LOCUS       V012244                11158 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V012244
VERSION     V012244
KEYWORDS    pCLIP-Cas9(D10a)-Nickase-hCMV-tRFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 11158)
  AUTHORS   transOMIC
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 11158)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11158
                     /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
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /label="2A peptide from porcine teschovirus-1 polyprotein"
                     /note="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7147..7839
                     /label="TurboRFP"
                     /note="red fluorescent protein from Entacmaea quadricolor"
     misc_feature    7867..8455
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             8660..8893
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        8989..9318
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(9407..9992)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(10166..11023)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(11024..11128)
                     /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 ggaccgatga gcgagctgat caaggagaac atgcacatga agctgtacat ggagggcacc
     7201 gtgaacaacc accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
     7261 cagaccatga agatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg
     7321 gctaccagct tcatgtacgg cagcaaagcc ttcatcaacc acacccaggg catccccgac
     7381 ttctttaagc agtccttccc tgagggcttc acatgggaga gaatcaccac atacgaagac
     7441 gggggcgtgc tgaccgctac ccaggacacc agcttccaga acggctgcat catctacaac
     7501 gtcaagatca acggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacacgc
     7561 ggctgggagg ccaacaccga gatgctgtac cccgctgacg gcggcctgag aggccacagc
     7621 cagatggccc tgaagctcgt gggcgggggc tacctgcact gctccttcaa gaccacatac
     7681 agatccaaga aacccgctaa gaacctcaag atgcccggct tccacttcgt ggaccacaga
     7741 ctggaaagaa tcaaggaggc cgacaaagag acctacgtcg agcagcacga gatggctgtg
     7801 gccaagtact gcgacctccc tagcaaactg gggcacagat aagaattcga tatcaagctt
     7861 atcggtaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     7921 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     7981 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     8041 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     8101 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     8161 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     8221 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     8281 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     8341 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     8401 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     8461 taccgtcgac ctcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct
     8521 accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc
     8581 acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     8641 ttaaaagaaa agaggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     8701 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     8761 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     8821 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     8881 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     8941 aaatgaatat cagagagtga gaggccttga cattgtttaa actggaatgt gtgtcagtta
     9001 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     9061 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     9121 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     9181 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     9241 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     9301 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattacatg tgagcaaaag
     9361 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     9421 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     9481 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     9541 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     9601 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     9661 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     9721 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     9781 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     9841 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     9901 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt gtttgcaagc
     9961 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    10021 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    10081 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    10141 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    10201 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    10261 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    10321 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    10381 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    10441 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    10501 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    10561 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    10621 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    10681 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    10741 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    10801 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    10861 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    10921 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    10981 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    11041 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    11101 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgt
//
