LOCUS       V000697                 8267 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V000697
VERSION     V000697
KEYWORDS    pLH-sgRNA1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8267)
  AUTHORS   Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T
  TITLE     Multiplexed labeling of genomic loci with dCas9 and engineered
            sgRNAs using CRISPRainbow.
  JOURNAL   Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.
   PUBMED   27088723
REFERENCE   2  (bases 1 to 8267)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8267)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat
            Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526."
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    51..72
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        87..117
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    125..141
                     /label="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     149..165
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        186..204
                     /label="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        232..458
                     /label="RSV promoter"
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             459..639
                     /label="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    686..811
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1304..1537
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             1722..1766
                     /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             1915..1956
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     promoter        2064..2304
                     /label="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     CDS             2653..2955
                     /label="ccdB"
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     misc_RNA        2979..3054
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     misc_feature    3103..3220
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        3269..3779
                     /label="hPGK promoter"
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             3869..4891
                     /label="HygR"
                     /note="aminoglycoside phosphotransferase from E. coli"
     primer_bind     complement(4918..4934)
                     /label="KS primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     LTR             5017..5250
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5322..5456
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      5483..5618
                     /label="SV40 ori"
                     /note="SV40 origin of replication"
     promoter        complement(5639..5657)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5667..5683)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     rep_origin      5825..6280
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6306..6410
                     /label="AmpR promoter"
     CDS             6411..7268
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      7442..8030
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     8184..8201
                     /label="L4440"
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
       61 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
      121 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
      181 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
      241 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
      301 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
      361 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
      421 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
      481 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      541 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      601 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
      661 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
      721 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
      781 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
      841 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
      901 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
      961 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1021 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1081 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1141 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1201 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1261 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1321 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1381 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1441 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1501 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1561 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1621 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     1681 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     1741 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     1801 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     1861 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     1921 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     1981 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
     2041 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata
     2101 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     2161 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     2221 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     2281 ctttatatat cttgtggaaa ggacgaaaca ccggtgtctt cgaggcttac taaaagccag
     2341 ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
     2401 tacccgaagt atgtcaaaaa gaggtgtgct atgaagcagc gtattacagt gacagttgac
     2461 agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca
     2521 caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg
     2581 aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca
     2641 gggactggtg aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
     2701 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg
     2761 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
     2821 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
     2881 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
     2941 atgttctggg gaatataaac cctgcaggac gaagacccgt ttgagagcta gaaatagcaa
     3001 gttcaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     3061 tgaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     3121 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     3181 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     3241 cagagatcca ctttggccgc ggctcgaggg ggttggggtt gcgccttttc caaggcagcc
     3301 ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg
     3361 accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta
     3421 cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc
     3481 ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag
     3541 acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg
     3601 gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt
     3661 gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc
     3721 cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg
     3781 gggatccacc ggatcaagct tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     3841 cagatctgat cccggggggc aatgagatat gaaaaagcct gaactcaccg cgacgtctgt
     3901 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     3961 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4021 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     4081 gctcccgatt ccggaagtgc ttgacattgg ggaatttagc gagagcctga cctattgcat
     4141 ctcccgccgt gcacagggtg tcacgttgca agatctgcct gaaaccgaac tgcccgctgt
     4201 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     4261 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     4321 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     4381 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     4441 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     4501 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     4561 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     4621 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     4681 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     4741 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     4801 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     4861 acgccccagc actcgtccga gggcaaagga atagagtaga tgccgaccgg gatctatcga
     4921 taccgtcgac ctcgaggggg ggcccggtac ctttaagacc aatgacttac aaggcagctg
     4981 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     5041 gaagaaaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     5101 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     5161 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     5221 ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt
     5281 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     5341 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5401 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     5461 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     5521 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     5581 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     5641 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     5701 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     5761 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     5821 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5881 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5941 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6001 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     6061 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6121 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     6181 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6241 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     6301 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6361 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6421 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6481 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6541 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6601 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6661 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6721 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6781 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6841 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6901 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6961 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7021 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7081 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     7141 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7201 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7261 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7321 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7381 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7441 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7501 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7561 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7621 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7681 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7741 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7801 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7861 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7921 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7981 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8041 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     8101 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     8161 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     8221 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagc
//
