LOCUS       Exported                8470 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor E2F5.
ACCESSION   .
VERSION     .
KEYWORDS    pE2F5.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8470)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8470)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8470
                     /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          697..924
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      936..945
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             945..1010
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1017..1037
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1062..5162
                     /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             5163..5210
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5238..5255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5244..5451
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5460..5600
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5460..5589
                     /label=AAV2 ITR
     rep_origin      5675..6130
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5762..5781)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5972..5993
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6147..6166)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6266..6288
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6326..6344)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6412..6516
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6517..7377
                     /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(6735..6754)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7548..8136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8037..8056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8198..8438
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8198..8218
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8369..8388
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8467..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 cggggggggg gggcggggcg gggcgaggcg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      841 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      901 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg
      961 acggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa
     1021 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
     1081 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     1141 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     1201 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     1261 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     1321 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     1381 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     1441 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     1501 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1561 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1621 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     1681 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     1741 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     1801 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     1861 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     1921 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     1981 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     2041 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     2101 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     2161 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     2221 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     2281 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     2341 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     2401 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     2461 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     2521 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2581 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     2641 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     2701 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     2761 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     2821 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     2881 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     2941 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     3001 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     3061 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     3121 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     3181 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     3241 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     3301 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3361 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     3421 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     3481 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3541 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     3601 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     3661 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     3721 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     3781 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     3841 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     3901 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     3961 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     4021 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     4081 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     4141 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4201 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     4261 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     4321 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     4381 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     4441 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     4501 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     4561 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     4621 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     4681 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     4741 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     4801 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     4861 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     4921 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     4981 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     5041 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     5101 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     5161 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc
     5221 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     5281 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     5341 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     5401 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca
     5461 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     5521 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     5581 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5641 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     5701 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     5761 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5821 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5881 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5941 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6001 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     6061 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     6121 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6181 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     6241 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6301 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     6361 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     6421 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     6481 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6541 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6601 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6661 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6721 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6781 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6841 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6901 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6961 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     7021 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     7081 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     7141 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     7201 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     7261 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7321 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     7381 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     7441 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7501 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     7561 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     7621 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7681 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7741 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7801 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7861 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7921 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7981 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     8041 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     8101 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     8161 cggttcctgg ccttttgctg gccttttgct cacatgtgag ggcctatttc ccatgattcc
     8221 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     8281 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     8341 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     8401 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgaat ctaataattt
     8461 agtatcgttt
//
