LOCUS       sequence-375074-        4938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-TBG-Cre-sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4938)
  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 4938)
  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: pGW03 AAV-TBG-Cre-sgRNA
FEATURES             Location/Qualifiers
     source          1..4938
                     /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        152..400
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     152..172
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     323..342
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        563..972
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin 
                     gene (Yan et al., 2012)"
     regulatory      991..1000
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1030..1050
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2077..2103
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2125..2173
                     /note="synthetic polyadenylation signal"
     repeat_region   2201..2341
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2201..2330
                     /label=AAV2 ITR
     rep_origin      2416..2871
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2503..2522)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2713..2734
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2888..2907)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3007..3029
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3067..3085)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3153..3257
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3258..3326,3327..4118)
                     /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(3476..3495)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4289..4877
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4778..4797
                     /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 gcgttctaga agagggccta tttcccatga ttccttcata
      181 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      241 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      301 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      361 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggaagagcga gctcttctgt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt ggtaccgcgg cctctagact cgaggggctg gaagctacct
      541 ttgacatcat ttcctctgcg aatgcatgta taatttctac agaacctatt agaaaggatc
      601 acccagcctc tgcttttgta caactttccc ttaaaaaact gccaattcca ctgctgtttg
      661 gcccaatagt gagaactttt tcctgctgcc tcttggtgct tttgcctatg gcccctattc
      721 tgcctgctga agacactctt gccagcatgg acttaaaccc ctccagctct gacaatcctc
      781 tttctctttt gttttacatg aagggtctgg cagccaaagc aatcactcaa agttcaaacc
      841 ttatcatttt ttgctttgtt cctcttggcc ttggttttgt acatcagctt tgaaaatacc
      901 atcccagggt taatgctggg gttaatttat aactaagagt gctctagttt tgcaatacag
      961 gacatgctat aaaaatggaa agataccggt gccaccatgg ccccaaaggt taaccgtacg
     1021 gccaccatgc ccaagaagaa gaggaaggtg tccaatctcc tgactgttca ccagaacctc
     1081 cctgcgctgc cagtagatgc cactagcgat gaggtcagga aaaatctcat ggatatgttt
     1141 agggatagac aggcgttttc tgaacacacc tggaaaatgc tgcttagcgt gtgccgatcc
     1201 tgggcagcct ggtgtaagct gaacaatcgc aaatggttcc ccgccgagcc ggaggacgtg
     1261 cgcgattacc tgctgtatct ccaggcaaga gggctggctg tcaagactat ccagcagcac
     1321 ttgggccaac tgaatatgct gcatcgacgc agcgggctcc cccggcctag cgattcaaac
     1381 gcagtctccc ttgttatgag gagaattaga aaggaaaacg tagatgcggg tgagagggct
     1441 aagcaggctc tcgcttttga gcggactgat ttcgaccagg tcagatccct gatggagaac
     1501 agcgatcggt gccaggacat caggaacctc gcatttctgg gaattgcata taacacactt
     1561 ctgcgcatag ctgagatcgc ccggatcaga gtgaaagaca tcagtcgaac ggacggcggc
     1621 cggatgctta ttcatattgg acgcacaaag acattggtca gcaccgctgg cgttgaaaag
     1681 gccttgtccc tgggcgtaac gaagctggtg gaaagatgga tctcagtgtc cggcgtggct
     1741 gacgacccta ataattactt gttctgtcga gtgagaaaaa acggagtcgc cgcgccctct
     1801 gccaccagcc aattgagtac acgggccctt gaagggatct ttgaggcaac ccaccgactc
     1861 atatacggag ccaaggatga cagtggccag aggtatctcg cctggtcagg tcattctgct
     1921 agggtggggg ccgcacgaga catggcgcgg gcaggagtct ccataccaga gattatgcaa
     1981 gctggaggtt ggacaaatgt gaacatcgtt atgaactata tccgcaatct tgactctgaa
     2041 accggggcca tggtgagact gctcgaagat ggtgactacc catacgatgt tccagattac
     2101 gcttaagaat tcgatatcaa gcttaataaa agatctttat tttcattaga tctgtgtgtt
     2161 ggttttttgt gtggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     2221 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     2281 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     2341 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     2401 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2461 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     2521 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2581 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     2641 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2701 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     2761 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     2821 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     2881 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     2941 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3001 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3061 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3121 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3181 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3241 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3301 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3361 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3421 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3481 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3541 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3601 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3661 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3721 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3781 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3841 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3901 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3961 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4021 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4081 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4141 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4201 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4261 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4321 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4381 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4441 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4501 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4561 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4621 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4681 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4741 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4801 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4861 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4921 ggccttttgc tcacatgt
//
