LOCUS       sequence-183193-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human KMT5A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  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 7301)
  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: KMT5A H1.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..74
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    191..212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(227..232,233..250,251..257)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    265..281
                     /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     270..292
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     289..305
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     289..305
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     324..344
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        326..344
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        372..598
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             599..779
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    826..951
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1444..1677
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1862..1906
                     /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        2084..2332
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2084..2104
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2255..2274
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2487..2604
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2673..2884
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2910..3509
                     /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(2910..2929)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3406..3426
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3525..4113
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3578..3598)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3996..4007)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4185..4418
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4496..4617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4533..4552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4587..4606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4657..4792
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4812..4831)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4813..4831)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4841..4858)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4841..4857)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4850..4872)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4999..5454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5086..5105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5296..5317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5480..5584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5585..5653,5654..6445)
                     /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(5803..5822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6616..7204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7105..7124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
       61 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
      121 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
      181 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
      241 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
      301 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
      361 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
      421 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
      481 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
      541 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
      601 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      661 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      721 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      781 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      841 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      901 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      961 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1021 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1081 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1141 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1201 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1261 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1321 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1381 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1441 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1501 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1561 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1621 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1681 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1741 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1801 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1861 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1921 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1981 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2041 attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat
     2101 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     2161 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2221 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2281 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtccaagc
     2341 cccgcgcggt gggtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag
     2401 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt ctagatcttg
     2461 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2521 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2581 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
     2641 cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2701 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     2761 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2821 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2881 acaggtgtcg tgacgcggga tccgccacca tgaccgagta caagcccacg gtgcgcctcg
     2941 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     3001 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     3061 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     3121 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     3181 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     3241 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
     3301 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     3361 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     3421 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     3481 ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt
     3541 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3601 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3661 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3721 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3781 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3841 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3901 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3961 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     4021 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     4081 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
     4141 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     4201 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     4261 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     4321 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     4381 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     4441 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     4501 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4561 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4621 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4681 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4741 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     4801 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     4861 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4921 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4981 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5041 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5101 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5161 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5221 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5281 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5341 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5401 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5461 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5521 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5581 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5641 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5701 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5761 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5821 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5881 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5941 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6001 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6061 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6121 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6181 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6241 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6301 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6361 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6421 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7261 catgttcttt cctgcgttat cccctgattc tgtggataac c
//
