LOCUS       pET-dCas9-VP64-6        9544 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of dCas9-VP64-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    pET-dCas9-VP64-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9544)
  AUTHORS   Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder
            ML, Joung JK, Chen ZY, Liu DR
  TITLE     Cationic lipid-mediated delivery of proteins enables efficient 
            protein-based genome editing in vitro and in vivo.
  JOURNAL   Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 
            2014 Oct 30.
  PUBMED    25357182
REFERENCE   2  (bases 1 to 9544)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9544)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.3081"; 
            journalName: "Nat Biotechnol"; date: "2015-01"; volume: "33"; issue:
            "1"; pages: "73-80"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             71..4174
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             4181..4201
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4208..4273
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4304..4453
                     /label=VP64
                     /note="tetrameric repeat of the minimal activation domain
                     of herpes simplex virus VP16 (Beerli et al., 1998)"
     CDS             4454..4471
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             4490..4534
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      4586..4633
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4670..5125
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5243..6100)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6101..6193)
                     /label=AmpR promoter
     rep_origin      6274..6862
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7016..7033
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(7048..7190)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7276..7298
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7295..7483)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(8021..8042)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(8058..9137)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(9138..9215)
                     /label=lacI promoter
     primer_bind     9421..9440
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     promoter        9528..9544
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg
      121 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
      181 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
      241 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
      301 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
      361 ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc
      421 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
      481 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
      541 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
      601 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
      661 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
      721 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
      781 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
      841 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
      901 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
      961 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
     1021 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
     1081 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
     1141 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
     1201 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
     1261 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
     1321 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg
     1381 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg
     1441 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
     1501 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
     1561 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
     1621 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
     1681 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
     1741 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
     1801 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
     1861 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
     1921 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
     1981 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
     2041 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
     2101 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
     2161 ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg
     2221 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
     2281 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
     2341 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
     2401 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
     2461 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
     2521 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
     2581 tgtcgatgcc attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
     2641 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
     2701 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
     2761 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
     2821 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
     2881 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
     2941 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
     3001 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
     3061 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
     3121 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
     3181 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
     3241 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
     3301 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa
     3361 aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga
     3421 caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc
     3481 aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt
     3541 aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa
     3601 tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa
     3661 actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc
     3721 cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata
     3781 tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt
     3841 gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc
     3901 taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca
     3961 tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa
     4021 tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac
     4081 gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga
     4141 aacacgcatt gatttgagtc agctaggagg tgacggttct cccaagaaga agaggaaagt
     4201 ctcgagcgac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     4261 tgacgatgac aaggctgcag gaggcggtgg aagcgggcgc gccgacgcgc tggacgattt
     4321 cgatctcgac atgctgggtt ctgatgccct cgatgacttt gacctggata tgttgggaag
     4381 cgacgcattg gatgactttg atctggacat gctcggctcc gatgctctgg acgatttcga
     4441 tctcgatatg ttacatcacc accaccatca ctaatgactc gagtctggta aagaaaccgc
     4501 tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta
     4561 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     4621 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     4681 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4741 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4801 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4861 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4921 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4981 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5041 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5101 aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga
     5161 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5221 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5281 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5341 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5401 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5461 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5521 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5581 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5641 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5701 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5761 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5821 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5881 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5941 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6001 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6061 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatcat
     6121 gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6181 aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6241 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6301 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6361 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6421 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6481 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6541 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6601 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6661 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6721 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6781 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6841 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6901 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6961 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7021 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     7081 cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     7141 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     7201 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     7261 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     7321 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     7381 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     7441 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     7501 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     7561 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     7621 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     7681 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     7741 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     7801 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     7861 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     7921 acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag ctgactgggt
     7981 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     8041 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     8101 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     8161 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     8221 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     8281 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     8341 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     8401 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     8461 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     8521 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     8581 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     8641 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     8701 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     8761 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     8821 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     8881 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     8941 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     9001 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     9061 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     9121 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     9181 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     9241 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     9301 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     9361 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     9421 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     9481 gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac
     9541 tata
//
