggtgcag gccagtaccc
     6001 aaaaagcggg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     6061 tgaaaaagtg gcaccgagtc ggtgctaaca aagcaccagt ggtctagtgg tagaatagta
     6121 ccctgccacg gtacagaccc gggttcgatt cccggctggt gcagggccag tacccaaaaa
     6181 gcggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     6241 agtggcaccg agtcggtgct tttttgccta cctggagcct gagagttgtt caataaaata
     6301 aaaatgtttc gtttttttgc tttcgccagt atttattatt tttcatcaat atgtattcaa
     6361 tttggtatgt atttagtaat tgtaatatat agacaatggt tttccgttga cgtacataca
     6421 tctgacgtgt gtttatttag acataatagt tatgttttca catcttttta atgttcgctt
     6481 aatgcgtatg cattctagac aattgtgctc ggcaacagta tatttgtggt gtgccaacca
     6541 acaacctgca ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     6601 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6661 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6721 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6781 taatgaatcg gccaacgcgc ggggagaggc ggttt
//

