LOCUS       V012237                10693 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V012237
VERSION     V012237
KEYWORDS    pCLIP-Cas9-Nuclease-EFS-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10693)
  AUTHORS   transOMIC
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 10693)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10693
                     /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    2093..2210
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2303..2514
                     /label="EF-1-alpha core promoter"
                     /note="core promoter for human elongation factor
                     EF-1-alpha"
     CDS             2539..6642
                     /label="Cas9"
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             6643..6690
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label="bipartite nuclear localization signal from nucl"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6691..6714
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             6724..6780
                     /label="P2A"
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             6781..7374
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     misc_feature    7402..7990
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             8195..8428
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        8524..8853
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(8942..9527)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(9701..10558)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(10559..10663)
                     /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 atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa
     2101 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     2161 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     2221 acagggacag cagagatcca gtttggttaa ttagctagct aggtcttgaa aggagtggga
     2281 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     2341 gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     2401 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     2461 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg
     2521 ttctagagcg ctgccaccat ggacaagaag tacagcatcg gcctggacat cggcaccaac
     2581 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     2641 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     2701 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     2761 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     2821 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     2881 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     2941 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     3001 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     3061 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     3121 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     3181 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     3241 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     3301 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     3361 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     3421 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     3481 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     3541 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     3601 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     3661 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     3721 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     3781 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     3841 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     3901 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     3961 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     4021 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     4081 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     4141 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     4201 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     4261 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     4321 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     4381 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     4441 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     4501 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     4561 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     4621 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     4681 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     4741 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     4801 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     4861 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     4921 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     4981 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     5041 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac
     5101 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     5161 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     5221 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     5281 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     5341 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     5401 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     5461 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     5521 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     5581 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     5641 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     5701 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     5761 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     5821 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     5881 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     5941 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     6001 aagaaactga agagtgtgaa agagctactg gggatcacca tcatggaaag aagcagcttc
     6061 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     6121 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     6181 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     6241 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     6301 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     6361 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     6421 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     6481 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     6541 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     6601 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc tgccgccaca
     6661 aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga taagggatcc
     6721 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
     6781 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     6841 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     6901 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     6961 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     7021 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     7081 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     7141 gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg tctgggcagc
     7201 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     7261 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     7321 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagaa
     7381 ttcgatatca agcttatcgg taatcaacct ctggattaca aaatttgtga aagattgact
     7441 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     7501 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     7561 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     7621 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     7681 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     7741 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     7801 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     7861 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     7921 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     7981 gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt
     8041 agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag
     8101 gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta
     8161 gatcttagcc actttttaaa agaaaagagg ggactggaag ggctaattca ctcccaacga
     8221 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     8281 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     8341 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     8401 ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat
     8461 ttataacttg caaagaaatg aatatcagag agtgagaggc cttgacattg tttaaactgg
     8521 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     8581 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     8641 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     8701 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     8761 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     8821 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     8881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     8941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     9001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     9061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     9121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     9181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     9241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     9301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     9361 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     9421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt
     9481 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     9541 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     9601 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     9661 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     9721 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     9781 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     9841 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     9901 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     9961 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
    10021 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
    10081 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
    10141 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
    10201 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
    10261 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
    10321 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
    10381 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
    10441 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
    10501 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
    10561 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
    10621 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
    10681 tgccacctga cgt
//
