LOCUS       sequence-375232-        5291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-TBG-Cre-sgKmt2d.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5291)
  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 5291)
  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: AAV-TBG-Cre-sgKmt2d
FEATURES             Location/Qualifiers
     source          1..5291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   30..159
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        174..422
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        443..518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        532..780
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        801..876
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        945..1354
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     regulatory      1373..1382
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1412..1432
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2459..2485
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2507..2555
                     /note="synthetic polyadenylation signal"
     repeat_region   2583..2723
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2583..2712
                     /label=AAV2 ITR
     rep_origin      2798..3253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2885..2904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3095..3116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3270..3289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3389..3411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3449..3467)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3535..3639
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3640..3708,3709..4500)
                     /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(3858..3877)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4671..5259
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5160..5179
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac
       61 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
      121 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg cgtgagggcc
      181 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
      241 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
      301 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
      361 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
      421 ccgtgtaata gctcctgcat gggttttaga gctagaaata gcaagttaaa ataaggctag
      481 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttctaga agagggccta
      541 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
      601 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      661 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      721 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      781 gtgtgtgaga catgtgacaa gttttagagc tagaaatagc aagttaaaat aaggctagtc
      841 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttggtaccgc ggcctctaga
      901 ctcgaggggc tggaagctac ctttgacatc atttcctctg cgaatgcatg tataatttct
      961 acagaaccta ttagaaagga tcacccagcc tctgcttttg tacaactttc ccttaaaaaa
     1021 ctgccaattc cactgctgtt tggcccaata gtgagaactt tttcctgctg cctcttggtg
     1081 cttttgccta tggcccctat tctgcctgct gaagacactc ttgccagcat ggacttaaac
     1141 ccctccagct ctgacaatcc tctttctctt ttgttttaca tgaagggtct ggcagccaaa
     1201 gcaatcactc aaagttcaaa ccttatcatt ttttgctttg ttcctcttgg ccttggtttt
     1261 gtacatcagc tttgaaaata ccatcccagg gttaatgctg gggttaattt ataactaaga
     1321 gtgctctagt tttgcaatac aggacatgct ataaaaatgg aaagataccg gtgccaccat
     1381 ggccccaaag gttaaccgta cggccaccat gcccaagaag aagaggaagg tgtccaatct
     1441 cctgactgtt caccagaacc tccctgcgct gccagtagat gccactagcg atgaggtcag
     1501 gaaaaatctc atggatatgt ttagggatag acaggcgttt tctgaacaca cctggaaaat
     1561 gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag ctgaacaatc gcaaatggtt
     1621 ccccgccgag ccggaggacg tgcgcgatta cctgctgtat ctccaggcaa gagggctggc
     1681 tgtcaagact atccagcagc acttgggcca actgaatatg ctgcatcgac gcagcgggct
     1741 cccccggcct agcgattcaa acgcagtctc ccttgttatg aggagaatta gaaaggaaaa
     1801 cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt gagcggactg atttcgacca
     1861 ggtcagatcc ctgatggaga acagcgatcg gtgccaggac atcaggaacc tcgcatttct
     1921 gggaattgca tataacacac ttctgcgcat agctgagatc gcccggatca gagtgaaaga
     1981 catcagtcga acggacggcg gccggatgct tattcatatt ggacgcacaa agacattggt
     2041 cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta acgaagctgg tggaaagatg
     2101 gatctcagtg tccggcgtgg ctgacgaccc taataattac ttgttctgtc gagtgagaaa
     2161 aaacggagtc gccgcgccct ctgccaccag ccaattgagt acacgggccc ttgaagggat
     2221 ctttgaggca acccaccgac tcatatacgg agccaaggat gacagtggcc agaggtatct
     2281 cgcctggtca ggtcattctg ctagggtggg ggccgcacga gacatggcgc gggcaggagt
     2341 ctccatacca gagattatgc aagctggagg ttggacaaat gtgaacatcg ttatgaacta
     2401 tatccgcaat cttgactctg aaaccggggc catggtgaga ctgctcgaag atggtgacta
     2461 cccatacgat gttccagatt acgcttaaga attcgatatc aagcttaata aaagatcttt
     2521 attttcatta gatctgtgtg ttggtttttt gtgtggtaac cacgtgcgga ccgagcggcc
     2581 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     2641 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     2701 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     2761 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     2821 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     2881 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2941 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3001 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3061 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3121 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     3181 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3241 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3301 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3361 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3421 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3481 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3541 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3601 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3661 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3721 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3781 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3841 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3901 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3961 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4021 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4081 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4141 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4201 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4261 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4321 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4381 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4441 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4501 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4561 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4621 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4681 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4741 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4801 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4861 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4921 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4981 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5041 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5101 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5161 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5221 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5281 ttacggttcc t
//
