LOCUS       sequence-375249-        5739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T cell CRISPR AAV-SB vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5739)
  AUTHORS   Ye L, Park JJ, Dong MB, Yang Q, Chow RD, Peng L, Du Y, Guo J, Dai X,
            Wang G, Errami Y, Chen S
  TITLE     In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty 
            hybrid vectors identifies membrane targets for improving 
            immunotherapy for glioblastoma.
  JOURNAL   Nat Biotechnol. 2019 Nov;37(11):1302-1313. doi: 
            10.1038/s41587-019-0246-4. Epub 2019 Sep 23.
  PUBMED    31548728
REFERENCE   2  (bases 1 to 5739)
  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: 
            "10.1038/s41587-019-0246-4"; journalName: "Nat Biotechnol"; date: 
            "2019-11"; volume: "37"; issue: "11"; pages: "1302-1313"
COMMENT     Sequence Label: pLY017SB_pAAV-U6sg(BbsI)-EFS-Thy1.1-P2A-SB100X
FEATURES             Location/Qualifiers
     source          1..5739
                     /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             1093..1578
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     CDS             1588..1641
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1642..2664
                     /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    2681..2729
                     /note="synthetic polyadenylation signal"
     repeat_region   3002..3142
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3002..3131
                     /label=AAV2 ITR
     rep_origin      3217..3672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3304..3323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3514..3535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3689..3708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3808..3830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3868..3886)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3954..4058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4059..4127,4128..4919)
                     /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(4277..4296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5090..5678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5579..5598
                     /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 gctcgagcca ccatgaaccc agccatcagc gtcgctctcc tgctctcagt cttgcaggtg
     1141 tcccgagggc agaaggtgac cagcctgaca gcctgcctgg tgaaccaaaa ccttcgcctg
     1201 gactgccgcc atgagaataa caccaaggat aactccatcc agcatgagtt cagcctgacc
     1261 cgagagaaga ggaagcacgt gctctcaggc acccttggga tacccgagca cacgtaccgc
     1321 tcccgcgtca ccctctccaa ccagccctat atcaaggtcc ttaccctagc caacttcacc
     1381 accaaggatg agggcgacta cttttgtgag cttcgcgtgt cgggcgcgaa tcccatgagc
     1441 tccaataaaa gtatcagtgt gtatagagac aagctggtca agtgtggcgg cataagcctg
     1501 ctggttcaga acacatcctg gatgctgctg ctgctgcttt ccctctccct cctccaagcc
     1561 ctggacttca tttctctggg cagtggagag ggcagaggaa gtctgctaac atgcggtgac
     1621 gtcgaggaga atcctggccc gatgggaaaa tcaaaagaaa tcagccaaga cctcagaaaa
     1681 agaattgtag acctccacaa gtctggttca tccttgggag caatttccaa acgcctggcg
     1741 gtaccacgtt catctgtaca aacaatagta cgcaagtata aacaccatgg gaccacgcag
     1801 ccgtcatacc gctcaggaag gagacgcgtt ctgtctccta gagatgaacg tactttggtg
     1861 cgaaaagtgc aaatcaatcc cagaacaaca gcaaaggacc ttgtgaagat gctggaggaa
     1921 acaggtacaa aagtatctat atccacagta aaacgagtcc tatatcgaca taacctgaaa
     1981 ggccactcag caaggaagaa gccactgctc caaaaccgac ataagaaagc cagactacgg
     2041 tttgcaactg cacatgggga caaagatcgt actttttgga gaaatgtcct ctggtctgat
     2101 gaaacaaaaa tagaactgtt tggccataat gaccatcgtt atgtttggag gaagaagggg
     2161 gaggcttgca agccgaagaa caccatccca accgtgaagc acgggggtgg cagcatcatg
     2221 ttgtgggggt gctttgctgc aggagggact ggtgcacttc acaaaataga tggcatcatg
     2281 gacgcggtgc agtatgtgga tatattgaag caacatctca agacatcagt caggaagtta
     2341 aagcttggtc gcaaatgggt tttccaacac gacaatgacc ccaagcatac ttccaaagtt
     2401 gtggcaaaat ggcttaagga caacaaagtc aaggtattgg agtggccatc acaaagccct
     2461 gacctcaatc ctatagaaaa tttgtgggca gaactgaaaa agcgtgtgcg agcaaggagg
     2521 cctacaaacc tgactcagtt acaccagctc tgtcaggagg aatgggccaa aattcaccca
     2581 aattattgtg ggaagcttgt ggaaggctac ccgaaacgtt tgacccaagt taaacaattt
     2641 aaaggcaatg ctaccaaata ctaggggccc taaccgcggg aataaaagat ctttattttc
     2701 attagatctg tgtgttggtt ttttgtgtga attcttgagt gtatgtaaac ttctgaccca
     2761 ctgggaatgt gatgaaagaa ataaaagctg aaatgaatca ttctctctac tattattctg
     2821 atatttcaca ttcttaaaat aaagtggtga tcctaactga cctaagacag ggaattttta
     2881 ctaggattaa atgtcaggaa ttgtgaaaaa gtgagtttaa atgtatttgg ctaaggtgta
     2941 tgtaaacttc cgacttcaac tgtataggca tgcggtaacc acgtgcggac cgagcggccg
     3001 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3061 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3121 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3181 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     3301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3421 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3541 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     3601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3661 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3721 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3841 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3901 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4021 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4081 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4141 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4201 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4261 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4321 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4381 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4441 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4501 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4561 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4621 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4681 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4741 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4801 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4861 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4921 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4981 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5041 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5101 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5161 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5221 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5281 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5341 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5401 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5461 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5521 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5581 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5641 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5701 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
