LOCUS       sequence-487904-        8452 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA CTEVp chain encoding (N to C): 
            TNFR2SS-3xFLAG-TNFR2ECD-mCD28TMD-CTEVp(190K)-PRS(M)-VP64-ZF6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8452)
  AUTHORS   Edelstein HI, Cosio A, Ezekiel ML, Corcoran WK, Morris AH, Leonard 
            JN
  TITLE     Conversion of natural cytokine receptors into orthogonal synthetic 
            biosensors.
  JOURNAL   Nat Chem Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1.
  PUBMED    40846994
REFERENCE   2  (bases 1 to 8452)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1."
COMMENT     Sequence Label: L4219 TNFR2 CTEVp chain in PolyTX-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..8452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        359..1541
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          590..1532
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1489..1509
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1543..1567
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1648..1657
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1720..1785
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2950..2970
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2989..3009
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3016..3165
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     polyA_signal    3529..3753
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3896..4690
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3950..3969)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4560..4579
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4721..4815
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4836..5215
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5216..5419
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5370..5390
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5464..5483
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5464..5482
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5502..5511
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6255..6479
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6525..6953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6612..6631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6822..6843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6976..7172
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7023..7158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7085..7104
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    7292..7413
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7329..7348)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7383..7402
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7462..7478)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7462..7478)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7475..7497)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7486..7502
                     /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(7510..7516,7517..7534,7535..7540))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7555..7576
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7693..7710)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7864..8452)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7944..7963)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cgatcgaata
      181 ttagatctga gacccaattg acgcgtgagt aattcataca aaaggactcg cccctgcctt
      241 ggggaatccc agggaccgtc gttaaactcc cactaacgta gaacccagag atcgctgcgt
      301 tcccgccccc tcacccgccc gctctcgtca tcactgaggt ggagaagagc atgcgtgagg
      361 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      421 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      481 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      541 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
      601 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
      661 ttccacgccc ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
      721 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
      781 ggcttgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
      841 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
      901 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
      961 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     1021 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     1081 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     1141 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     1201 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     1261 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     1321 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     1381 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     1441 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     1501 cagtggttca aagttttttt cttccatttc aggtgtcgtg agagctcgtt tagtgaaccg
     1561 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagag aacgggaccg
     1621 atccaaattc tcgaggctag ctgtacagcc accatggctc ccgtggctgt ttgggctgct
     1681 ctggctgttg gactggaact gtgggctgcc gctcacgccg attacaagga tcacgacggc
     1741 gactataagg accacgacat cgattacaaa gacgacgacg acaagctgcc cgctcaggtg
     1801 gcctttacac cttacgctcc agagcctggc agcacctgta gactgagaga gtactacgac
     1861 cagacagccc agatgtgctg ctccaagtgt tctcctggac agcacgccaa ggtgttctgc
     1921 accaagacca gcgacaccgt gtgcgacagc tgcgaggata gcacctacac tcagctgtgg
     1981 aactgggtgc ccgagtgtct gtcttgcggc agcagatgta gcagcgacca ggtggaaaca
     2041 caggcctgca ccagagagca gaaccggatc tgcacatgca gacccggctg gtactgtgcc
     2101 ctgtctaagc aagagggatg cagactgtgc gcccctctga gaaagtgcag acctggcttc
     2161 ggagtggcca gaccaggcac cgagacatct gatgtcgtgt gcaagccttg cgctcccggc
     2221 acctttagca ataccaccag cagcaccgac atctgccggc ctcaccaaat ctgcaacgtg
     2281 gtggccattc ctggcaacgc ctctatggat gccgtgtgta ccagcacaag ccccactcgg
     2341 tctatggctc caggtgctgt tcatctgccc cagcctgtgt ctaccagaag ccagcacaca
     2401 cagcccacac ctgagccttc tacagcccct agcaccagct ttctgctgcc tatgggacct
     2461 tctccaccag ccgaaggcag caccggcgat accggtcttg tcgtcgttgc aggggtgctc
     2521 ttctgttatg gtctgttggt gacagtcgca ctgtgtgtag gaggcggtag cggaggcggc
     2581 gccaagagca tgtctagcat ggtgtcagac actagttgca cattcccttc atctgatggc
     2641 atattctgga agcattggat tcaaaccaag gatgggcagt gtggcagtcc attagtatca
     2701 actagagatg ggttcattgt tggtatacac tcagcatcga atttcaccaa cacaaacaat
     2761 tatttcacaa gcgtgccgaa aaacttcatg gaattgaaga caaatcagga ggcgcagcag
     2821 tgggttagtg gttggcgatt aaatgctgac tcagtattgt gggggggcca taaagttttc
     2881 atggtgaaac ctgaagagcc ttttcagcca gttaaggaag cgactcaact catgaatctt
     2941 aagggctcgg agaacctgta ttttcagatg gggggaggca gcgggggccc taagaaaaag
     3001 cgcaaagtct ccggagacgc tctggatgac tttgatctgg acatgcttgg ttccgatgcc
     3061 ttggacgatt ttgacctcga tatgctgggc tccgacgctc tcgacgattt cgacctcgac
     3121 atgctgggta gcgatgcact ggatgacttt gatctggata tgctcggatc cggtggtggc
     3181 gggtctggag gaggaggtag tggtggaggt ggctctggta ccgctagacc cggcgaaaga
     3241 cctttccagt gccggatctg catgaggaac ttcagcaagg gcgagagact cgtgcggcac
     3301 accagaacac acacaggcga gaagcccttc cagtgtagaa tctgtatgcg caacttcagc
     3361 cggatggaca acctgagcac ccacctgaga acccataccg gggagaagcc atttcaatgc
     3421 cgcatctgta tgagaaattt ttcccggaag gacgccctga accggcacct gaaaacacac
     3481 ctgagaggca gctgagcggc cgcgtttaaa cccgctgatc agcctcgact gtgccttcta
     3541 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3601 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3661 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagtcaata
     3721 gcaggcatgc tggggatgcg gtgggctcta tgggacgtcg gaattgcaag ttgggccgcc
     3781 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     3841 ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3901 tgaacaagat ggattgcacg caggttctcc ggcggcttgg gtggagaggc tattcggcta
     3961 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4021 gggtcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4081 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcgg ctgtgctcga
     4141 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4201 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4261 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4321 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4381 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgtatgc ccgacggcga
     4441 ggatctcgtc gtgacccacg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4501 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4561 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4621 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4681 gttcttctga gcgggactct ggggttcgag aattggcttg gactcctgtt gatagatcca
     4741 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     4801 tttattggtg agaatccaag cactagccat gaccagacat tgattattga ctagttatta
     4861 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     4921 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4981 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     5041 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     5101 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     5161 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     5221 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     5281 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     5341 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     5401 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     5461 aattaatacg actcactata gggagtccca agctgactag cgccaccatg gtttccaagg
     5521 gcgaagaact gatcaaagaa aacatgcaca tgaagctcta catggaaggc accgtggaca
     5581 accaccactt caagtgtacc agcgaaggcg agggcaagcc ttacgagggc acccagacca
     5641 tgagaatcaa ggtggtggaa ggcggccctc tgcctttcgc ctttgatatc ctggccacca
     5701 gctttctgta cggcagcaag accttcatca atcacaccca gggcatcccc gatttcttca
     5761 agcagagctt ccccgagggc ttcacctggg agagagtgac cacatacgag gatggcggcg
     5821 tgctgacagc cacacaggat acaagcctcc aggacggctg cctgatctac aacgtgaaga
     5881 tccggggcgt gaacttcacc agcaacggcc ccgtgatgca gaagaaaacc cttggctggg
     5941 aagccttcac cgagacactg tatcctgccg atggcggcct ggaaggcaga aacgatatgg
     6001 ccctgaagct cgtcggcggc tctcacctga ttgccaatgc caagaccacc tacagaagca
     6061 agaagcccgc caagaacctg aagatgcccg gcgtgtacta cgtggactac cggctggaaa
     6121 gaatcaaaga ggccaacaac gagacatacg tggaacagca cgaggtggcc gtggccagat
     6181 actgtgatct gccttctaag ctgggccaca agctgaactg agcggccaag tttaaacccg
     6241 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     6301 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     6361 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     6421 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatggc
     6481 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     6541 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6601 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6661 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6721 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6781 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6841 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6901 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     6961 tggaatgtgt gtcagttagg ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     7021 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     7081 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     7141 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     7201 tcggaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     7261 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     7321 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     7381 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     7441 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     7501 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     7561 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     7621 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     7681 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     7741 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     7801 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     7861 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     7921 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7981 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     8041 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     8101 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     8161 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     8221 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     8281 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     8341 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     8401 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aa
//
