LOCUS       sequence-332488-        8100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TOX in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8100)
  AUTHORS   Seo H, Chen J, Gonzalez-Avalos E, Samaniego-Castruita D, Das A, Wang
            YH, Lopez-Moyado IF, Georges RO, Zhang W, Onodera A, Wu CJ, Lu LF, 
            Hogan PG, Bhandoola A, Rao A
  TITLE     TOX and TOX2 transcription factors cooperate with NR4A transcription
            factors to impose CD8(+) T cell exhaustion.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Jun 18;116(25):12410-12415. doi: 
            10.1073/pnas.1905675116. Epub 2019 May 31.
  PUBMED    31152140
REFERENCE   2  (bases 1 to 8100)
  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: 
            "10.1073/pnas.1905675116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-06-18- 18"; volume: "116"; issue: "25"; pages: 
            "12410-12415"
COMMENT     Sequence Label: MSCV-Tox-IRES-eGFP
FEATURES             Location/Qualifiers
     source          1..8100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1034..1052
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1090..1112)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1212..1231
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1425..1447
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1575..1594
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             1794..2310
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    2374..2715
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             2782..3198
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     3128..3150
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     3166..3182
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      3211..3220
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3220..3285
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4886..5453
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4903..5455
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5047..5064)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5274..5293
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5467..5469,5470..5472,5473..6186)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5512..5533)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5773..5792)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6120..6141
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             6288..6802
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    6964..6980
                     /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(6988..6994,6995..7012,7013..7018))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7033..7054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7171..7188)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7342..7930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7422..7441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     1021 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     1081 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     1141 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     1201 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     1261 accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
     1321 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     1381 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     1441 taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     1501 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     1561 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     1621 gtgatgccgg ccacgatgcg tccggcgtag aggcgattag tccaatttgt taaagacagg
     1681 atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa gcctatagag
     1741 tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa
     1801 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa
     1861 atacataact gagaatagag aagttcagat caaggttagg aacagagaga cagcagaata
     1921 tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga
     1981 tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg
     2041 gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc
     2101 tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac
     2161 tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtattcc caataaagcc
     2221 tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt gggagggtct cctcagattg
     2281 attgactgcc cacctcgggg gtctttcatt tggaggttcc accgagattt ggagacccct
     2341 gcccagggac caccgacccc cccgccggga ggtaagctgg ccagcggtcg tttcgtgtct
     2401 gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt tgcgcctgcg tctgtactag
     2461 ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc
     2521 ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga
     2581 ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag
     2641 aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc
     2701 gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt
     2761 ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt
     2821 cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt
     2881 tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc
     2941 acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat
     3001 ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct
     3061 ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg
     3121 tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc
     3181 actccttctc taggcgccgg aattagatct gccaccatgg actacaaaga ccatgacggt
     3241 gattataaag atcatgacat cgattacaag gatgacgatg acaagcttgc ggccgcgatg
     3301 gacgtaagat tttatcctcc tccagcccag cccgctgctg cgcccgccgc tccttgtctg
     3361 ggaccttctc cctgcctgga cccctactat tgcaacaagt ttgacgggga gaacatgtat
     3421 atgagcatga cagagccgag ccaggactat gtgccagcca gccagtccta ccctggacca
     3481 agtctggaaa gtgaagattt taacatccca cccatcacgc ctccctccct ccctgaccac
     3541 tctctggtgc acctgaatga ggtagaatcc ggttaccact ctctgtgtca ccccatgaat
     3601 cacaatggcc tgctcccgtt ccatccacaa accatggacc tgccagagat cacggtgtcc
     3661 aacatgctgg gccaagatgg cgcactgctc tccaattcca tctctgtgat gcaagagatt
     3721 gggaacgcag aaggagctca gtacagctcc catcctcaga tggcagccat gaggccgaga
     3781 ggccagccta cagacatcag acagcaggca agcatgatgc agcccggcca gctgaccacc
     3841 atcaaccagt cgcagctgag cgcacagttg ggcttgaaca tgggaggaac caatgtggcc
     3901 cacaactccc cttctccacc cgggagcaag tctgcaaccc cttccccatc cagctcagtg
     3961 catgaggatg agtgtgagga tgcctccaag atcaatggag gagagaagcg gcctgcctct
     4021 gatatgggga aaaaacccaa aactcccaag aagaagaaga agaaggatcc taatgagccc
     4081 cagaagcctg tgtctgccta tgctttgttc tttcgtgata cccaggctgc cattaagggc
     4141 cagaatccaa atgctacttt tggtgaagta tcgaagattg tggcatcaat gtgggatggc
     4201 ctaggagaag aacagaaaca ggtctataag aagaaaaccg aggctgcaaa gaaggaatac
     4261 ctgaagcagc tagcagcata cagagccagc cttgtatcca agagctacac tgatcctgtt
     4321 gatgttaaga catctcagcc accccagctg gtcaactcaa agccgtcagt attccatggg
     4381 cccagccagg cccactcagc tctgtaccta agttctcact atcaccaaca accaggaatg
     4441 actcctcagc tcaccgccat gcatccgagc ctccccagaa acatagcacc taagcccaat
     4501 aaccaaatgc cagtgactgt ctccatagcc aacatggctg tgtccccacc accgcccctt
     4561 cagatcagcc cacctcttca tcagcatctc agcatgcagc agcatcagtc gcttgccatg
     4621 cagcagcccc ttgggagtca gctccccatg caggtccaga ctgccttaca ctcgcccacc
     4681 atgcagcaag gatttactct tcaacccgac tatcagacta ttatcaaccc tacatctaca
     4741 gctgcacaag ttgtcaccca agcgatggag tacgtgcgtt ctgggtgcag aaatccgccc
     4801 ccacagcccg tggactggag taccgactac tgcagtagtg ggggcatgca gagggacaaa
     4861 gcgctgtatc tcacctgata agaattccgc cccccccccc taacgttact ggccgaagcc
     4921 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt
     4981 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc
     5041 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc
     5101 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc
     5161 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg
     5221 cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct
     5281 cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat
     5341 ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc
     5401 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc
     5461 acaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     5521 ctggacggcg acgtaaacgg ccacaagttc agcgtgtctg gcgagggcga gggcgatgcc
     5581 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     5641 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     5701 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     5761 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     5821 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     5881 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     5941 aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     6001 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     6061 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     6121 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     6181 aagtaatgaa ttaattaaga attatcaagc ttatcgatac cgtcgacctg cagccaagct
     6241 tatcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     6301 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaatacata
     6361 actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga atatgggcca
     6421 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc
     6481 cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc
     6541 aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc
     6601 tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggcgc
     6661 gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt atccaataaa ccctcttgca
     6721 gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
     6781 cccgtcagcg ggggtctttc atgggtaaca gtttcttgaa gttggagaac aacattctga
     6841 gggtaggagt cgaatattaa gtaatcctga ctcaattagc cactgttttg aatccacata
     6901 ctccaatact cctgaaatag ttcattatgg acagcgcaga aagagctggg gagaattgtg
     6961 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     7021 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     7081 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     7141 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     7201 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     7261 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7321 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     7381 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     7441 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     7501 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     7561 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     7621 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     7681 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7741 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     7801 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7861 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7921 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7981 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     8041 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
//
