LOCUS       ER50-SpCas9-ER50       10000 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses SpCas9 fused to ER50 domain on both N- and C-termini in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    ER50-SpCas9-ER50.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10000)
  AUTHORS   Maji B, Moore CL, Zetsche B, Volz SE, Zhang F, Shoulders MD, 
            Choudhary A
  TITLE     Multidimensional chemical control of CRISPR-Cas9.
  JOURNAL   Nat Chem Biol. 2017 Jan;13(1):9-11. doi: 10.1038/nchembio.2224. Epub
            2016 Oct 31.
  PUBMED    27820801
REFERENCE   2  (bases 1 to 10000)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10000)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nchembio.2224"; journalName: "Nat Chem Biol"; date: 
            "2017-01"; volume: "13"; issue: "1"; pages: "9-11"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1245..1254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1254..1319
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
     CDS             1326..1346
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             1671..2033
                     /gene="ESR1"
                     /label=ESR1
                     /note="Estrogen receptor from Macaca mulatta. Accession#: 
                     P49886"
     CDS             2130..6230
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             6231..6278
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    7047..7254
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7263..7403
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      7478..7933
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7950..7969)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     8069..8091
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8129..8147)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        8215..8319
                     /label=AmpR promoter
     CDS             8320..9177
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      9351..9939
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
     1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc ggatccagcc
     1381 ttgccctgtc acttacagcc gaccagatgg tttccgcgct tctcgacgct gaacctccaa
     1441 ttctctattc cgaatacgac ccaaccaggc cgttctccga ggcatctatg atgggtctgc
     1501 tgacaaatct ggcagacagg gaactggtgc acatgatcaa ttgggcgaag cgcgtacccg
     1561 gattcgtcga tcttgcactc catgatcagg tgcacttgct ggagtgcgct tggatggaga
     1621 tcctcatgat cgggctggtg tggcggagta tggaacaccc cggcaagttg ctgtttgcgc
     1681 ctaacctcct gttggacagg aaccagggga aatgtgtgga gggcggtgtg gaaatctttg
     1741 acatgctcct cgctacctca agccggttta ggatgatgaa tctgcagggc gaagagttcg
     1801 tgtgtctcaa atctatcata ctgttgaaca gcggagtcta caccttcctc tccagtactc
     1861 tgaaatctct ggaggagaaa gatcatatcc atcgcgtgct ggacaagata accgacacgt
     1921 tgattcactt gatggccaaa gctgggctca ctctgcaaca acaacatcag cgactggcac
     1981 agctgttgct gattttgagc cacattcggc acatgtccag caagagaatg gagcacctct
     2041 atagtatgaa gtgcaagaac gtcgtacccc tgtcagatct gcttcttgaa atgcttgatg
     2101 cccaccggct gggaagcgga agcggatccg acaagaagta cagcatcggc ctggacatcg
     2161 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     2221 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     2281 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     2341 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     2401 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     2461 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     2521 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     2581 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     2641 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     2701 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     2761 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     2821 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     2881 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     2941 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3001 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3061 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3121 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     3181 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     3241 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     3301 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     3361 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     3421 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     3481 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     3541 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     3601 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     3661 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     3721 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     3781 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     3841 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     3901 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     3961 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     4021 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     4081 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     4141 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     4201 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     4261 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     4321 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     4381 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     4441 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     4501 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     4561 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     4621 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     4681 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     4741 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     4801 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     4861 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     4921 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     4981 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     5041 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     5101 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     5161 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     5221 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     5281 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     5341 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     5401 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     5461 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     5521 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     5581 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     5641 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     5701 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     5761 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     5821 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     5881 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     5941 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     6001 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     6061 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     6121 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     6181 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg
     6241 cggccacgaa aaaggccggc caggcaaaaa agaaaaagag ccttgccctg tcacttacag
     6301 ccgaccagat ggtttccgcg cttctcgacg ctgaacctcc aattctctat tccgaatacg
     6361 acccaaccag gccgttctcc gaggcatcta tgatgggtct gctgacaaat ctggcagaca
     6421 gggaactggt gcacatgatc aattgggcga agcgcgtacc cggattcgtc gatcttgcac
     6481 tccatgatca ggtgcacttg ctggagtgcg cttggatgga gatcctcatg atcgggctgg
     6541 tgtggcggag tatggaacac cccggcaagt tgctgtttgc gcctaacctc ctgttggaca
     6601 ggaaccaggg gaaatgtgtg gagggcggtg tggaaatctt tgacatgctc ctcgctacct
     6661 caagccggtt taggatgatg aatctgcagg gcgaagagtt cgtgtgtctc aaatctatca
     6721 tactgttgaa cagcggagtc tacaccttcc tctccagtac tctgaaatct ctggaggaga
     6781 aagatcatat ccatcgcgtg ctggacaaga taaccgacac gttgattcac ttgatggcca
     6841 aagctgggct cactctgcaa caacaacatc agcgactggc acagctgttg ctgattttga
     6901 gccacattcg gcacatgtcc agcaagagaa tggagcacct ctatagtatg aagtgcaaga
     6961 acgtcgtacc cctgtcagat ctgcttcttg aaatgcttga tgcccaccgg ctgtaagaat
     7021 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     7081 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     7141 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     7201 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     7261 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     7321 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     7381 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     7441 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     7501 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     7561 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     7621 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     7681 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     7741 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     7801 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     7861 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     7921 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     7981 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     8041 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     8101 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     8161 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     8221 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     8281 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     8341 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     8401 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     8461 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     8521 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     8581 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     8641 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     8701 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     8761 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     8821 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     8881 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     8941 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     9001 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     9061 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     9121 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     9181 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     9241 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     9301 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     9361 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     9421 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     9481 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     9541 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     9601 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     9661 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     9721 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     9781 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     9841 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     9901 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     9961 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
