LOCUS       V012245                11059 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V012245
VERSION     V012245
KEYWORDS    pCLIP-Cas9(D10a)-Nickase-hCMV-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 11059)
  AUTHORS   transOMIC
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 11059)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..404
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     LTR             411..668
                     /label="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    715..840
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1333..1566
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             1751..1795
                     /label="gp41 peptide"
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
                     al., 2013)"
     CDS             1944..1985
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    2057..2174
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     enhancer        2238..2541
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2542..2745
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             2905..7008
                     /label="Cas9(D10A)"
                     /note="nickase mutant of the Cas9 endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             7009..7056
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label="bipartite nuclear localization signal from nucl"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7057..7080
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             7090..7146
                     /label="P2A"
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             7147..7740
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     misc_feature    7768..8356
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             8561..8794
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        8890..9219
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(9308..9893)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(10067..10924)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(10925..11029)
                     /label="AmpR promoter"
ORIGIN
        1 gacggatcgg gagatcctcg cgcgttgaca ttgattattg actagttatt aatagtaatc
       61 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      121 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      181 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      241 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      301 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      361 tcctacttgg cagtacatct acgtattagt catcgctatt accagctagc gaggcgtggc
      421 ctgggcggga ctggggagtg gcgagccctc agatcctgca tataagcagc tgctttttgc
      481 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
      541 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
      601 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
      661 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
      721 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
      781 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
      841 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
      901 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
      961 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     1021 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     1081 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     1141 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     1201 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     1261 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     1321 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     1381 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     1441 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     1501 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     1561 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     1621 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     1681 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     1741 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     1801 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     1861 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     1921 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga
     1981 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg
     2041 atctcgacgg tatcgatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2101 gaatagtaga cataatagca acagacatac aaactaaaga actacaaaaa caaattacaa
     2161 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg aattcatcga
     2221 taagcttggg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     2281 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     2341 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     2401 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     2461 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta
     2521 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag
     2581 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     2641 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     2701 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
     2761 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgactctag
     2821 ctagaggatc cactagtcca gtgtggtgga attctgcaga tatccagcac agtggcggcc
     2881 gctcgagtct agagcgctgc caccatggac aagaagtaca gcatcggcct ggccatcggc
     2941 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     3001 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     3061 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     3121 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     3181 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     3241 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     3301 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     3361 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     3421 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     3481 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     3541 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     3601 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     3661 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     3721 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     3781 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     3841 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     3901 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     3961 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     4021 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     4081 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     4141 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     4201 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     4261 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     4321 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     4381 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     4441 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     4501 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     4561 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     4621 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     4681 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     4741 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     4801 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     4861 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     4921 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     4981 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     5041 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     5101 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     5161 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     5221 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     5281 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     5341 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     5401 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     5461 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     5521 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     5581 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     5641 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     5701 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     5761 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     5821 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     5881 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     5941 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     6001 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     6061 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     6121 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     6181 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     6241 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     6301 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     6361 aagtccaaga aactgaagag tgtgaaagag ctactgggga tcaccatcat ggaaagaagc
     6421 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     6481 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     6541 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     6601 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     6661 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     6721 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     6781 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     6841 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     6901 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     6961 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc
     7021 gccacaaaga aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag
     7081 ggatccggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct
     7141 ggaccgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     7201 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     7261 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     7321 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     7381 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     7441 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     7501 gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg
     7561 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     7621 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     7681 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     7741 tgagaattcg atatcaagct tatcggtaat caacctctgg attacaaaat ttgtgaaaga
     7801 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     7861 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     7921 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     7981 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     8041 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     8101 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     8161 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     8221 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     8281 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     8341 tgggccgcct ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc
     8401 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
     8461 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
     8521 gctgtagatc ttagccactt tttaaaagaa aagaggggac tggaagggct aattcactcc
     8581 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     8641 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     8701 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     8761 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     8821 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggccttg acattgttta
     8881 aactggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     8941 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     9001 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     9061 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     9121 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     9181 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     9241 tccattacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     9301 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     9361 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     9421 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     9481 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     9541 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     9601 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     9661 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     9721 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     9781 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     9841 gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     9901 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     9961 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
    10021 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
    10081 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
    10141 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
    10201 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
    10261 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
    10321 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
    10381 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
    10441 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
    10501 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
    10561 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
    10621 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
    10681 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
    10741 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
    10801 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
    10861 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
    10921 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
    10981 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
    11041 gaaaagtgcc acctgacgt
//
