LOCUS       40924_595        9355 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector dCas9g1pac, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9355)
  AUTHORS   McInally SG, Hagen KD, Nosala C, Williams J, Nguyen K, Booker J, 
            Jones K, Dawson SC.
  TITLE     Robust and stable transcriptional repression in Giardia using 
            CRISPRi
  JOURNAL   Mol. Biol. Cell, mbcE18090605 (2018) In press
  PUBMED    30379614
REFERENCE   2  (bases 1 to 9355)
  AUTHORS   Hagen KD, McInally SG, Dawson SC.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-MAR-2018) Dept of Microbiology and Molecular Genetics,
            University of California, Davis, One Shields Ave, Davis, CA 95616, 
            USA
REFERENCE   3  (bases 1 to 9355)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9355)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Biol. 
            Cell, mbcE18090605 (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (08-MAR-2018) Dept of Microbiology and Molecular Genetics, 
            University of California, Davis, One Shields Ave, Davis, CA 95616, 
            USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    893..914
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        929..959
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    967..983
                     /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     991..1007
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1025..1043
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     5'UTR           1064..1204
                     /gene="malate dehydrogenase"
     gene            1064..1204
                     /gene="malate dehydrogenase"
                     /label=malate dehydrogenase
     CDS             1205..5308
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     misc_feature    5315..5335
                     /gene="dCas9"
                     /label=SV40 NLS
                     /note="SV40 NLS"
     CDS             5315..5335
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5350..5366)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5378..5404
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             5408..5434
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             5438..5464
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     misc_feature    5489..5590
                     /gene="dCas9"
                     /label=Giardia NLS
                     /note="Giardia NLS"
     3'UTR           5603..5727
                     /gene="alpha tubulin"
     gene            5603..5727
                     /gene="alpha tubulin"
                     /label=alpha tubulin
     5'UTR           5735..6008
                     /gene="gamma giardin"
     gene            5735..6008
                     /gene="gamma giardin"
                     /label=gamma giardin
     CDS             6015..6608
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     3'UTR           6618..6736
                     /gene="glutamate dehydrogenase"
     gene            6618..6736
                     /gene="glutamate dehydrogenase"
                     /label=glutamate dehydrogenase
     misc_feature    6949..6962
                     /label=region with BbsI sites for cloning
                     /note="region with BbsI sites for cloning"
     misc_RNA        6965..7040
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7267..7285)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7292..7308)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(7449..7904)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        8236..8340
                     /label=AmpR promoter
     CDS             8341..9198
                     /label=AmpR
                     /note="beta-lactamase"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      841 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      901 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      961 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1021 agctatttag gtgacactat agaatactca agcttggcgc gccctattag taagttgatc
     1081 tggcaccctt gtttctataa tgataagaat ctaagtagat atatacgtaa attgtaatta
     1141 gcagcatatc gtttgcattc gctaaatata gtatacgccc ttaaagccga aaattaaaat
     1201 tcccatggat aaaaagtatt ctattggttt agccatcggc actaattccg ttggatgggc
     1261 tgtcataacc gatgaataca aagtaccttc aaagaaattt aaggtgttgg ggaacacaga
     1321 ccgtcattcg attaaaaaga atcttatcgg tgccctccta ttcgatagtg gcgaaacggc
     1381 agaggcgact cgcctgaaac gaaccgctcg gagaaggtat acacgtcgca agaaccgaat
     1441 atgttactta caagaaattt ttagcaatga gatggccaaa gttgacgatt ctttctttca
     1501 ccgtttggaa gagtccttcc ttgtcgaaga ggacaagaaa catgaacggc accccatctt
     1561 tggaaacata gtagatgagg tggcatatca tgaaaagtac ccaacgattt atcacctcag
     1621 aaaaaagcta gttgactcaa ctgataaagc ggacctgagg ttaatctact tggctcttgc
     1681 ccatatgata aagttccgtg ggcactttct cattgagggt gatctaaatc cggacaactc
     1741 ggatgtcgac aaactgttca tccagttagt acaaacctat aatcagttgt ttgaagagaa
     1801 ccctataaat gcaagtggcg tggatgcgaa ggctattctt agcgcccgcc tctctaaatc
     1861 ccgacggcta gaaaacctga tcgcacaatt acccggagag aagaaaaatg ggttgttcgg
     1921 taaccttata gcgctctcac taggcctgac accaaatttt aagtcgaact tcgacttagc
     1981 tgaagatgcc aaattgcagc ttagtaagga cacgtacgat gacgatctcg acaatctact
     2041 ggcacaaatt ggagatcagt atgcggactt atttttggct gccaaaaacc ttagcgatgc
     2101 aatcctccta tctgacatac tgagagttaa tactgagatt accaaggcgc cgttatccgc
     2161 ttcaatgatc aaaaggtacg atgaacatca ccaagacttg acacttctca aggccctagt
     2221 ccgtcagcaa ctgcctgaga aatataagga aatattcttt gatcagtcga aaaacgggta
     2281 cgcaggttat attgacggcg gagcgagtca agaggaattc tacaagttta tcaaacccat
     2341 attagagaag atggatggga cggaagagtt gcttgtaaaa ctcaatcgcg aagatctact
     2401 gcgaaagcag cggactttcg acaacggtag cattccacat caaatccact taggcgaatt
     2461 gcatgctata cttagaaggc aggaggattt ttatccgttc ctcaaagaca atcgtgaaaa
     2521 gattgagaaa atcctaacct ttcgcatacc ttactatgtg ggacccctgg cccgagggaa
     2581 ctctcggttc gcatggatga caagaaagtc cgaagaaacg attactccct ggaattttga
     2641 ggaagttgtc gataaaggtg cgtcagctca atcgttcatc gagaggatga ccaactttga
     2701 caagaattta ccgaacgaaa aagtattgcc taagcacagt ttactttacg agtatttcac
     2761 agtgtacaat gaactcacga aagttaagta tgtcactgag ggcatgcgta aacccgcctt
     2821 tctaagcgga gaacagaaga aagcaatagt agatctgtta ttcaagacca accgcaaagt
     2881 gacagttaag caattgaaag aggactactt taagaaaatt gaatgcttcg attctgtcga
     2941 gatctccggg gtagaagatc gatttaatgc gtcacttggt acgtatcatg acctcctaaa
     3001 gataattaaa gataaggact tcctggataa cgaagagaat gaagatatct tagaagatat
     3061 agtgttgact cttaccctct ttgaagatcg ggaaatgatt gaggaaagac taaaaacata
     3121 cgctcacctg ttcgacgata aggttatgaa acagttaaag aggcgtcgct atacgggctg
     3181 gggacgattg tcgcggaaac ttatcaacgg gataagagac aagcaaagtg gtaaaactat
     3241 tctcgatttt ctaaagagcg acggcttcgc caataggaac tttatgcagc tgatccatga
     3301 tgactcttta accttcaaag aggatataca aaaggcacag gtttccggac aaggggactc
     3361 attgcacgaa catattgcga atcttgctgg ttcgccagcc atcaaaaagg gcatactcca
     3421 gacagtcaaa gtagtggatg agctagttaa ggtcatggga cgtcacaaac cggaaaacat
     3481 tgtaatcgag atggcacgcg aaaatcaaac gactcagaag gggcaaaaaa acagtcgaga
     3541 gcggatgaag agaatagaag agggtattaa agaactgggc agccagatct taaaggagca
     3601 tcctgtggaa aatacccaat tgcagaacga gaaactttac ctctattacc tacaaaatgg
     3661 aagggacatg tatgttgatc aggaactgga cataaaccgt ttatctgatt acgacgtcga
     3721 tgccattgta ccccaatcct ttttgaagga cgattcaatc gacaataaag tgcttacacg
     3781 ctcggataag aaccgaggga aaagtgacaa tgttccaagc gaggaagtcg taaagaaaat
     3841 gaagaactat tggcggcagc tcctaaatgc gaaactgata acgcaaagaa agttcgataa
     3901 cttaactaaa gctgagaggg gtggcttgtc tgaacttgac aaggccggat ttattaaacg
     3961 tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca cagatactag attcccgaat
     4021 gaatacgaaa tacgacgaga acgataagct gattcgggaa gtcaaagtaa tcactttaaa
     4081 gtcaaaattg gtgtcggact tcagaaagga ttttcaattc tataaagtta gggagataaa
     4141 taactaccac catgcgcacg acgcttatct taatgccgtc gtagggaccg cactcattaa
     4201 gaaatacccg aagctagaaa gtgagtttgt gtatggtgat tacaaagttt atgacgtccg
     4261 taagatgatc gcgaaaagcg aacaggagat aggcaaggct acagccaaat acttctttta
     4321 ttctaacatt atgaatttct ttaagacgga aatcactctg gcaaacggag agatacgcaa
     4381 acgaccttta attgaaacca atggggagac aggtgaaatc gtatgggata agggccggga
     4441 cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc aacatagtaa agaaaactga
     4501 ggtgcagacc ggagggtttt caaaggaatc gattcttcca aaaaggaata gtgataagct
     4561 catcgctcgt aaaaaggact gggacccgaa aaagtacggt ggcttcgata gccctacagt
     4621 tgcctattct gtcctagtag tggcaaaagt tgagaaggga aaatccaaga aactgaagtc
     4681 agtcaaagaa ttattgggga taacgattat ggagcgctcg tcttttgaaa agaaccccat
     4741 cgacttcctt gaggcgaaag gttacaagga agtaaaaaag gatctcataa ttaaactacc
     4801 aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg atgttggcta gcgccggaga
     4861 gcttcaaaag gggaacgaac tcgcactacc gtctaaatac gtgaatttcc tgtatttagc
     4921 gtcccattac gagaagttga aaggttcacc tgaagataac gaacagaagc aactttttgt
     4981 tgagcagcac aaacattatc tcgacgaaat catagagcaa atttcggaat tcagtaagag
     5041 agtcatccta gctgatgcca atctggacaa agtattaagc gcatacaaca agcacaggga
     5101 taaacccata cgtgagcagg cggaaaatat tatccatttg tttactctta ccaacctcgg
     5161 cgctccagcc gcattcaagt attttgacac aacgatagat cgcaaacgat acacttctac
     5221 caaggaggtg ctagacgcga cactgattca ccaatccatc acgggattat atgaaactcg
     5281 gatagatttg tcacagcttg ggggtgacgg atcccccaag aagaagagga aagtctcgag
     5341 caagcttatc gataccgtcg acctcgagct taaggaatat ccttatgacg tcccagacta
     5401 cgcggagtac ccatacgatg tacctgatta tgctgagtac ccatacgacg taccagatta
     5461 cgctggatcc gtggtgatac cggtcctccc agagaagaag cggtccaagt cctccaagtc
     5521 ctcacgcaaa gagaaaaaac gccacgagga cgagggtatg aaggatcgta gagttagtaa
     5581 aattaagagc taaagcggcc gcagtgaatt ctttccgtcg acctttgcat catcgttgct
     5641 cggtcgtctg cctcgtttac gacctgcttg ttcctgggcc tcaaatggtt agtggttttc
     5701 cttcttccaa cggcgttggt ccatcctcgg ccgcgtgatc aaatgccttc gagcgtatgt
     5761 ctgacctgtt tgtgaatgac gatccgtggc tttactcaga ttcctgtgtt tgtgaataag
     5821 aaatccgtga atttgttggg gtggaacggc gaagtggcct ctgctacaat aagaactgga
     5881 gagtgtgttc agaggcgaat gcgcaacgaa ctttgtcgtt acacgacgtg tgcgcctgtg
     5941 gaattcgtgc ggtttccgcg cggaattttg cgccttttct aaaaaactaa ccctaaaaca
     6001 agaaaatcat gggcaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     6061 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     6121 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     6181 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     6241 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     6301 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     6361 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     6421 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     6481 ctgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     6541 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     6601 ccggtgccct cgagtgaggg ctgaagtgaa tatttacctt ttccgggttc gacgggcgtg
     6661 cttgacgatg cggaagtcgc tgtccgttcc aacggctatc tgccccagga cccccatccc
     6721 ggcgccgatc agctggtacc ttataatgat tgcaatagca aacagtgtct atagtctaat
     6781 tgtggacaac agagggctta ttgcaacgtt gatgaccaag ttcaacaagg gcgtcgcctc
     6841 ctatgagcag gtcattcgga atttcgacga tcggtagcgt ccccagagta aaccatttta
     6901 aattgaaata ggcggttgga aataaaagcg cggcgtggtt aacaaagggt cttcgagaag
     6961 acctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     7021 aagtggcacc gagtcggtgc tccttttctc aatcttcgcc ggccggaccc gctggcgctc
     7081 cctactcgca ggggcgggtg tagacgaagt cccccctgtc ccagctgcct gcgattatgg
     7141 tgtcttggtc cagccacaga gcgcaggcga tcccagccgt ctggtaggtc tggtcgtcgg
     7201 gtacggcccg gcagacaccg atgctgtggc tgatgttgtc ttataaggta ccgagctcga
     7261 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     7321 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     7381 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     7441 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     7501 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     7561 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     7621 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     7681 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     7741 agggagcccc cgatttagag cttgacgggg acagccggcg aacgtggcga gaaaggaagg
     7801 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     7861 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcctgat gcggtatttt
     7921 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     7981 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     8041 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     8101 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     8161 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     8221 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     8281 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     8341 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     8401 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     8461 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     8521 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     8581 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     8641 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     8701 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     8761 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     8821 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     8881 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     8941 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     9001 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     9061 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     9121 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     9181 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     9241 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     9301 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaa
//
