LOCUS       V012242                10596 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V012242
VERSION     V012242
KEYWORDS    pCLIP-Cas9(D10a)-Nickase-TRE3G-Blast
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10596)
  AUTHORS   transOMIC
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 10596)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10596
                     /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    1337..1570
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             1755..1799
                     /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             1948..1989
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    2061..2178
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2244..2622
                     /label="TRE3G promoter"
                     /note="3rd-generation Tet-responsive promoter that can be
                     activated by binding of Tet-On(R) 3G"
     CDS             2640..6743
                     /label="Cas9(D10A)"
                     /note="nickase mutant of the Cas9 endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             6744..6791
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label="bipartite nuclear localization signal from nucl"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6792..6815
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             6825..6881
                     /label="P2A"
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             6882..7277
                     /label="BSD"
                     /note="blasticidin S deaminase"
     misc_feature    7305..7893
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             8098..8331
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        8427..8756
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(8845..9430)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(9604..10461)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(10462..10566)
                     /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 ccggccgctg atcttcagac
     1201 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     1261 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     1321 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     1381 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     1441 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     1501 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     1561 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     1621 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     1681 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     1741 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     1801 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     1861 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     1921 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
     1981 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     2041 acggatctcg acggtatcgc ctttaaaaga aaagggggga ttggggggta cagtgcaggg
     2101 gaaagaatag tagacataat agcaacagac atacaaacta aagaactaca aaaacaaatt
     2161 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt ttatcgataa
     2221 ccccgtggaa ttatcacctc gaggagttta ctccctatca gtgatagaga acgtatgaag
     2281 agtttactcc ctatcagtga tagagaacgt atgcagactt tactccctat cagtgataga
     2341 gaacgtataa ggagtttact ccctatcagt gatagagaac gtatgaccag tttactccct
     2401 atcagtgata gagaacgtat ctacagttta ctccctatca gtgatagaga acgtatatcc
     2461 agtttactcc ctatcagtga tagagaacgt ataagcttta ggcgtgtacg gtgggcgcct
     2521 ataaaagcag agctcgttta gtgaaccgtc agatcgcctg gagcaattcc acaacacttt
     2581 tgtcttatac caactttccg taccacttcc taccctcgta aatctagagc gctgccacca
     2641 tggacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga
     2701 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     2761 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     2821 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     2881 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     2941 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     3001 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     3061 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     3121 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     3181 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     3241 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     3301 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     3361 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     3421 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     3481 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     3541 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     3601 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     3661 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     3721 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     3781 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     3841 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     3901 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     3961 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     4021 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     4081 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     4141 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     4201 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     4261 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     4321 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     4381 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     4441 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     4501 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     4561 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     4621 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     4681 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     4741 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     4801 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     4861 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     4921 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     4981 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     5041 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     5101 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     5161 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     5221 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     5281 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     5341 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     5401 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     5461 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     5521 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     5581 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     5641 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     5701 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     5761 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     5821 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     5881 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     5941 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     6001 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     6061 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     6121 aagagctact ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     6181 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     6241 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     6301 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     6361 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     6421 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     6481 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     6541 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     6601 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     6661 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     6721 acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta
     6781 agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc
     6841 tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggccaag cctttgtctc
     6901 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     6961 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     7021 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     7081 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     7141 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     7201 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     7261 gttatgtgtg ggagggctaa gaattcgata tcaagcttat cggtaatcaa cctctggatt
     7321 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     7381 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     7441 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     7501 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     7561 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     7621 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     7681 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     7741 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     7801 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     7861 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgagacctag
     7921 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     7981 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     8041 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag aggggactgg
     8101 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     8161 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     8221 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     8281 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     8341 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     8401 ggccttgaca ttgtttaaac tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     8461 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     8521 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     8581 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     8641 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     8701 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     8761 tcccgggagc ttgtatatcc attacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     8821 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     8881 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     8941 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     9001 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     9061 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     9121 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     9181 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     9241 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     9301 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     9361 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     9421 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     9481 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     9541 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     9601 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     9661 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     9721 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     9781 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     9841 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     9901 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     9961 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
    10021 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
    10081 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
    10141 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
    10201 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
    10261 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
    10321 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
    10381 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
    10441 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
    10501 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
    10561 tccgcgcaca tttccccgaa aagtgccacc tgacgt
//
