LOCUS       Exported                8473 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor SPDEF.
ACCESSION   .
VERSION     .
KEYWORDS    pSPDEF.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8473)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8473)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8473
                     /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          699..926
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      938..947
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             947..1012
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1019..1039
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1064..5164
                     /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             5165..5212
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5240..5257)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5246..5453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5462..5602
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5462..5591
                     /label=AAV2 ITR
     rep_origin      5677..6132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5764..5783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5974..5995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6149..6168)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6268..6290
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6328..6346)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6414..6518
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6519..7379
                     /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(6737..6756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7550..8138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8039..8058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8200..8440
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8200..8220
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8371..8390
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8470..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 gggggcgggg cggggcgagg cggagaggtg
      601 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
      661 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
      721 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
      781 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
      841 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
      901 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
      961 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     1021 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     1081 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     1141 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     1201 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     1261 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     1321 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     1381 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     1441 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     1501 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     1561 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     1621 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     1681 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     1741 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     1801 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     1861 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     1921 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     1981 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     2041 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     2101 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     2161 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     2221 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     2281 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     2341 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     2401 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     2461 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     2521 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     2581 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     2641 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     2701 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     2761 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     2821 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     2881 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     2941 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     3001 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     3061 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     3121 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     3181 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     3241 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     3301 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     3361 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     3421 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     3481 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     3541 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     3601 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     3661 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     3721 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     3781 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     3841 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     3901 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     3961 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     4021 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     4081 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     4141 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     4201 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     4261 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     4321 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     4381 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     4441 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     4501 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     4561 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     4621 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     4681 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     4741 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     4801 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     4861 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     4921 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     4981 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     5041 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     5101 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     5161 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt
     5221 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5281 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     5341 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     5401 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     5461 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5521 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5581 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5641 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5701 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5761 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5821 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5881 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5941 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     6001 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     6061 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6121 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6181 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6241 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6301 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6361 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6421 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6481 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6541 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6601 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6661 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6721 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6781 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6841 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6901 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6961 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7021 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7081 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7141 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7201 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7261 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7321 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7381 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7441 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7501 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7561 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7621 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7681 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7741 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7801 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7861 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7921 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7981 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8041 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8101 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8161 tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt
     8221 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     8281 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     8341 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     8401 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc tgggccaggc
     8461 actcagatgg ttt
//
