LOCUS       sequence-319095-        6637 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Retroviral expression plasmid of sgRNA with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6637)
  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 6637)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-mCherry
FEATURES             Location/Qualifiers
     source          1..6637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1406..1654
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1406..1426
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1577..1596
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1818..1935
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2004..2215
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2235..2244
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2244..2267
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2268..2978
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2413..2431)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     LTR             3030..3544
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3706..3722
                     /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(3730..3736,3737..3754,3755..3760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3775..3796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3913..3930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4084..4672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4164..4183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4843..5634,5635..5703))
                     /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     5466..5485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5704..5808)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5876..5894
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5932..5954)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6054..6073
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6267..6289
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6417..6436
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6636..6637,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccgggaggg cctatttccc atgattcctt catatttgca
     1441 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     1501 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     1561 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     1621 ggctttatat atcttgtgga aaggacgaaa caccggggtc ttcgagaaga cctgtttaag
     1681 agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc aactttgctg
     1741 gaaacagcaa agtggcaccg agtcggtgct ttttttgaat tctagatctt gagacaaatg
     1801 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     1861 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     1921 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc
     1981 gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     2041 ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga
     2101 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     2161 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc
     2221 gtgacgcggg atccgccacc atggattaca aagacgatga cgataagatg gtgagcaagg
     2281 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     2341 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     2401 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     2461 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     2521 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2581 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     2641 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaaa
     2701 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2761 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     2821 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2881 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2941 gccactccac cggcggcatg gacgagctgt acaagtaaag cttatcgata aaataaaaga
     3001 ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc
     3061 tagcttaagt aacgccattt tgcaaggcat ggaaaataca taactgagaa tagagaagtt
     3121 cagatcaagg ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta
     3181 agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg gtcccgccct
     3241 cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc
     3301 tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct
     3361 ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgatagac
     3421 tgcgtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg
     3481 tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt
     3541 tcatgggtaa cagtttcttg aagttggaga acaacattct gagggtagga gtcgaatatt
     3601 aagtaatcct gactcaatta gccactgttt tgaatccaca tactccaata ctcctgaaat
     3661 agttcattat ggacagcgca gaaagagctg gggagaattg tgaaattgtt atccgctcac
     3721 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3781 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3901 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3961 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4021 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4081 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4141 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4201 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4261 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4321 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4381 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4441 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4501 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4561 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4621 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4681 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4741 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4801 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4861 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4921 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4981 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5041 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5101 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5161 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5221 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5281 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5341 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5401 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5461 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5521 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5581 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5641 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5701 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5761 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5821 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5881 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     5941 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     6001 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     6061 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     6121 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     6181 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     6241 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggcgcaagg
     6301 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6361 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6421 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6481 cgtccggcgt agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct
     6541 ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa
     6601 taaaagattt tatttagtct ccagaaaaag gggggaa
//
