LOCUS       Exported                8475 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor TOX2.
ACCESSION   .
VERSION     .
KEYWORDS    pTOX2.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8475)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8475)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8475
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        169..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          701..928
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      940..949
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             949..1014
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1021..1041
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1066..5166
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5167..5214
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5242..5259)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5248..5455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5464..5604
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5464..5593
                     /label=AAV2 ITR
     rep_origin      5679..6134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5766..5785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5976..5997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6151..6170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6270..6292
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6330..6348)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6416..6520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6521..7381
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6739..6758)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7552..8140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8041..8060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8202..8442
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8202..8222
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8373..8392
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8472..72
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
       61 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      121 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact
      181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
      481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      541 attttgtgca gcgatggggg cggggggngg cgaggggcgg ggcggggcga ggcggagagg
      601 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg
      661 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct
      721 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc
      781 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag
      841 caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga
      901 gcacctgcct gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac
      961 cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc
     1021 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc
     1081 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     1141 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     1201 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     1261 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     1321 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     1381 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     1441 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     1501 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     1561 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     1621 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     1681 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     1741 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     1801 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     1861 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     1921 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     1981 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     2041 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     2101 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     2161 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     2221 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     2281 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     2341 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     2401 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     2461 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     2521 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     2581 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     2641 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     2701 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     2761 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     2821 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     2881 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     2941 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     3001 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     3061 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     3121 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     3181 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     3241 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     3301 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     3361 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     3421 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     3481 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     3541 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     3601 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     3661 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     3721 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     3781 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     3841 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     3901 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     3961 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     4021 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     4081 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     4141 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     4201 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     4261 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     4321 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     4381 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     4441 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
     4501 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     4561 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     4621 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     4681 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
     4741 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     4801 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     4861 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     4921 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     4981 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     5041 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
     5101 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     5161 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa
     5221 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     5281 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5341 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     5401 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc
     5461 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     5521 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     5581 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     5641 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     5701 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     5761 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5821 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5881 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5941 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6001 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     6061 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6121 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6181 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     6241 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     6301 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     6361 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     6421 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6481 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6541 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6601 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6661 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6721 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6781 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6841 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6901 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6961 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     7021 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     7081 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     7141 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     7201 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     7261 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     7321 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     7381 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     7441 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7501 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7561 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7621 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7681 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7741 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7801 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7861 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7921 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7981 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     8041 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     8101 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     8161 tttacggttc ctggcctttt gctggccttt tgctcacatg tgagggccta tttcccatga
     8221 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     8281 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     8341 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     8401 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gtagggaggc
     8461 gggattaggt ggttt
//
