LOCUS       sequence-375128-        5289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-TBG-Cre-sgP53-sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5289)
  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 5289)
  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: pGW02 AAV-TBG-Cre-sgP53-sgRNA
FEATURES             Location/Qualifiers
     source          1..5289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        503..751
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        770..845
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        914..1323
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     regulatory      1342..1351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1381..1401
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2428..2454
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2476..2524
                     /note="synthetic polyadenylation signal"
     repeat_region   2552..2692
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2552..2681
                     /label=AAV2 ITR
     rep_origin      2767..3222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2854..2873)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3064..3085
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3239..3258)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3358..3380
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3418..3436)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3504..3608
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3609..3677,3678..4469)
                     /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(3827..3846)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4640..5228
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5129..5148
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgtgtaat agctcctgca tgggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag aagagggcct atttcccatg attccttcat atttgcatat
      541 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      601 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      661 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      721 tttatatatc ttgtggaaag gacgaaacac cggaagagcg agctcttctg ttttagagct
      781 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      841 ggtgcttttt tggtaccgcg gcctctagac tcgaggggct ggaagctacc tttgacatca
      901 tttcctctgc gaatgcatgt ataatttcta cagaacctat tagaaaggat cacccagcct
      961 ctgcttttgt acaactttcc cttaaaaaac tgccaattcc actgctgttt ggcccaatag
     1021 tgagaacttt ttcctgctgc ctcttggtgc ttttgcctat ggcccctatt ctgcctgctg
     1081 aagacactct tgccagcatg gacttaaacc cctccagctc tgacaatcct ctttctcttt
     1141 tgttttacat gaagggtctg gcagccaaag caatcactca aagttcaaac cttatcattt
     1201 tttgctttgt tcctcttggc cttggttttg tacatcagct ttgaaaatac catcccaggg
     1261 ttaatgctgg ggttaattta taactaagag tgctctagtt ttgcaataca ggacatgcta
     1321 taaaaatgga aagataccgg tgccaccatg gccccaaagg ttaaccgtac ggccaccatg
     1381 cccaagaaga agaggaaggt gtccaatctc ctgactgttc accagaacct ccctgcgctg
     1441 ccagtagatg ccactagcga tgaggtcagg aaaaatctca tggatatgtt tagggataga
     1501 caggcgtttt ctgaacacac ctggaaaatg ctgcttagcg tgtgccgatc ctgggcagcc
     1561 tggtgtaagc tgaacaatcg caaatggttc cccgccgagc cggaggacgt gcgcgattac
     1621 ctgctgtatc tccaggcaag agggctggct gtcaagacta tccagcagca cttgggccaa
     1681 ctgaatatgc tgcatcgacg cagcgggctc ccccggccta gcgattcaaa cgcagtctcc
     1741 cttgttatga ggagaattag aaaggaaaac gtagatgcgg gtgagagggc taagcaggct
     1801 ctcgcttttg agcggactga tttcgaccag gtcagatccc tgatggagaa cagcgatcgg
     1861 tgccaggaca tcaggaacct cgcatttctg ggaattgcat ataacacact tctgcgcata
     1921 gctgagatcg cccggatcag agtgaaagac atcagtcgaa cggacggcgg ccggatgctt
     1981 attcatattg gacgcacaaa gacattggtc agcaccgctg gcgttgaaaa ggccttgtcc
     2041 ctgggcgtaa cgaagctggt ggaaagatgg atctcagtgt ccggcgtggc tgacgaccct
     2101 aataattact tgttctgtcg agtgagaaaa aacggagtcg ccgcgccctc tgccaccagc
     2161 caattgagta cacgggccct tgaagggatc tttgaggcaa cccaccgact catatacgga
     2221 gccaaggatg acagtggcca gaggtatctc gcctggtcag gtcattctgc tagggtgggg
     2281 gccgcacgag acatggcgcg ggcaggagtc tccataccag agattatgca agctggaggt
     2341 tggacaaatg tgaacatcgt tatgaactat atccgcaatc ttgactctga aaccggggcc
     2401 atggtgagac tgctcgaaga tggtgactac ccatacgatg ttccagatta cgcttaagaa
     2461 ttcgatatca agcttaataa aagatcttta ttttcattag atctgtgtgt tggttttttg
     2521 tgtggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     2581 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     2641 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     2701 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     2761 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2821 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     2881 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2941 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3001 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3061 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     3121 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     3181 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3241 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3301 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3361 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3421 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3481 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3541 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3601 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3661 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3721 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3781 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3841 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3901 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3961 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4021 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4081 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4141 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4201 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4261 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4321 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4381 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4441 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4501 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4561 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4621 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4681 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4741 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4801 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4861 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4921 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4981 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5041 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5101 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5161 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5221 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5281 ctcacatgt
//
