LOCUS       Exported                8508 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF331.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF331.1.0-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 12, 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"
     intron          734..961
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      973..982
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             982..1047
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1054..1074
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1099..5199
                     /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             5200..5247
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5275..5292)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5281..5488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5497..5637
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5497..5626
                     /label=AAV2 ITR
     rep_origin      5712..6167
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5799..5818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6009..6030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6184..6203)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6303..6325
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6363..6381)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6449..6553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6554..7414
                     /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(6772..6791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7585..8173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8074..8093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8235..8475
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8235..8255
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8406..8425
                     /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 gcgccrggsg gggsgggggg
      601 gggggggggg gggggggggg sggggsggrg gggtgcggcg gcagccaatc agagcggcgc
      661 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      721 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
      781 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
      841 cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt
      901 ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca
      961 ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat
     1021 attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc
     1081 cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct
     1141 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     1201 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     1261 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     1321 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     1381 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     1441 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     1501 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     1561 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     1621 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     1681 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     1741 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     1801 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     1861 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     1921 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     1981 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     2041 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     2101 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     2161 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     2221 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     2281 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     2341 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     2401 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     2461 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     2521 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     2581 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     2641 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     2701 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     2761 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     2821 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     2881 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     2941 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     3001 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     3061 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     3121 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     3181 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     3241 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     3301 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     3361 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     3421 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     3481 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     3541 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     3601 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     3661 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     3721 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     3781 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     3841 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     3901 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     3961 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     4021 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     4081 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     4141 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     4201 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     4261 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     4321 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     4381 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     4441 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     4501 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     4561 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     4621 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     4681 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     4741 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     4801 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     4861 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     4921 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     4981 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     5041 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     5101 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     5161 tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa
     5221 aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga tcagcctcga
     5281 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5341 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5401 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5461 gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg
     5521 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     5581 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     5641 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     5701 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5761 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     5821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     5941 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     6001 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     6061 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     6121 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     6181 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     6241 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     6301 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     6361 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     6421 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     6481 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     6541 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     6601 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6661 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6721 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6781 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6841 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6901 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6961 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     7021 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     7081 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     7141 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     7201 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     7261 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     7321 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     7381 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     7441 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     7501 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     7561 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7621 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7681 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7741 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7801 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7861 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7921 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7981 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     8041 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     8101 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     8161 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     8221 ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca
     8281 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     8341 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     8401 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     8461 tcttgtggaa aggacgaaac accgaaaagg ctcttcaact gttggttt
//
