LOCUS       40924_47263        8395 bp DNA     circular SYN 13-MAY-2021
DEFINITION  This lentiviral vector can be used to assay Cas9 activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8395)
  AUTHORS   Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, 
            Sullender M, Ebert BL, Xavier RJ, Root DE
  TITLE     Rational design of highly active sgRNAs for CRISPR-Cas9-mediated 
            gene inactivation.
  JOURNAL   Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026.
  PUBMED    25184501
REFERENCE   2  (bases 1 to 8395)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8395)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..507
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(519..538)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also
                     called puro-variant-R"
     primer_bind     1015..1035
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1155..1220
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             1227..1943
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    1964..2552
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             2624..2857
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2935..3069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      3096..3231
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(3252..3270)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3280..3296)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3438..3893
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3919..4023
                     /label=AmpR promoter
     CDS             4024..4881
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5055..5643
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5797..5814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5931..5952
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5967..5997
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6005..6021
                     /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     6029..6045
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6066..6084
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6112..6338
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6339..6519
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6566..6691
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    7184..7417
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7602..7646
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     promoter        7824..8064
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    8226..8343
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        8395
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 cccgggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatccaccat gaccgagtac aagcccaccg
      541 tgcgcctcgc cacccgcgac gacgtgccca gggccgtccg caccctcgcc gccgctttcg
      601 ccgactaccc cgccacccgc cacaccgtcg atcccgaccg ccacatcgag cgggtcaccg
      661 agctgcaaga actcttcctc acccgcgtcg ggctcgacat cggcaaggtg tgggtcgctg
      721 acgacggcgc cgcagtggct gtctggacca cacccgagag cgtcgaagct ggggcggtgt
      781 tcgccgagat cggccctcgc atggccgagt tgagcggttc ccggctggcc gctcagcaac
      841 agatggaagg cctcctggct ccacaccggc ccaaggagcc cgcatggttc ctggccaccg
      901 tcggcgtctc tcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
      961 tggaggctgc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgca ccccgcaacc
     1021 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtggaggtg cccgaaggac
     1081 cacgcacctg gtgcatgacc cgcaagcccg gtgccctgca gggatctggc gaagccagac
     1141 acaaacagaa aattgtgaaa cagactttga attttgacct tctcaagttg gcgggagacg
     1201 tggagtccaa cccagggccc gctagcatgg tgagcaaggg cgaggagctg ttcaccgggg
     1261 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1321 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1381 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1441 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1501 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1561 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1621 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1681 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1741 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1801 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1861 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1921 tcggcatgga cgagctgtac aagtcctaac gcgttaagtc gacaatcaac ctctggatta
     1981 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2041 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2101 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2161 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2221 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     2281 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2341 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     2401 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     2461 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2521 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     2581 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     2641 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     2701 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     2761 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     2821 ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat
     2881 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     2941 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3001 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3061 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3121 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3181 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     3241 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     3301 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3361 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3421 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3481 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3541 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3601 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3661 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3721 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3781 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3841 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3901 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3961 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4021 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4081 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4141 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4201 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4261 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4321 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4381 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4441 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4501 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4561 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4621 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4681 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4741 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4801 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4861 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4921 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4981 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5041 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5101 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5161 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5221 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5281 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5341 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5401 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5461 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5521 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5581 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5641 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5701 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5761 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5821 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5881 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     5941 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     6001 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     6061 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
     6121 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
     6181 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
     6241 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
     6301 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
     6361 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     6421 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     6481 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     6541 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     6601 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     6661 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     6721 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     6781 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     6841 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     6901 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     6961 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     7021 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     7081 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     7141 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     7201 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     7261 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     7321 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     7381 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     7441 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     7501 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     7561 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     7621 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     7681 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     7741 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     7801 tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata
     7861 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
     7921 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     7981 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     8041 ctttatatat cttgtggaaa ggacgaaaca ccggtgaacc gcatcgagct gagtttaaga
     8101 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa
     8161 gtggcaccga gtcggtgctt ttttgaattc tagatcttga gacaaatggc agtattcatc
     8221 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     8281 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     8341 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga ggggg
//
