LOCUS       Exported                8463 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ISL1.
ACCESSION   .
VERSION     .
KEYWORDS    pISL1.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8463)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8463)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8463
                     /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          689..916
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      928..937
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             937..1002
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1009..1029
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1054..5154
                     /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             5155..5202
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5230..5247)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5236..5443
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5452..5592
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5452..5581
                     /label=AAV2 ITR
     rep_origin      5667..6122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5754..5773)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5964..5985
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6139..6158)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6258..6280
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6318..6336)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6404..6508
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6509..7369
                     /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(6727..6746)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7540..8128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8029..8048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8190..8430
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8190..8210
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8361..8380
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8460..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 cggggcgagg cggagaggtg cggcggcagc
      601 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
      661 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
      721 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      781 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
      841 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
      901 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac
      961 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag
     1021 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac
     1081 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     1141 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     1201 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     1261 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     1321 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     1381 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     1441 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     1501 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     1561 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     1621 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     1681 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     1741 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     1801 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     1861 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     1921 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     1981 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     2041 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     2101 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     2161 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     2221 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     2281 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     2341 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     2401 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     2461 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     2521 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     2581 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     2641 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     2701 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     2761 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     2821 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     2881 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     2941 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     3001 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     3061 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     3121 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     3181 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     3241 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     3301 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     3361 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     3421 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     3481 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     3541 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     3601 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     3661 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     3721 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     3781 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     3841 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     3901 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     3961 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     4021 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     4081 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     4141 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     4201 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     4261 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     4321 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     4381 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     4441 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     4501 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     4561 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     4621 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     4681 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     4741 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     4801 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     4861 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     4921 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     4981 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     5041 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     5101 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg
     5161 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt cctagagctc
     5221 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     5281 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     5341 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     5401 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc
     5461 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     5521 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     5581 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5641 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5701 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     5761 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5821 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5881 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5941 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     6001 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     6061 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     6121 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     6181 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6241 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6301 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     6361 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6421 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6481 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6541 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6601 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6661 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6721 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6781 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6841 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6901 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6961 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     7021 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     7081 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     7141 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     7201 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     7261 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7321 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7381 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7441 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7501 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7561 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7621 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7681 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7741 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7801 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7861 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7921 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7981 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     8041 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     8101 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     8161 ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt
     8221 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     8281 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     8341 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     8401 tcttggcttt atatatcttg tggaaaggac gaaacaccga atgttcctca tgcctcaatg
     8461 ttt
//
