LOCUS       Exported                8508 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor SMAD3.
ACCESSION   .
VERSION     .
KEYWORDS    pSMAD3.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8508)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8508)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8508
                     /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"
     promoter        456..733
                     /label=chicken beta-actin promoter
     intron          734..962
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      974..983
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             983..1048
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1055..1075
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1100..5200
                     /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             5201..5248
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5276..5293)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5282..5489
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5498..5638
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5498..5627
                     /label=AAV2 ITR
     rep_origin      5713..6168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5800..5819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6010..6031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6185..6204)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6304..6326
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6364..6382)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6450..6554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6555..7415
                     /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(6773..6792)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7586..8174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8075..8094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8236..8476
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8236..8256
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8407..8426
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8505..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 gggggggcgc gcgccaggcg gggcggggcg
      601 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      661 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      721 cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg
      781 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
      841 acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt
      901 tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc
      961 aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga
     1021 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat
     1081 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc
     1141 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     1201 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     1261 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     1321 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     1381 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     1441 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     1501 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     1561 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     1621 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     1681 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     1741 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     1801 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     1861 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     1921 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     1981 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     2041 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     2101 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     2161 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     2221 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     2281 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     2341 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     2401 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     2461 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     2521 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     2581 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     2641 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     2701 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     2761 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     2821 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     2881 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     2941 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     3001 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     3061 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     3121 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     3181 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     3241 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     3301 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     3361 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     3421 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     3481 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     3541 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     3601 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     3661 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     3721 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     3781 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     3841 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     3901 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     3961 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     4021 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     4081 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     4141 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     4201 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     4261 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     4321 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     4381 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     4441 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     4501 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     4561 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     4621 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     4681 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     4741 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     4801 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     4861 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     4921 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     4981 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     5041 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     5101 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     5161 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa
     5221 aaaggccggc caggcaaaaa agaaaaagta agaattccta gagctcgctg atcagcctcg
     5281 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5341 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     5401 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     5461 tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg
     5521 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     5581 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     5641 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     5701 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5761 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5821 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5881 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     5941 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     6001 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     6061 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6121 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     6181 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     6241 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6301 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     6361 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6421 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6481 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6541 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6601 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6661 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6721 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6781 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6841 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6901 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6961 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     7021 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7081 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7141 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7201 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7261 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7321 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7381 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7441 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     7501 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     7561 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     7621 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     7681 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     7741 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7801 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7861 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7921 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7981 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     8041 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     8101 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     8161 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     8221 cttttgctca catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac
     8281 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     8341 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     8401 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     8461 atcttgtgga aaggacgaaa cacctctaag acacactgga acaggttt
//
