LOCUS       sequence-375247-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T cell CRISPR AAV vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5185)
  AUTHORS   Dong MB, Wang G, Chow RD, Ye L, Zhu L, Dai X, Park JJ, Kim HR, 
            Errami Y, Guzman CD, Zhou X, Chen KY, Renauer PA, Du Y, Shen J, Lam 
            SZ, Zhou JJ, Lannin DR, Herbst RS, Chen S
  TITLE     Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo
            CRISPR Screens in CD8 T Cells.
  JOURNAL   Cell. 2019 Aug 22;178(5):1189-1204.e23. doi: 
            10.1016/j.cell.2019.07.044.
  PUBMED    31442407
REFERENCE   2  (bases 1 to 5185)
  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.cell.2019.07"; journalName: "Cell"; date: "2019-08-22- 
            22"; volume: "178"; issue: "5"; pages: "1189-1204.e23"
COMMENT     Sequence Label: pLY005SB_pAAV-U6sg(BbsI)-EFS-SB100X
FEATURES             Location/Qualifiers
     source          1..5185
                     /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        449..697
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     449..469
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     620..639
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        716..791
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        869..1080
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1088..2110
                     /codon_start=1
                     /product="hyperactive variant of the Sleeping Beauty 
                     transposase (Mates et al., 2009)"
                     /label=SB100X
                     /translation="MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRK
                     YKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVK
                     RVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHN
                     DHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVDIL
                     KQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENL
                     WAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY
                     "
     polyA_signal    2127..2175
                     /note="synthetic polyadenylation signal"
     repeat_region   2448..2588
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2448..2577
                     /label=AAV2 ITR
     rep_origin      2663..3118
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2750..2769)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2960..2981
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3135..3154)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3254..3276
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3314..3332)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3400..3504
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3505..3573,3574..4365)
                     /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(3723..3742)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4536..5124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5025..5044
                     /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 ctatacagtt gaagtcggaa gtttacatac
      181 acttaagttg gagtcattaa aactcgtttt tcaactactc cacaaatttc ttgttaacaa
      241 acaatagttt tggcaagtca gttaggacat ctactttgtg catgacacaa gtcatttttc
      301 caacaattgt ttacagacag attatttcac ttataattca ctgtatcaca attccagtgg
      361 gtcagaagtt tacatacact aagttgactg tgcctttaaa cagcttggaa aattccagaa
      421 aatgatgtca tggctttaga gaggatccga gggcctattt cccatgattc cttcatattt
      481 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
      541 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
      601 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
      661 cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa gacctgtttt
      721 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
      781 cgagtcggtg cttttttggc tagctggccg cgtttaaacg tcgactaggt cttgaaagga
      841 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
      901 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
      961 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     1021 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     1081 gctcgagatg ggaaaatcaa aagaaatcag ccaagacctc agaaaaagaa ttgtagacct
     1141 ccacaagtct ggttcatcct tgggagcaat ttccaaacgc ctggcggtac cacgttcatc
     1201 tgtacaaaca atagtacgca agtataaaca ccatgggacc acgcagccgt cataccgctc
     1261 aggaaggaga cgcgttctgt ctcctagaga tgaacgtact ttggtgcgaa aagtgcaaat
     1321 caatcccaga acaacagcaa aggaccttgt gaagatgctg gaggaaacag gtacaaaagt
     1381 atctatatcc acagtaaaac gagtcctata tcgacataac ctgaaaggcc actcagcaag
     1441 gaagaagcca ctgctccaaa accgacataa gaaagccaga ctacggtttg caactgcaca
     1501 tggggacaaa gatcgtactt tttggagaaa tgtcctctgg tctgatgaaa caaaaataga
     1561 actgtttggc cataatgacc atcgttatgt ttggaggaag aagggggagg cttgcaagcc
     1621 gaagaacacc atcccaaccg tgaagcacgg gggtggcagc atcatgttgt gggggtgctt
     1681 tgctgcagga gggactggtg cacttcacaa aatagatggc atcatggacg cggtgcagta
     1741 tgtggatata ttgaagcaac atctcaagac atcagtcagg aagttaaagc ttggtcgcaa
     1801 atgggttttc caacacgaca atgaccccaa gcatacttcc aaagttgtgg caaaatggct
     1861 taaggacaac aaagtcaagg tattggagtg gccatcacaa agccctgacc tcaatcctat
     1921 agaaaatttg tgggcagaac tgaaaaagcg tgtgcgagca aggaggccta caaacctgac
     1981 tcagttacac cagctctgtc aggaggaatg ggccaaaatt cacccaaatt attgtgggaa
     2041 gcttgtggaa ggctacccga aacgtttgac ccaagttaaa caatttaaag gcaatgctac
     2101 caaatactag gggccctaac cgcgggaata aaagatcttt attttcatta gatctgtgtg
     2161 ttggtttttt gtgtgaattc ttgagtgtat gtaaacttct gacccactgg gaatgtgatg
     2221 aaagaaataa aagctgaaat gaatcattct ctctactatt attctgatat ttcacattct
     2281 taaaataaag tggtgatcct aactgaccta agacagggaa tttttactag gattaaatgt
     2341 caggaattgt gaaaaagtga gtttaaatgt atttggctaa ggtgtatgta aacttccgac
     2401 ttcaactgta taggcatgcg gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     2461 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     2521 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     2581 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2641 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2701 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     2761 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2821 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     2881 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2941 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     3001 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     3061 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     3121 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3181 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3241 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3301 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     3361 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3421 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3481 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3541 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3601 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3661 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     3721 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     3781 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     3841 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3901 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3961 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4021 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4081 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4141 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4201 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4261 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4321 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4381 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4441 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4501 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4561 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4621 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4681 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4741 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4801 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4861 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4921 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4981 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5041 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5101 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5161 ttttgctggc cttttgctca catgt
//
