LOCUS       sequence-183229-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human EZH2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the
            Diversity of p53-Dependent Responses to Cell-Cycle Defects.
  JOURNAL   Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 
            10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.
  PUBMED    28216383
REFERENCE   2  (bases 1 to 7302)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.devcel.2017.01.012"; journalName: "Dev Cell"; date: 
            "2017-02-27- 27"; volume: "40"; issue: "4"; pages: "405-420.e2"
COMMENT     Sequence Label: EZH2 E10.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    127..148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(163..168,169..186,187..193)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    201..217
                     /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     206..228
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     225..241
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     225..241
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     260..280
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        262..280
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        308..534
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             535..715
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    762..887
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1380..1613
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1798..1842
                     /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        2020..2268
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2020..2040
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2191..2210
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2424..2541
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2610..2821
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2847..3446
                     /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(2847..2866)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3343..3363
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3462..4050
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3515..3535)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3933..3944)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4122..4355
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4433..4554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4470..4489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4524..4543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4594..4729
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4749..4768)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4750..4768)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4778..4795)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4778..4794)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4787..4809)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4936..5391
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5023..5042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5233..5254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5417..5521
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5522..5590,5591..6382)
                     /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(5740..5759)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6553..7141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7042..7061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7295..7302,1..10)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
       61 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      121 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      181 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      241 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
      301 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
      361 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      421 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      481 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      541 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      601 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      661 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      721 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      781 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      841 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      901 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      961 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1021 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1081 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1141 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1201 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1261 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1321 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1381 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1441 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1501 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1561 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1621 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1681 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1741 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1801 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1861 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1921 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1981 tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt
     2041 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
     2101 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     2161 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     2221 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg agctcattgc
     2281 gcgggactag tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc
     2341 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga
     2401 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2461 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2521 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc
     2581 cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2641 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
     2701 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2761 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2821 ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa gcccacggtg cgcctcgcca
     2881 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2941 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3001 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3061 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3121 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3181 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     3241 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3301 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     3361 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3421 gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca
     3481 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3541 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3601 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3661 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3721 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3781 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3841 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     3901 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3961 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4021 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     4081 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4141 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     4201 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     4261 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     4321 cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct
     4381 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4441 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4501 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4561 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4621 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4681 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4741 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4801 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4861 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4921 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4981 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5041 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5101 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5161 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5221 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5281 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5341 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5401 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5461 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5521 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5581 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5641 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5701 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5761 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5821 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5881 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5941 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6001 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6061 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6121 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6181 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6241 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6301 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6361 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6421 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6481 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6541 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6601 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6661 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6721 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6781 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6841 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6901 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6961 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7021 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7081 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7141 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7201 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7261 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tc
//
