LOCUS       V006930                 9583 bp    DNA     circular SYN 03-JAN-2022
DEFINITION  Exported.
ACCESSION   V006930
VERSION     V006930
KEYWORDS    pgRNA-CKB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9583)
  AUTHORS   Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
            AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen
            TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP,
            Weissman JS, Qi LS, So PL, Conklin BR
  TITLE     CRISPR Interference Efficiently Induces Specific and Reversible Gene
            Silencing in Human iPSCs.
  JOURNAL   Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi:
            10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
   PUBMED   26971820
REFERENCE   2  (bases 1 to 9583)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9583)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1016/j.stem.2016.01.022"; journalName: "Cell Stem Cell"; date:
            "2016-04-7- 7"; volume: "18"; issue: "4"; pages: "541-53"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             160..201
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    309..426
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     intron          1636..2652
                     /label="chimeric intron"
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     CDS             2717..2737
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             2741..2761
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             2765..2785
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             2801..3496
                     /label="mKate2"
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
     CDS             3551..3970
                     /gene="bsr"
                     /label="Blasticidin-S deaminase"
                     /note="Blasticidin-S deaminase from Bacillus cereus.
                     Accession#: P33967"
     protein_bind    complement(4248..4281)
                     /label="loxP"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4337..4925
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     primer_bind     complement(4928..4944)
                     /label="KS primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     LTR             5454..5634
                     /label="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5696..6284)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(6458..7315)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(7316..7420)
                     /label="AmpR promoter"
     enhancer        7686..8065
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8066..8268
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     misc_feature    8510..8635
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    9132..9365
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             join(9550..9583,1..11)
                     /label="gp41 peptide"
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
                     al., 2013)"
ORIGIN
        1 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
       61 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
      121 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
      181 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
      241 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
      301 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
      361 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
      421 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
      481 tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacgga cttgtgggag
      541 aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag taactataga
      601 ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct acattttaca
      661 tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt ataaacagca
      721 caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat aagtatccct
      781 tggagaacca ccttgttggg gagacggacg tctccgttta agagctatgc tggaaacagc
      841 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      901 ctttttttct cgagtactag gatccattag gcggccgcgt ggataaccgt attaccgcca
      961 tgcatcccgg gtgactgact gagcggccgc actagttatt aatagtaatc aattacgggg
     1021 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     1081 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     1141 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1201 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1261 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1321 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     1381 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     1441 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccrgg sggggsgggg
     1501 sggggsgrgg ggsggggsgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     1561 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     1621 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
     1681 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     1741 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
     1801 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
     1861 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc
     1921 ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag
     1981 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc
     2041 tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg
     2101 caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg
     2161 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     2221 gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc
     2281 ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa
     2341 tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg
     2401 aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag
     2461 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag
     2521 cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
     2581 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
     2641 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
     2701 gaattccgcc accatgccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt
     2761 agatccaaaa aagaagagaa aggtacacgt gtcaacgcgt atggtgagcg agctgattaa
     2821 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg
     2881 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggcggt
     2941 cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag
     3001 caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt ccttccccga
     3061 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     3121 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     3181 cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct ccaccgagac
     3241 cctgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga agctcgtggg
     3301 cgggggccac ctgatctgca acttgaagac cacatacaga tccaagaaac ccgctaagaa
     3361 cctcaagatg cccggcgtct actatgtgga cagaagactg gaaagaatca aggaggccga
     3421 caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag
     3481 caaactgggg cacagagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa
     3541 tcccggccct atgaagacct tcaacatctc ccagcaggat ctagaattag tagaagtagc
     3601 gacagagaag attacaatgc tttatgagga taataaacat catgtgggag cggcaattcg
     3661 tacgaaaaca ggagaaatca tttcggcagt acatattgaa gcgtatatag gacgagtaac
     3721 tgtttgtgca gaagccattg cgattggtag tgcagtttcg aatggacaaa aggattttga
     3781 cacgattgta gctgttagac acccttattc tgacgaagta gatagaagta ttcgagtggt
     3841 aagtccttgt ggtatgtgta gggagttgat ttcagactat gcaccagatt gttttgtgtt
     3901 aatagaaatg aatggcaagt tagtcaaaac tacgattgaa gaactcattc cactcaaata
     3961 tacccgaaat taatcgacat gataagatac attgatgagt ttggacaaac cacaactaga
     4021 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtgaaa
     4081 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4141 acaattgcat tcattttatg tttcaggttc agagggaggt gtgggaggtt ttttaaagca
     4201 agtaaaacct ctacaaatgt ggtagatcga attcgtcgag ggacctaata acttcgtata
     4261 gcatacatta tacgaagtta tacatgttta agggttccgg ttccactagg tacaattcga
     4321 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     4381 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     4441 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     4501 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     4561 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4621 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     4681 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     4741 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     4801 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4861 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4921 cccgcatcga taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta
     4981 gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg
     5041 tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag
     5101 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     5161 gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga
     5221 actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag
     5281 taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac
     5341 accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg
     5401 acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc
     5461 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     5521 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     5581 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt
     5641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6121 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6421 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6481 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6541 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6601 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6661 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6721 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6781 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6841 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6901 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6961 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7021 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7081 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7141 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7201 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7261 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7321 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7381 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7441 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7501 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7561 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     7621 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7681 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     7741 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     7801 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     7861 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     7921 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     7981 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     8041 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     8101 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     8161 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     8221 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta
     8281 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     8341 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     8401 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     8461 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
     8521 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
     8581 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
     8641 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
     8701 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
     8761 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     8821 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     8881 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     8941 gcaaaacaaa agtaagacca ccgcacagca agcggccggc cgctgatctt cagacctgga
     9001 ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt
     9061 gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga
     9121 gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc
     9181 gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag
     9241 cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg
     9301 ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag
     9361 ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat
     9421 gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg
     9481 gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac
     9541 cagcaagaaa agaatgaaca agaattattg gaattagata aat
//
