LOCUS       sequence-326092-        8088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TOX2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8088)
  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 8088)
  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-Tox2-IRES-eGFP
FEATURES             Location/Qualifiers
     source          1..8088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(455..1246,1247..1315))
                     /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     1078..1097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1316..1420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1488..1506
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1544..1566)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1666..1685
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1879..1901
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2029..2048
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             2248..2764
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    2828..3169
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             3236..3652
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     3582..3604
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     3620..3636
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      3665..3674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3674..3739
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    5328..5895
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    5345..5897
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5489..5506)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5716..5735
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5909..5911,5912..5914,5915..6628)
                     /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(5954..5975)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6215..6234)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6562..6583
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             6730..7244
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    7406..7422
                     /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(7430..7436,7437..7454,7455..7460))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7475..7496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7613..7630)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7784..8088,1..284))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7864..7883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
       61 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
      121 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
      181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
      241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
      301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
      361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
      421 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
      481 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
      541 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
      601 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
      661 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
      721 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
      781 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
      841 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
      901 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
      961 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     1021 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     1081 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     1141 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     1201 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     1261 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     1321 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     1381 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     1441 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     1501 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1561 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1621 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     1681 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     1741 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     1801 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     1861 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc
     1921 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     1981 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     2041 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     2101 gtagaggcga ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt
     2161 gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat
     2221 tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct
     2281 agcttaagta acgccatttt gcaaggcatg gaaaatacat aactgagaat agagaagttc
     2341 agatcaaggt taggaacaga gagacagcag aatatgggcc aaacaggata tctgtggtaa
     2401 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tcccgccctc
     2461 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
     2521 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
     2581 cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgatagact
     2641 gcgtcgcccg ggtacccgta ttcccaataa agcctcttgc tgtttgcatc cgaatcgtgg
     2701 actcgctgat ccttgggagg gtctcctcag attgattgac tgcccacctc gggggtcttt
     2761 catttggagg ttccaccgag atttggagac ccctgcccag ggaccaccga cccccccgcc
     2821 gggaggtaag ctggccagcg gtcgtttcgt gtctgtctct gtctttgtgc gtgtttgtgc
     2881 cggcatctaa tgtttgcgcc tgcgtctgta ctagttagct aactagctct gtatctggcg
     2941 gacccgtggt ggaactgacg agttctgaac acccggccgc aaccctggga gacgtcccag
     3001 ggactttggg ggccgttttt gtggcccgac ctgaggaagg gagtcgatgt ggaatccgac
     3061 cccgtcagga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc
     3121 tgaatttttg ctttcggttt ggaaccgaag ccgcgcgtct tgtctgctgc agcgctgcag
     3181 catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa attagggcca
     3241 gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg agcggatcgc
     3301 tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct ctgcagaatg
     3361 gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc tcatcaccca
     3421 ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg tcccctacat
     3481 cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct ttgtacaccc
     3541 taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac ctcctcgttc
     3601 gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg ccggaattag
     3661 atctgccacc atggactata aggatcacga tggtgactat aaagatcatg acattgacta
     3721 caaggatgac gacgacaagc tcgccgcagc aatgcaacaa cacttgaccg aggcagtggc
     3781 aggggccttt agcagatgtc ttgggttccg gggcatgaga ctccggctgc tgactcgcca
     3841 ttggtgcata gccggggcat ctttgcagaa gttcgatgga gatagtgcct atgtgggtat
     3901 gtccgatggt aacccagaac ttctcagtac cagccagacc tacaatagtc agggtgagag
     3961 caacgaggac tatgaaattc ctcccattac ccccccaaat ctgcccgaac cttccttgct
     4021 tcacctgggt gaccacgagg ctggttacca cagtctgtgt catggactgg ctccaaacgg
     4081 cttgctccca gcttatagct accaagcaat ggacctgcct gctatcatgg tcagcaatat
     4141 gctggcccag gacggacacc tgctgtcagg acagctccca acaattcaag agatggtgca
     4201 cagtgaagtg gccgcatatg attccgggag gccaggcccc ttgcttggaa gacccgccat
     4261 gcttgcctca catatgtcag ccttgagcca aagtcaactg atcagccaaa tgggcatacg
     4321 ctctggcatt gctcacggca gccccagtcc acccggttct aaatccgcta cacccagtcc
     4381 aagtagcagc actcaagagg aggagtctga cgcccatttt aagatctcag gggagaagcg
     4441 gccaagtgtg gaccctggta agaaggccaa gaatccaaag aaaaagaaga aaaaagaccc
     4501 taatgagcca cagaaaccag tgtccgctta cgccctcttc ttcagagata cccaagcagc
     4561 tataaaaggt cagaacccct ccgcaacctt tggtgatgtc tcaaaaatag tagcttctat
     4621 gtgggatagc ttgggagaag agcagaaaca ggcctacaaa cggaagaccg aggctgccaa
     4681 gaaggagtac ttgaaggccc ttgctgcata cagagcttct ttggtatcaa aatccccacc
     4741 cgaccagggc gaggctaaaa acactcaggc caatcctccc gctaagatgc ttccccctaa
     4801 gcaaccaatg tacgcaatgc ccggcttggc ttcttttttg acaccaagcg accttcaggc
     4861 tttcagaagc ggagcaagcc cagcatctct cgccaggacc cttggttcca aagctctgtt
     4921 gcccggtctg tcaacttcac ccccaccacc aagcttcccc ttgtcaccct cactgcatca
     4981 acaattgcca ctgccccctc atgcacaagg aacacttttg tcccctcctc ttagcatgtc
     5041 acccgcccct caaccccctg tattgccagc ctccatggca ctccaagttc agctggctat
     5101 gtccccctca cctcccggcc ctcaggactt tccccatatt tcagacttta gtagcgggtc
     5161 cggatcacga agtcctggtc cttccaatcc ctcatcatct ggcgactggg acggttccta
     5221 cccatcaggc gagcgcggac tggggacctg tagactgtgc agaggctctc caccccctac
     5281 aacctcaccc aaaaatcttc aggagccctc cgcacgctga taagaattcc gccccccccc
     5341 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     5401 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     5461 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     5521 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     5581 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     5641 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     5701 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     5761 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     5821 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     5881 aaaacacgat gataatatgg ccacaaccat ggtgagcaag ggcgaggagc tgttcaccgg
     5941 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     6001 tggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     6061 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     6121 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     6181 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     6241 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     6301 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     6361 ctatatcatg gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa
     6421 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     6481 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     6541 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     6601 tctcggcatg gacgagctgt acaagtaatg aattaattaa gaattatcaa gcttatcgat
     6661 accgtcgacc tgcagccaag cttatcgata aaataaaaga ttttatttag tctccagaaa
     6721 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt
     6781 tgcaaggcat ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag
     6841 agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
     6901 agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca
     6961 tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac
     7021 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag
     7081 cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt
     7141 gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg
     7201 gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa cagtttcttg
     7261 aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct gactcaatta
     7321 gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat ggacagcgca
     7381 gaaagagctg gggagaattg tgaaattgtt atccgctcac aattccacac aacatacgag
     7441 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     7501 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     7561 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     7621 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     7681 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     7741 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     7801 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     7861 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     7921 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     7981 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     8041 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatc
//
