LOCUS       Exported                8323 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDK4.
ACCESSION   .
VERSION     .
KEYWORDS    CDK4 gRNA (BRDN0001145730)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8323)
  AUTHORS   Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, 
            Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, 
            Root DE
  TITLE     Optimized sgRNA design to maximize activity and minimize off-target 
            effects of CRISPR-Cas9.
  JOURNAL   Nat Biotechnol. 2016 Jan 18. doi: 10.1038/nbt.3437.
  PUBMED    26780180
REFERENCE   2  (bases 1 to 8323)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8323
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        87..1265
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          318..1256
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1213..1233
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1278..1877
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1278..1297)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1774..1794
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1893..2481
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1946..1966)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2364..2375)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2553..2786
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2864..2985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2901..2920)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2955..2974
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3025..3160
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3087..3106
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3180..3199)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3181..3199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3209..3226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3209..3225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3367..3822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3454..3473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3664..3685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3848..3952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3953..4813
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4171..4190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4984..5572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5473..5492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5726..5743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5860..5881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5896..5926
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5934..5950
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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     5939..5961
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5958..5974
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5958..5974
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5993..6013
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5995..6013
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6041..6267
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6268..6448
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6495..6620
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7113..7346
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7531..7575
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        7753..7993
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     7753..7773
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7924..7943
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8023..8098
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    8154..8271
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
       61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat
      121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa
      181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg
      361 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc
      421 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
      481 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
      541 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
      601 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
      661 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
      721 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
      781 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
      841 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
      901 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
      961 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     1021 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     1081 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     1141 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     1201 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     1261 cgtgacgtac ggccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     1321 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1381 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1441 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg
     1501 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1561 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1621 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc
     1681 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1741 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1801 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1861 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
     1921 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     1981 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     2041 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     2101 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     2161 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     2221 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     2281 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     2341 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     2401 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     2461 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
     2521 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     2581 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     2641 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     2701 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     2761 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     2821 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     2881 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2941 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     3001 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3061 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3121 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     3181 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     3241 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3301 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3361 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3421 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3481 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3541 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3601 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3661 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3721 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3781 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3841 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3901 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3961 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4021 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4081 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4141 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4201 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4261 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4321 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4381 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4441 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4501 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4561 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4621 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4681 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4741 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4801 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4861 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4921 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4981 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5041 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5101 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5161 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5221 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5281 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5341 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5401 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5461 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5521 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5581 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5641 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5701 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     5761 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5821 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     5881 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5941 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     6001 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
     6061 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
     6121 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
     6181 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
     6241 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
     6301 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     6361 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     6421 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     6481 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     6541 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     6601 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     6661 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     6721 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     6781 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     6841 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     6901 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     6961 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     7021 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     7081 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     7141 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     7201 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     7261 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     7321 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     7381 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     7441 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     7501 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     7561 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     7621 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     7681 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     7741 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     7801 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     7861 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     7921 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     7981 ttgtggaaag gacgaaacac cggtccacat atgcaacacc tggttttaga gctagaaata
     8041 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     8101 ttttaagctt ggcgtaacta gatcttgaga caaatggcag tattcatcca caattttaaa
     8161 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     8221 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     8281 tacagggaca gcagagatcc actttggcgc cggctcgagg ggg
//
