LOCUS       sequence-183189-        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 H2.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    64..85
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(100..105,106..123,124..130)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    138..154
                     /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     143..165
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     162..178
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     162..178
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     197..217
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        199..217
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        245..471
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             472..652
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    699..824
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1317..1550
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1735..1779
                     /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        1957..2205
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1957..1977
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2128..2147
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2360..2477
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2546..2757
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2783..3382
                     /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(2783..2802)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3279..3299
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3398..3986
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3451..3471)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3869..3880)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4058..4291
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4369..4490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4406..4425)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4460..4479
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4530..4665
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4685..4704)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4686..4704)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4714..4731)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4714..4730)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4723..4745)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4872..5327
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4959..4978)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5169..5190
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5353..5457
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5458..5526,5527..6318)
                     /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(5676..5695)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6489..7077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6978..6997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7231..7248
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
       61 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      121 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      181 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      241 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      301 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      361 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      421 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      481 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      541 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      601 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      661 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      721 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      781 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      841 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      901 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      961 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     1021 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1081 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1141 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1201 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1261 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1321 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1381 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1441 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1501 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1561 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1621 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1681 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1741 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1801 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1861 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1921 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1981 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     2041 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2101 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2161 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgaaga tgtccaagcc
     2221 ccgcggttta agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta
     2281 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa
     2341 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     2401 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2461 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc
     2521 tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     2581 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2641 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2701 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg
     2761 tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     2821 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     2881 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     2941 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3001 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3061 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3121 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     3181 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     3241 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     3301 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     3361 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     3421 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3481 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3541 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3601 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3661 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3721 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3781 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     3841 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3901 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3961 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     4021 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     4081 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     4141 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     4201 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     4261 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     4321 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4381 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4441 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4501 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4561 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4621 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4681 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4741 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4801 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4861 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4921 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4981 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5041 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5101 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5161 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5221 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5281 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5341 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5401 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5461 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5521 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5581 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5641 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5701 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5761 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5821 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5881 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5941 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6001 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6061 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6121 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6181 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6241 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6301 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6361 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6421 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6481 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6541 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6601 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6661 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6721 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6781 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6841 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6901 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6961 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7021 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7081 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7141 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7201 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7261 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga t
//
