LOCUS       Exported                8451 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ATF4.
ACCESSION   .
VERSION     .
KEYWORDS    pATF4.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8451
                     /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          677..904
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      916..925
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             925..990
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             997..1017
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1042..5142
                     /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             5143..5190
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5218..5235)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5224..5431
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5440..5580
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5440..5569
                     /label=AAV2 ITR
     rep_origin      5655..6110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5742..5761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5952..5973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6127..6146)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6246..6268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6306..6324)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6392..6496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6497..7357
                     /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(6715..6734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7528..8116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8017..8036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8178..8418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8178..8198
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8349..8368
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8448..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 gggaggtgcg gcggcagcca atcagagcgg
      601 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
      661 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
      721 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
      781 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg
      841 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt
      901 tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat
      961 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt
     1021 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac
     1081 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     1141 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     1201 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     1261 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     1321 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     1381 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     1441 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     1501 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     1561 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     1621 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     1681 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     1741 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     1801 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     1861 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     1921 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     1981 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     2041 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     2101 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     2161 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     2221 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     2281 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     2341 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     2401 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     2461 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     2521 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     2581 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     2641 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     2701 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     2761 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     2821 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     2881 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     2941 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     3001 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     3061 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     3121 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     3181 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     3241 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     3301 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     3361 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     3421 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     3481 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     3541 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac
     3601 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     3661 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     3721 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     3781 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     3841 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     3901 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     3961 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     4021 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     4081 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     4141 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     4201 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     4261 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     4321 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     4381 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     4441 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     4501 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     4561 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     4621 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     4681 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     4741 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     4801 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     4861 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     4921 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     4981 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     5041 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     5101 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg
     5161 aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct
     5221 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     5281 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5341 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5401 attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
     5461 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     5521 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     5581 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5641 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5701 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     5761 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5821 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5881 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5941 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     6001 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     6061 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     6121 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     6181 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6241 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     6301 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     6361 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6421 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6481 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6541 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6601 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6661 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6721 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6781 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6841 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6901 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6961 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     7021 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     7081 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     7141 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     7201 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7261 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7321 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7381 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7441 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7501 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7561 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7621 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7681 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7741 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7801 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7861 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7921 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7981 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     8041 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     8101 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     8161 gccttttgct cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     8221 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     8281 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     8341 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     8401 atatcttgtg gaaaggacga aacaccgtcc cctagttgag gatagtcgtt t
//
