LOCUS       Exported                8481 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor DDIT3.
ACCESSION   .
VERSION     .
KEYWORDS    pDDIT3.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8481)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8481)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8481
                     /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          708..935
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      947..956
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             956..1021
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1028..1048
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1073..5173
                     /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             5174..5221
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5249..5266)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5255..5462
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5471..5611
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5471..5600
                     /label=AAV2 ITR
     rep_origin      5686..6141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5773..5792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5983..6004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6158..6177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6277..6299
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6337..6355)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6423..6527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6528..7388
                     /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(6746..6765)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7559..8147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8048..8067
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8209..8449
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8209..8229
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8380..8399
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8478..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 gggggggcga ggggcggggc ggggcgaggc
      601 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      661 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
      721 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
      781 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
      841 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
      901 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
      961 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     1021 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     1081 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1141 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1201 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1261 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1321 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1381 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1441 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1501 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1561 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1621 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1681 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1741 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     1801 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     1861 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     1921 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     1981 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2041 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2101 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2161 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2221 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2281 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2341 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2401 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2461 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2521 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2581 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2641 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     2701 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     2761 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     2821 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     2881 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     2941 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3001 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3061 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3121 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3181 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3241 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3301 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3361 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3421 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3481 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3541 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3601 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3661 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3721 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     3781 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     3841 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     3901 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     3961 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4021 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4081 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4141 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4201 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4261 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4321 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4381 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4441 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4501 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4561 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4621 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4681 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4741 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     4801 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     4861 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     4921 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     4981 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5041 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5101 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5161 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     5221 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5281 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5341 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5401 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     5461 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     5521 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     5581 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     5641 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     5701 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5761 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5821 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5881 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     5941 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6001 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     6061 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6121 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     6181 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6241 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6301 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6361 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6421 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6481 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6541 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6601 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6661 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6721 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6781 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6841 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6901 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6961 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     7021 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     7081 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     7141 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7201 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     7261 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7321 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7381 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7441 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7501 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7561 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7621 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7681 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7741 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7801 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7861 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7921 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7981 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     8041 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     8101 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     8161 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt
     8221 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     8281 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     8341 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     8401 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacacctgc
     8461 accaagcatg aacaattgtt t
//
