LOCUS       sequence-453642-        8309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses estrogen receptor linked KLF2 and mCherry under EF1a 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8309)
  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 8309)
  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-ER-KLF2-T2A-mCHERRY
FEATURES             Location/Qualifiers
     source          1..8309
                     /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             1700..2635
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             3713..3766
                     /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             3773..4477
                     /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(3915..3933)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4183..4202
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4514..4533)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     LTR             4702..5216
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5378..5394
                     /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(5402..5408,5409..5426,5427..5432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5447..5468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5585..5602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5756..6344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5836..5855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6515..7306,7307..7375))
                     /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     7138..7157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7376..7480)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7548..7566
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7604..7626)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7726..7745
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7939..7961
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8089..8108
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8308..8309,1..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 ctggagacat gagagctgcc aacctttggc caagcccgct
     1741 catgatcaaa cgctctaaga agaacagcct ggccttgtcc ctgacggccg accagatggt
     1801 cagtgccttg ttggatgctg agccccccat actctattcc gagtatgatc ctaccagacc
     1861 cttcagtgaa gcttcgatga tgggcttact gaccaacctg gcagacaggg agctggttca
     1921 catgatcaac tgggcgaaga gggtgccagg ctttgtggat ttgaccctcc atgatcaggt
     1981 ccaccttcta gaatgtgcct ggctagagat cctgatgatt ggtctcgtct ggcgctccat
     2041 ggagcaccca gtgaagctac tgtttgctcc taacttgctc ttggacagga accagggaaa
     2101 atgtgtagag ggcatggtgg agatcttcga catgctgctg gctacatcat ctcggttccg
     2161 catgatgaat ctgcagggag aggagtttgt gtgcctcaaa tctattattt tgcttaattc
     2221 tggagtgtac acatttctgt ccagcaccct gaagtctctg gaagagaagg accatatcca
     2281 ccgagtcctg gacaagatca cagacacttt gatccacctg atggccaagg caggcctgac
     2341 cctgcagcag cagcaccagc ggctggccca gctcctcctc atcctctccc acatcaggca
     2401 catgagtaac aaaggcatgg agcatctgta cagcatgaag tgcaagaacg tggtgcccct
     2461 ctatgacctg ctgctggagg cggcggacgc ccaccgccta catgcgccca ctagccgtgg
     2521 aggggcatcc gtggaggaaa cggaccaaag ccacttggcc actgcgggct ctacttcatc
     2581 gcattccttg caaaagtatt acatcacggg ggaggcagag ggtttccctg ccacagctgt
     2641 cgacatggcg ctcagcgagc ctatcttgcc tagttttgcc acttttgcat ccccctgtga
     2701 acgaggattg caggaacgct ggcctcgcaa cgaaccagaa gctggaggga ccgacgaaga
     2761 cctcaacaat gtgttggatt ttatcctgag tatggggctc gatggcttgg gggccgaaaa
     2821 cccaccagaa ccacctccac agccccctcc accagccttt tattacccag aacccggagc
     2881 accaccacca tactcaatcc ccgccgcttc acttggtaca gaactcctca ggccagattt
     2941 ggatcctcca cagggtcccg cattgcacgg acgcttcctt ttggcccctc cagggcgact
     3001 ggtcaaggcc gagcctcctg aagtggatgg tggaggctat gggtgcgctc cagggctggc
     3061 tcacgggcca agaggcttga aacttgaggg cgctccaggt gcaaccggag cctgcatgcg
     3121 gggtcccgcc ggccgccccc cgccgccccc ggacacgccg ccgctcagcc ccgacggccc
     3181 cctgcgcatc ccggcgtccg gtccacgcaa cccgttcccg ccgcccttcg gtcccggccc
     3241 cagcttcggc ggccccggcc ccgcgttgca ctacgggcct cccgcgcctg gcgccttcgg
     3301 tcttttcgag gacgcggcgg cagcggcggc ggcgctgggc ttggctccac ctgccacgcg
     3361 cggtctcctc acgccgccct cgtccccgct ggagctgctg gaggccaagc ccaaacgcgg
     3421 ccgccgctcc tggccccgca agcgcgccgc cacacatact tgcagctaca ccaactgcgg
     3481 caagacctac accaagagct cgcacctaaa ggcgcatctg cgtacacaca caggtgagaa
     3541 gccttatcat tgcaactggg aaggatgcgg ctggaagttc gcgcgctctg acgagcttac
     3601 ccgccactac cgaaagcaca caggtcacag accctttcag tgccacttgt gcgaccgggc
     3661 cttctccagg tccgaccacc tggccttgca catgaagcga cacatggcta gcgagggcag
     3721 aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctggta ccgtgagcaa
     3781 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     3841 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     3901 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     3961 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     4021 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     4081 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     4141 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     4201 gaccatgggc tgggatgcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     4261 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     4321 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     4381 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     4441 ccgccactcc accggcggca tggacgagct gtacaagacc ggttagtaat gagtttaaac
     4501 gggggaggct aactgaaaca cggaaggaga caataccgga aggggccgcg actctagagt
     4561 cgacctgcag gcatgcaagc ttcaggtagc cggctaacgt taacaaccgg tacctctaga
     4621 actatagcta gcatgcgcaa atttaaagcg ctgatatcga taaaataaaa gattttattt
     4681 agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa
     4741 gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa
     4801 ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc
     4861 ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt
     4921 tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
     4981 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
     5041 tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc
     5101 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
     5161 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt
     5221 aacagtttct tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc
     5281 ctgactcaat tagccactgt tttgaatcca catactccaa tactcctgaa atagttcatt
     5341 atggacagcg cagaaagagc tggggagaat tgtgaaattg ttatccgctc acaattccac
     5401 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5461 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5521 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5581 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5641 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5701 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5761 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5821 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5881 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5941 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6001 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6061 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6121 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6181 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6241 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6301 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6361 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6421 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6481 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6541 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6601 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6661 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6721 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6781 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6841 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6901 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6961 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7021 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7081 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7141 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7201 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7261 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7321 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7381 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7441 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7501 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7561 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     7621 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcaaacaa gcccgtcagg
     7681 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     7741 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     7801 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     7861 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     7921 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc
     7981 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     8041 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     8101 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     8161 gtagaggcga ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt
     8221 gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat
     8281 tttatttagt ctccagaaaa aggggggaa
//
