LOCUS       Exported                8768 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses R1335K mutant SpCas9 fused to ZFP-TS2 in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    pCSDest2-SpCas9-MT3-NLS-3XHA-NLS-ZFP_TS2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8768)
  AUTHORS   Bolukbasi MF, Gupta A, Oikemus S, Derr AG, Garber M, Brodsky MH, Zhu
            LJ, Wolfe SA
  TITLE     DNA-binding-domain fusions enhance the targeting range and precision
            of Cas9.
  JOURNAL   Nat Methods. 2015 Oct 19. doi: 10.1038/nmeth.3624.
  PUBMED    26480473
REFERENCE   2  (bases 1 to 8768)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8768
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        37..55
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     37..54
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    110..134
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      138..147
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             144..1862
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             2731..2742
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4260..4280
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4281..4370
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4383..4409
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     promoter        complement(4797..4814)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    4819..4953
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4843..4862)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4897..4916
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5066..5086)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5066..5084)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5105..5121)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5105..5121)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5118..5140)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5129..5145
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5153..5183)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5198..5219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5336..5353)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5507..6095)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5587..5606)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6266..7126)
                     /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     6889..6908
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7127..7231)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7257..7712)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7394..7415)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7606..7625
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7786..2
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca
       61 agctacttgt tctttttgca ggatcccatc gattcgaatt caaggatcaa caagtttgta
      121 caaaaaagca ggctggcgcc accatggaca agaagtactc cattgggctc gatatcggta
      181 ccaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
      241 aagttctggg caataccgat cgccacagca taaagaagaa cctcattgga gccctcctgt
      301 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
      361 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
      421 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
      481 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
      541 caaccatata tcatctgagg aagaagctgg tagacagtac tgataaggct gacttgcggt
      601 tgatctatct cgcgctggcg cacatgatca aatttcgggg acacttcctc atcgaggggg
      661 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca
      721 atcagctttt cgaggagaac ccgatcaacg catccggcgt tgacgccaaa gcaatcctga
      781 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
      841 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
      901 aatctaactt cgacctggcc gaagatgcca agctgcaact gagcaaagac acctacgatg
      961 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
     1021 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
     1081 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
     1141 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
     1201 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
     1261 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
     1321 tgaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
     1381 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
     1441 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
     1501 gccccctcgc tcggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
     1561 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
     1621 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
     1681 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
     1741 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
     1801 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
     1861 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa
     1921 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg
     1981 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg
     2041 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga
     2101 gacgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggc atccgagaca
     2161 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact
     2221 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag
     2281 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt agcccagcta
     2341 tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa gtaatgggaa
     2401 ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact acccagaagg
     2461 gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa gaactggggt
     2521 cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag aagctctacc
     2581 tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac atcaaccggt
     2641 tgtccgacta cgacgtggat catatcgtgc cccaaagctt tctcaaagat gattctattg
     2701 ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac gtcccctcag
     2761 aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc aaactgatca
     2821 cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct gagttggata
     2881 aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag cacgtggccc
     2941 aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg attcgagagg
     3001 tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac tttcagtttt
     3061 ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg aatgcagtgg
     3121 taggcactgc acttatcaaa aaatatccca agctggaatc tgaatttgtt tacggagact
     3181 ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata ggcaaggcca
     3241 ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag attacactgg
     3301 ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg
     3361 tgtgggacaa gggtagggat ttcgcgacag tccgcaaggt cctgtccatg ccgcaggtga
     3421 acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt atcctcccga
     3481 aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag aaatacggcg
     3541 gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg gagaaaggga
     3601 agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg gagcgatcca
     3661 gcttcgagaa aaaccccatc gactttctcg aagcgaaagg atataaagag gtcaaaaaag
     3721 acctcatcat taagctgccc aagtactctc tctttgagct tgaaaacggc cggaaacgaa
     3781 tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc tctaaatacg
     3841 ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc gaagataatg
     3901 agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc atcgagcaaa
     3961 taagcgagtt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag gtgctttctg
     4021 cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt atccacttgt
     4081 ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc accatagaca
     4141 gaaagaagta cacctctaca aaggaggtcc tggacgccac actgattcat cagtcaatta
     4201 cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacggc accggcgggc
     4261 ccaagaagaa gaggaaggta tacccatacg atgttcctga ctatgcgggc tatccctatg
     4321 acgtcccgga ctatgcagga tcgtatcctt atgacgttcc agattacgct ggatccgccg
     4381 ctccggcagc taagaaaaag aaactggatt tcgaatccgg aaagccctat aaatgtcctg
     4441 aatgtggcaa gtccttctcg gagaagagcc acctgacacg gcaccaacgc acgcacactg
     4501 gtgagaagcc atacgcgtgt cctgtcgagt cctgtgaccg ccgcttcagt cggagcgacc
     4561 acctgacaca gcacatccgc attcacacag ggcaaaaacc gtttcaatgc cgcatctgca
     4621 tgaggaactt cagcgacaag ggccacctga cccggcacat ccgcacccac acaggagaaa
     4681 agcccttcgc ctgtgacatc tgcggcagga agttcgcgcg gagcgacgac ctgacacggc
     4741 acaccaagat ccacctccgt cagaaagacc ccgggtaatg actcgagcct ctagaactat
     4801 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     4861 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4921 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4981 tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc gccaatgcat
     5041 tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     5101 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5161 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5221 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5281 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5341 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5401 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5461 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5521 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5581 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5641 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5701 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5761 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5821 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5881 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5941 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6001 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6061 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6121 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6181 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6241 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6301 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6361 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6421 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6481 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6541 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6601 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6661 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6721 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6781 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6841 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6901 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6961 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7021 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7081 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7141 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7201 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     7261 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     7321 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     7381 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     7441 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     7501 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     7561 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     7621 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     7681 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     7741 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgacca tagccaattc
     7801 aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct gtgccaattc
     7861 aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc cagccaattc
     7921 aatatggcgg acttggcacc atgccaattc aatatggcgg acttggcact gtgccaactg
     7981 gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt
     8041 ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat atgccaggat
     8101 caatatatag gcaatatcca atatggccct atgccaatat ggctattggc caggttcaat
     8161 actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga
     8221 tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata
     8281 gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg
     8341 ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc
     8401 gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg
     8461 ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc
     8521 ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa
     8581 ggacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga
     8641 tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt
     8701 tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg
     8761 tttaggga
//
