LOCUS       sequence-375229-        7344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lenti-sgKmt2d-sg3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7344)
  AUTHORS   Wang G, Chow RD, Zhu L, Bai Z, Ye L, Zhang F, Renauer PA, Dong MB, 
            Dai X, Zhang X, Du Y, Cheng Y, Niu L, Chu Z, Kim K, Liao C, Clark P,
            Errami Y, Chen S
  TITLE     CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major 
            Modulator of Immune Checkpoint Blockade.
  JOURNAL   Cancer Discov. 2020 Dec;10(12):1912-1933. doi: 
            10.1158/2159-8290.CD-19-1448. Epub 2020 Sep 4.
  PUBMED    32887696
REFERENCE   2  (bases 1 to 7344)
  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: 
            "10.1158/2159-8290.CD-19-1448"; journalName: "Cancer Discov"; date: 
            "2020-12"; volume: "10"; issue: "12"; pages: "1912-1933"
COMMENT     Sequence Label: Lenti-sgKmt2d-sg3
FEATURES             Location/Qualifiers
     source          1..7344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..273
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             299..898
                     /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(299..318)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     795..815
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    914..1502
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(967..987)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1385..1396)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1574..1807
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1885..2006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1922..1941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1976..1995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2046..2181
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2201..2220)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2202..2220)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2230..2247)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2230..2246)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2239..2261)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2388..2843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2475..2494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2685..2706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2869..2973
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2974..3042,3043..3834)
                     /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(3192..3211)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4005..4593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4494..4513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4747..4764
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4881..4902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4917..4922,4923..4940,4941..4947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4955..4971
                     /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     4960..4982
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4979..4995
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4979..4995
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5014..5034
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5016..5034
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5062..5288
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5289..5469
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5516..5641
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6134..6367
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6552..6596
                     /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        6774..7022
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6774..6794
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6945..6964
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7044..7119
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    7175..7292
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccggggaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
       61 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
      121 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
      181 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
      241 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagacgta cggccaccat
      301 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
      361 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
      421 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
      481 cggcaaggtg tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag
      541 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
      601 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
      661 cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag
      721 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
      781 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
      841 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
      901 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
      961 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     1021 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     1081 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     1141 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     1201 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     1261 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     1321 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     1381 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     1441 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     1501 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     1561 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     1621 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     1681 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     1741 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     1801 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     1861 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     1921 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1981 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     2041 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2101 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     2161 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     2221 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2281 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2341 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     2401 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2461 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2521 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2581 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2641 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2701 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2761 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2821 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2881 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2941 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3001 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3061 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3121 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3181 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     3241 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3301 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3361 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3421 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3481 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3541 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3601 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3661 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3721 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3781 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3841 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3901 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3961 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4021 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4081 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4141 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4201 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4261 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4321 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4381 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4441 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4501 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4561 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4621 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4681 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4741 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     4801 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4861 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     4921 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     4981 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
     5041 aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg
     5101 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
     5161 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
     5221 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
     5281 acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5341 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     5401 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     5461 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     5521 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     5581 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     5641 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     5701 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     5761 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     5821 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     5881 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     5941 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     6001 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     6061 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     6121 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     6181 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     6241 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     6301 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     6361 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     6421 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     6481 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     6541 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     6601 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     6661 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     6721 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc
     6781 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     6841 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     6901 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     6961 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     7021 ccggtgtgtg agacatgtga caagttttag agctagaaat agcaagttaa aataaggcta
     7081 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttaagct tggcgtaact
     7141 agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
     7201 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     7261 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
     7321 cactttggcg ccggctcgag gggg
//
