LOCUS       sequence-445714-        7883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse TBET and mCherry under EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7883)
  AUTHORS   Fagerberg E, Attanasio J, Dien C, Singh J, Kessler EA, Abdullah L, 
            Shen J, Hunt BG, Connolly KA, De Brouwer E, He J, Iyer NR, Buck J, 
            Borr ER, Damo M, Foster GG, Giles JR, Huang YH, Tsang JS, 
            Krishnaswamy S, Cui W, Joshi NS
  TITLE     KLF2 maintains lineage fidelity and suppresses CD8 T cell exhaustion
            during acute LCMV infection.
  JOURNAL   Science. 2025 Jan 2;387(6735):eadn2337. doi: 
            10.1126/science.adn2337. Epub 2025 Feb 14.
  PUBMED    39946463
REFERENCE   2  (bases 1 to 7883)
  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"; journalName: "Science."; 
            date: "2025-01-2"; volume: "387(6735):eadn2337. doi"; pages: " 
            10.1126/science.adn2337. Epub 2025 Feb 14"
COMMENT     Sequence Label: EF1a-TBET-T2A-mCHERRY
FEATURES             Location/Qualifiers
     source          1..7883
                     /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        1466..1677
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1691..1700
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3287..3340
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3347..4051
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWDASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3489..3507)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3757..3776
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4088..4107)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     LTR             4276..4790
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4952..4968
                     /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(4976..4982,4983..5000,5001..5006))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5021..5042
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5159..5176)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5330..5918)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5410..5429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6089..6880,6881..6949))
                     /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     6712..6731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6950..7054)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7122..7140
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7178..7200)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7300..7319
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7513..7535
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7663..7682
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7882..7883,1..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 gccggaattg cttcgcgatg tacgggccag gaaaggagtg
     1441 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     1501 ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg
     1561 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     1621 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggac
     1681 cggttctaga gccaccatgg gcatcgtgga gccgggctgc ggagacatgc tgaccggcac
     1741 cgagccgatg ccgagtgacg agggccgggg gcccggagcg gaccaacagc atcgtttctt
     1801 ctatcccgag ccgggcgcac aggacccgac cgatcgccgc gcaggtagca gcctggggac
     1861 gccctactct gggggcgccc tggtgcctgc cgcgccgggt cgcttccttg gatccttcgc
     1921 ctacccgccc cgggctcagg tggctggctt tcccgggcct ggcgagttct tcccgccgcc
     1981 cgcgggtgcg gagggctacc cgcccgtgga tggctaccct gcccctgacc cgcgcgcggg
     2041 gctctaccca gggccgcgcg aggactacgc attgcccgcg gggttggagg tgtctgggaa
     2101 gctgagagtc gcgctcagca accacctgtt gtggtccaag ttcaaccagc accagacaga
     2161 gatgatcatc actaagcaag gacggcgaat gttcccattc ctgtccttca ccgtggccgg
     2221 gctggagccc acaagccatt acaggatgtt tgtggatgtg gtcttggtgg accagcacca
     2281 ctggcggtac cagagcggca agtgggtgca gtgtggaaag gcagaaggca gcatgccagg
     2341 gaaccgctta tatgtccacc cagactcccc caacaccgga gcccactgga tgcgccagga
     2401 agtttcattt gggaagctaa agctcaccaa caacaagggg gcttccaaca atgtgaccca
     2461 gatgatcgtc ctgcagtctc tccacaagta ccagccccgg ctgcacatcg tggaggtgaa
     2521 tgatggagag ccagaggctg cctgcagtgc ttctaacaca cacgtcttta ctttccaaga
     2581 gacccagttc attgcagtga ctgcctacca gaacgcagag atcactcagc tgaaaatcga
     2641 caacaacccc tttgccaaag gattccggga gaactttgag tccatgtacg catctgttga
     2701 tacgagtgtc ccctcgccac ctggacccaa ctgtcaactg cttgggggag accccttctc
     2761 acctcttcta tccaaccagt atcctgttcc cagccgtttc taccccgacc ttccaggcca
     2821 gcccaaggat atgatctcac agccttactg gctggggaca cctcgggaac acagttatga
     2881 agcggagttc cgagctgtga gcatgaagcc cacactccta ccctctgccc cggggcccac
     2941 tgtgccctac taccggggcc aagacgtcct ggcgcctgga gctggttggc ccgtggcccc
     3001 tcaatacccg cccaagatga gcccagctgg ctggttccgg cccatgcgaa ctctgcccat
     3061 ggacccgggc ctgggatcct cagaggaaca gggctcctcc ccctcgctgt ggcctgaggt
     3121 cacctccctc cagccggagc ccagcgactc aggactaggc gaaggagaca ctaagaggag
     3181 gaggatatcc ccctatcctt ccagtggcga cagctcctct cccgctgggg ccccttctcc
     3241 ttttgataag gaaaccgaag gccagtttta taattatttt cccaacgagg gcagaggaag
     3301 tcttctaaca tgcggtgacg tggaggagaa tcccggccct ggtaccgtga gcaagggcga
     3361 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     3421 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     3481 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     3541 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     3601 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     3661 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     3721 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     3781 gggctgggat gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     3841 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     3901 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     3961 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     4021 ctccaccggc ggcatggacg agctgtacaa gaccggttag taatgagttt aaacggggga
     4081 ggctaactga aacacggaag gagacaatac cggaaggggc cgcgactcta gagtcgacct
     4141 gcaggcatgc aagcttcagg tagccggcta acgttaacaa ccggtacctc tagaactata
     4201 gctagcatgc gcaaatttaa agcgctgata tcgataaaat aaaagatttt atttagtctc
     4261 cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg
     4321 ccattttgca aggcatggaa aatacataac tgagaataga gaagttcaga tcaaggttag
     4381 gaacagagag acagcagaat atgggccaaa caggatatct gtggtaagca gttcctgccc
     4441 cggctcaggg ccaagaacag atggtcccca gatgcggtcc cgccctcagc agtttctaga
     4501 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
     4561 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
     4621 aaagagccca caacccctca ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt
     4681 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
     4741 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat gggtaacagt
     4801 ttcttgaagt tggagaacaa cattctgagg gtaggagtcg aatattaagt aatcctgact
     4861 caattagcca ctgttttgaa tccacatact ccaatactcc tgaaatagtt cattatggac
     4921 agcgcagaaa gagctgggga gaattgtgaa attgttatcc gctcacaatt ccacacaaca
     4981 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5041 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5101 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5161 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5221 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5281 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5341 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5401 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5461 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5521 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5581 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5641 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5701 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5761 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5821 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5881 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5941 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6001 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6061 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6121 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6181 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6241 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6301 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6361 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6421 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6481 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6541 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6601 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6661 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6721 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6781 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6841 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6901 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6961 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7021 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7081 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     7141 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     7201 agacggtcac agcttgtctg taagcggatg ccgggagcaa acaagcccgt cagggcgcgt
     7261 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     7321 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     7381 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     7441 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     7501 cgccagggtt ttcccagtca cgacgttgta aaacgacggc gcaaggaatg gtgcatgcaa
     7561 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     7621 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     7681 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     7741 gcgattagtc caatttgtta aagacaggat atcagtggtc caggctctag ttttgactca
     7801 acaatatcac cagctgaagc ctatagagta cgagccatag ataaaataaa agattttatt
     7861 tagtctccag aaaaaggggg gaa
//
