LOCUS       sequence-183230-        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 E9.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    114..135
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(150..155,156..173,174..180)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    188..204
                     /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     193..215
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     212..228
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     212..228
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     247..267
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        249..267
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        295..521
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             522..702
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    749..874
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1367..1600
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1785..1829
                     /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        2007..2255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2007..2027
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2178..2197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2411..2528
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2597..2808
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2834..3433
                     /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(2834..2853)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3330..3350
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3449..4037
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3502..3522)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3920..3931)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4109..4342
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4420..4541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4457..4476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4511..4530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4581..4716
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4736..4755)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4737..4755)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4765..4782)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4765..4781)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4774..4796)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4923..5378
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5010..5029)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5220..5241
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5404..5508
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5509..5577,5578..6369)
                     /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(5727..5746)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6540..7128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7029..7048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7282..7299
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
       61 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      121 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      181 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      241 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
      301 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
      361 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
      421 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
      481 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
      541 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      601 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      661 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      721 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      781 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      841 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      901 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      961 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     1021 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1081 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1141 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1201 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1261 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1321 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1381 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1441 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1501 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1561 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1621 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1681 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1741 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1801 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1861 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1921 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1981 acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc
     2041 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2101 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2161 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2221 tggctttata tatcttgtgg aaaggacgaa acaccgacac gcttccgcca acaaacgttt
     2281 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     2341 aaaagtggca ccgagtcggt gctttttttg aattctagat cttgagacaa atggcagtat
     2401 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2461 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2521 aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc
     2581 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2641 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2701 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2761 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc
     2821 gggatccgcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     2881 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     2941 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3001 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3061 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3121 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3181 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     3241 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     3301 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     3361 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     3421 gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     3481 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3541 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3601 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3661 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3721 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3781 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3841 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3901 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3961 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     4021 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     4081 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     4141 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     4201 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     4261 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     4321 tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat
     4381 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     4441 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4501 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     4561 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4621 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4681 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     4741 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4801 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4861 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4921 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4981 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5041 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5101 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5161 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5221 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5281 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5341 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     5401 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5461 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5521 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5581 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5641 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5701 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5761 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5821 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5881 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5941 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     6001 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6061 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6121 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6181 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6241 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6301 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6361 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6421 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6481 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6541 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6601 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6661 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6721 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6781 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6841 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6901 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6961 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7021 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7081 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7141 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7201 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7261 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aa
//
