LOCUS       sequence-317503-        6619 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression plasmid of sgRNA with violet-excited 
            fluorescent protein (VEX).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6619)
  AUTHORS   Chen Z, Arai E, Khan O, Zhang Z, Ngiow SF, He Y, Huang H, Manne S, 
            Cao Z, Baxter AE, Cai Z, Freilich E, Ali MA, Giles JR, Wu JE, 
            Greenplate AR, Hakeem MA, Chen Q, Kurachi M, Nzingha K, Ekshyyan V, 
            Mathew D, Wen Z, Speck NA, Battle A, Berger SL, Wherry EJ, Shi J
  TITLE     In vivo CD8(+) T cell CRISPR screening reveals control by Fli1 in 
            infection and cancer.
  JOURNAL   Cell. 2021 Mar 4;184(5):1262-1280.e22. doi: 
            10.1016/j.cell.2021.02.019. Epub 2021 Feb 25.
  PUBMED    33636129
REFERENCE   2  (bases 1 to 6619)
  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.2021.02.019"; journalName: "Cell"; date: "2021-03-4-
            4"; volume: "184"; issue: "5"; pages: "1262-1280.e22"
COMMENT     Sequence Label: pSL21-VEX
FEATURES             Location/Qualifiers
     source          1..6619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1408..1656
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1408..1428
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1579..1598
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1820..1937
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2006..2217
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             join(2246..2248,2249..2251,2252..2962)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFIQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2291..2312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2552..2571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2899..2920
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3014..3528
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3690..3706
                     /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)."
     promoter        complement(join(3714..3720,3721..3738,3739..3744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3759..3780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3897..3914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4068..4656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4148..4167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4827..5618,5619..5687))
                     /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     5450..5469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5688..5792)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5860..5878
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5916..5938)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6038..6057
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6251..6273
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6401..6420
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccgggag ggcctatttc ccatgattcc ttcatatttg
     1441 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     1501 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     1561 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     1621 ttggctttat atatcttgtg gaaaggacga aacaccgggg tcttcgagaa gacctgttta
     1681 agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaactttgc
     1741 tggaaacagc aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa
     1801 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     1861 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     1921 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc
     1981 tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     2041 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2101 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2161 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg
     2221 tcgtgacgcg ggatccgcca ccatgatggt gagcaagggc gaggagctgt tcaccggggt
     2281 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2341 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2401 caagctgccc gtgccctggc ccaccctcgt gaccaccttc agctacggcg tgcagtgctt
     2461 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2521 ctacgtccag gagcgcacca tcagcttcaa ggacgacggc aactacaaga cccgcgccga
     2581 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2641 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2701 tatcacggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat
     2761 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2821 ccccgtgctg ctgcccgaca accactacct gttcatccag tccgccctga gcaaagaccc
     2881 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactca
     2941 cggcatggac gagctgtaca agagcttatc gataaaataa aagattttat ttagtctcca
     3001 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc
     3061 attttgcaag gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga
     3121 acagagagac agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     3181 gctcagggcc aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga
     3241 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     3301 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     3361 agagcccaca acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac
     3421 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     3481 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcatgg gtaacagttt
     3541 cttgaagttg gagaacaaca ttctgagggt aggagtcgaa tattaagtaa tcctgactca
     3601 attagccact gttttgaatc cacatactcc aatactcctg aaatagttca ttatggacag
     3661 cgcagaaaga gctggggaga attgtgaaat tgttatccgc tcacaattcc acacaacata
     3721 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3781 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3841 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3901 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3961 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4021 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4081 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4141 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4201 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4261 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4321 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4381 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4441 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4501 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4561 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4621 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4681 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4741 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4801 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4861 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4921 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4981 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5041 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5101 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5161 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5221 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5281 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5341 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5401 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5461 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5521 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5581 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5641 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5701 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5761 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5821 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     5881 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     5941 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     6001 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     6061 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6121 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     6181 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     6241 ccagggtttt cccagtcacg acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg
     6301 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     6361 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     6421 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagaggc
     6481 gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac
     6541 aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta
     6601 gtctccagaa aaagggggg
//
