LOCUS       sequence-324328-        6637 bp    DNA     circular SYN 15-DEC-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 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-mCherry
FEATURES             Location/Qualifiers
     source          1..6637
                     /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"
     regulatory      2237..2246
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2246..2269
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2270..2980
                     /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(2415..2433)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     LTR             3032..3546
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3708..3724
                     /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(3732..3738,3739..3756,3757..3762))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3777..3798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3915..3932)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4086..4674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4166..4185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4845..5636,5637..5705))
                     /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     5468..5487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5706..5810)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5878..5896
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5934..5956)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6056..6075
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6269..6291
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6419..6438
                     /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 ccatggatta caaagacgat gacgataaga tggtgagcaa
     2281 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     2341 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     2401 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     2461 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     2521 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     2581 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     2641 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     2701 aaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     2761 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     2821 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     2881 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     2941 ccgccactcc accggcggca tggacgagct gtacaagtaa agcttatcga taaaataaaa
     3001 gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa
     3061 gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag
     3121 ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg
     3181 taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc
     3241 ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac
     3301 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg
     3361 ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag
     3421 actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt
     3481 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     3541 tttcatgggt aacagtttct tgaagttgga gaacaacatt ctgagggtag gagtcgaata
     3601 ttaagtaatc ctgactcaat tagccactgt tttgaatcca catactccaa tactcctgaa
     3661 atagttcatt atggacagcg cagaaagagc tggggagaat tgtgaaattg ttatccgctc
     3721 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3781 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3841 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3901 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3961 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4021 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4081 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4141 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4201 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4261 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4321 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4381 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4441 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4501 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4561 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4621 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4681 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4741 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4801 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4861 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4921 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4981 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5041 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5101 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5161 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5221 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5281 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5341 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5401 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5461 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5521 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5581 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5641 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5701 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5761 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5821 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5881 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     5941 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     6001 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     6061 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     6121 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6181 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     6241 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggcgcaa
     6301 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6361 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6421 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     6481 tgcgtccggc gtagaggcga ttagtccaat ttgttaaaga caggatatca gtggtccagg
     6541 ctctagtttt gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa
     6601 aataaaagat tttatttagt ctccagaaaa agggggg
//
