LOCUS       sequence-375250-        6696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T cell CRISPR lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6696)
  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 6696)
  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: pSC008_pLKO-U6-BsmBI-chRNA(+85)-EFS-Thy11
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1982..2057
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2113..2230
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2344..2555
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2581..3069
                     /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"
     polyA_signal    3076..3124
                     /note="synthetic polyadenylation signal"
     LTR             3213..3446
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3524..3645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3561..3580)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3615..3634
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3685..3820
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3840..3859)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3841..3859)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3869..3886)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3869..3885)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3878..3900)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4027..4482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4114..4133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4324..4345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4508..4612
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4613..4681,4682..5473)
                     /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(4831..4850)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5644..6232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6133..6152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6386..6403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6520..6541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6556..6561,6562..6579,6580..6586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6594..6610
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6599..6621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6618..6634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6618..6634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6653..6673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6655..6673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cggacgtctc
     1981 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     2041 tggcaccgag tcggtgcttt tttaagcttg gcgtaactag atcttgagac aaatggcagt
     2101 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2161 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2221 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgaggg
     2281 ggcccgggga attcgctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
     2341 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2401 gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2461 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2521 ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagacg tacggccacc
     2581 atgaacccag ccatcagcgt cgctctcctg ctctcagtct tgcaggtgtc ccgagggcag
     2641 aaggtgacca gcctgacagc ctgcctggtg aaccaaaacc ttcgcctgga ctgccgccat
     2701 gagaataaca ccaaggataa ctccatccag catgagttca gcctgacccg agagaagagg
     2761 aagcacgtgc tctcaggcac ccttgggata cccgagcaca cgtaccgctc ccgcgtcacc
     2821 ctctccaacc agccctatat caaggtcctt accctagcca acttcaccac caaggatgag
     2881 ggcgactact tttgtgagct tcgcgtctcg ggcgcgaatc ccatgagctc caataaaagt
     2941 atcagtgtgt atagagacaa gctggtcaag tgtggcggca taagcctgct ggttcagaac
     3001 acatcctgga tgctgctgct gctgctttcc ctctccctcc tccaagccct ggacttcatt
     3061 tctctgtgaa gcgctaataa aagatcttta ttttcattag atctgtgtgt tggttttttg
     3121 tgtggtaact cgagacgtgc ggtcgacttt aagaccaatg acttacaagg cagctgtaga
     3181 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     3241 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     3301 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     3361 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     3421 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     3481 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     3541 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3601 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     3661 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3721 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3781 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     3841 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     3901 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3961 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4021 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4081 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4141 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4201 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4261 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4321 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4381 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4441 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     4501 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4561 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4621 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4681 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4741 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4801 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4861 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4921 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4981 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5041 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5101 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5161 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5221 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5281 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5341 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5401 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5461 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5521 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5581 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5761 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     6361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     6421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     6481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     6541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     6601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     6661 ccctcactaa agggaacaaa agctggagct gcaagc
//
